Stabilized and atomic layer deposition (ALD)-coated lipid assembly-free polynucleotide-containing microparticles and uses thereof

Abstract

Embodiments of the present disclosure provide novel compositions and methods for making and using thermostable coated therapeutic polynucleic acid-containing formulations. In certain embodiments, compositions and methods are disclosed for using lipid assembly-free, or naked therapeutic polynucleic acid sequence- or polynucleotide-containing formulations having improved stability, improved transfection and / or retaining or enhancing immunogenicity or therapeutic effect thereof. In certain embodiments, microparticles harboring stabilized lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations can be coated by one or more coating layer using atomic layer deposition to produce thermal stable, transportable, lipid assembly-free, or naked therapeutic polynucleic acid sequences for use in therapeutic treatments.

Description

STABILIZED AND ATOMIC LAYER DEPOSITION (ALD)-COATED LIPID ASSEMBLY-FREE POLYNUCLEOTIDE-CONTAINING MICROPARTICLES AND USES THEREOFPRIORITY

[0001] This International Application claims priority to U.S. Provisional Application No. 63 / 666,084 filed June 28, 2024 and U.S. Provisional Application No. 63 / 666,088 filed June 28, 2024. These applications are incorporated herein by reference in their entireties for all purposes.FIELD

[0002] Embodiments of the present disclosure provide novel compositions and methods for making and using atomic layer deposition (ALD) coated therapeutic polynucleotide- containing microparticles for delayed delivery and enhanced response. In certain embodiments, therapeutic polynucleotides sequestered in ALD coated microparticles are naked, lipid complex free or lipid decomplexed polynucleotides.BACKGROUND

[0003] Size and charge of mRNA prevents it from directly crossing cell membranes, and when outside of cells, mRNA is rapidly degraded by RNase enzymes. To allow mRNA to access cytosolic space within cells, and to prevent rapid extracellular degradation, a protective delivery vehicle is required to create effective mRNA formulations for vaccine uses. To date, lipid nanoparticles (LNPs) are the most common delivery vector used for mRNA-based vaccines.

[0004] However, mRNA vaccines that rely on LNPs as a delivery vehicle have several disadvantages. The complex mixtures of lipids typically used in LNP -based mRNA vaccines are expensive, difficult to manufacture, and contribute to the instability of the resulting vaccines and other formulation. To alleviate some of the instability problems with mRNA LNP vaccines, the vaccines must be transported and stored under low and sometimes extremely low temperature conditions. Naked mRNA formulations have been tested in clinical applications without much success due in part to low transfectivity (e.g., low ability to enter a cell), and increased degradation. Naked mRNA preparations tested to date have exhibited limited efficacy compared to LNP formulations.

[0005] Therefore, it is desirable to find improved, stabilized formulations and protections for storage and delivery of therapeutic mRNAs and other polynucleic acid molecules in therapeutic settings.SUMMARY

[0006] Embodiments of the present disclosure provide novel compositions and methods formaking and using thermostable therapeutic lipid assembly-free, lipid uncomplexed, lipid decomplexed, free, raw, or naked therapeutic polynucleotide-containing coated microparticles, referred herein as atomic layer deposition (ALD)-coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles. In accordance with these embodiments, the polynucleotide is not complexed with a lipid, has never been complexed with a lipid or lipid assembly, or has been de-complexed from a lipid or lipid assembly (e.g., de-complexed from a lipid nanoparticle (LNP)). In certain embodiments, compositions and methods are disclosed for adapting ALD-coated lipid assembly-free or naked therapeutic polynucleotide- containing microparticles (e.g., mRNA) for improved stability, improved transfectivity, and / or retaining or enhancing immunogenicity or efficacy of the therapeutic molecule(s). In accordance with these embodiments, thermostable therapeutic lipid assembly-free naked or raw, polynucleotide-containing formulations disclosed herein can be spray-dried to form particles and these spray-dried particles further coated using atomic layer deposition (ALD) of a metal oxide, metal alkoxide and / or aluminum-containing coating material to completely encase the particles with one or more ALD coating layers. In accordance with these embodiments, therapeutic polynucleotide containing formulations include polynucleic acids that are not complexed within lipid nanoparticles, lipid assemblies, or other assemblies and can be spray-dried and further embedded within glassy matrices or microparticles for coating by ALD to improve delivery, delayed delivery, storage and / or other benefits disclosed herein and observed by one of skill in the art.

[0007] In some embodiments and further to paragraph

[0006] above, an ALD-coated lipid assembly-free or naked therapeutic polynucleotide-containing microparticle can be created starting with a central or innermost lipid assembly-free, lipid de-complexed, lipid uncomplexed, naked, free, or raw therapeutic polynucleotide-containing glassy microparticle including, but not limited to, spray drying the at least one lipid assembly-free, lipid de- complexed, lipid uncomplexed, naked, free, or raw therapeutic polynucleotide and at least one glass-forming agent; and then applying one or more ALD coating layers of one or more of metal oxide, metal alkoxide, and an aluminum-based coating layer and combinations thereof covering the spray dried central or innermost microparticle. In certain embodiments, the at least one lipid assembly-free or naked therapeutic polynucleotide and at least one glassforming agent can further include at least one buffering salt. In some embodiments, the central or innermost lipid assembly-free or naked therapeutic polynucleotide-containing microparticles are completely encased by each ALD-applied coating layer. In accordance with these embodiments, the lipid assembly-free, naked therapeutic polynucleotide is not partof a lipid nanoparticle (LNP) or other lipid assembly. In yet other embodiments, the lipid assembly-free or naked therapeutic polynucleotide-containing microparticles disclosed herein can be absent or devoid of cholesterol, phospholipid, polyethylene glycol-modified lipids, and ionizable lipids.

[0008] In certain embodiments and further to paragraphs

[0006] -

[0007] above, the therapeutic polynucleotide of a lipid assembly-free, naked therapeutic polynucleotide can include, but is not limited to, single stranded (ss) or double-stranded (ds) DNA such as linear or circular DNA. In other embodiments, the lipid assembly-free, naked therapeutic polynucleotide can include, but is not limited to, RNA, messenger RNA (mRNA), siRNA, saRNA, or a chimeric molecule thereof, or a chimeric molecule including DNA, or other polynucleotide thereof. In some embodiments, the polynucleotide can be mRNA encoding a full length or fragment of a polypeptide or protein thereof, for example, of a targeted agent, antibody, or receptor molecule thereof (e.g., virus, bacteria, or other pathogen or other polynucleotide for treating a health condition).

[0009] In certain embodiments and further to paragraphs

[0006] -

[0008] above, the at least one glass-forming agent includes, but is not limited to, at least one polysaccharide. In accordance with these embodiments, the at least one glass-forming agent includes, but is not limited to, at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, glycine, cyclodextrin, and povidone and the like. In some embodiments, the at least one glass-forming agent is trehalose. In certain embodiments, the at least one glass-forming agent concentration in solutions that are spray dried to form glassy microparticles can be a weight-to-volume (w / v) concentration of about 0.1% to about 40%; or about 8% to about 15%. In other embodiments, a central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing microparticle formulation can further include at least one additional glass-forming agent or polysaccharide.

[0010] In certain embodiments and further to paragraphs

[0006] -

[0009] above, a central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle formulation can further include at least one surfactant and / or smoothing agent. In accordance with these embodiments, the at least one smoothing agent includes, but is not limited to, hydroxyethyl starch (HES), polyvinylpyrrolidone, serum albumin, dextran, hetastarch, plasma protein factor and the like. In some embodiments, the at least one surfactant includes a high molecular weight surfactant. In other embodiments, the at least one surfactant includes, but is not limited to, at least one non-ionic surfactant. In some embodiments, the at least one non-ionic surfactant includes, but is not limited to, at least onepoloxamer or other block copolymer (e.g., pluronic F127, F123, F68 or similar molecule), polysorbate 80, polysorbate 20, poloxamer 188, poloxamer 403, poloxamer 407, Tween 20, Tween 80, or combination thereof, or the like.

[0011] In certain embodiments and further to paragraphs

[0006] -

[0010] above, lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticles can be coated by ALD with at least one of a metal oxide, metal alkoxide, and / or an aluminum-based coating layer or mixture thereof or alternating pattern thereof. In accordance with these embodiments, the metal oxide, metal alkoxide, and / or the aluminum-based coating material can include, but is not limited to, one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCh), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCL), zirconium silicate or zircon (ZrSiCU) and hafnium oxide (HfCh) and combinations thereof. In certain embodiments, the ALD coating layers are made of the same metal oxide, metal alkoxide, or aluminum-based coating layer. In other embodiments, the ALD coating layers are made of a mixture of metal oxide, metal alkoxide, and aluminum-based materials. In yet other embodiments, the ALD coating layers are made of alternating metal oxide, metal alkoxide, and an aluminum-based coating layers applied over a central or innermost lipid assembly-free or naked therapeutic polynucleotide containing glassy microparticle or over another outer layer of a lipid assembly-free or naked therapeutic polynucleotide containing glassy microparticle.

[0012] In certain embodiments and further to paragraphs

[0006] -

[0011] above, a composition of ALD coated lipid assembly-free, naked therapeutic polynucleotide containing glassy microparticles can further include at least one amino acid. In some embodiments, the at least one amino acid can include, but is not limited to, alanine, arginine, glycine, proline, histidine or combination thereof or the like. In certain embodiments, the amino acid can include glycine or histidine.

[0013] In certain embodiments and further to paragraphs

[0006] -

[0012] above, the ALD coated microparticles disclosed herein can form part of a pharmaceutical composition and further include a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is an injectable formulation. In other embodiments, the pharmaceutical composition is in a form for direct application or administration to a condition or anatomical location on a subject to treat the condition. In accordance with these embodiments, the ALD coated microparticles harboring or protecting one or more lipid assembly-free or naked therapeutic polynucleotide can be systematically dissolved, such as layer by layer or other dissolution to expose the one or more lipid assembly-free, nakedtherapeutic polynucleotide to the subject after administration of a formulation to the subject.

[0014] In some embodiments and further to paragraphs

[0006] -

[0013] above, methods for making metal oxide, metal alkoxide, an aluminum-based or combinations thereof coated lipid assembly-free or naked therapeutic polynucleotide-containing microparticles are disclosed. In accordance with these embodiments, a method can include a) combining at least one lipid assembly-free, naked therapeutic polynucleotide with at least one glass-forming agent and optionally, at least one buffer salt; and b) spray-drying a) to make a glass formulation of essentially dried lipid assembly-free, naked therapeutic polynucleotide-containing microparticles to generate a central or innermost lipid assembly-free, naked therapeutic polynucleotide; and c) coating by ALD the essentially dried lipid assembly-free, naked therapeutic polynucleotide microparticles of b) with one or more coating layers of one or more of metal oxide, metal alkoxide, aluminum-based or combination thereof, and encasing a central or innermost essentially dried lipid assembly-free or naked therapeutic polynucleotide-containing microparticles. In other embodiments, the at least one lipid assembly-free, naked therapeutic polynucleotide in a) further includes at least one surfactant. In some embodiments, the at least one glass-forming agent includes at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, glycine, cyclodextrin, and povidone and the like.

[0015] In certain embodiments and further to paragraphs

[0006] -

[0014] above, a composition of ALD coated lipid assembly-free or naked therapeutic polynucleotide containing glassy microparticles can further include at least one buffer. In accordance with these embodiments, the buffer can be a buffer salt. In some embodiments, the buffer salt can include, but is not limited to, at least one of succinate, acetate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate, phosphate, and the like.

[0016] In certain embodiments and further to paragraphs

[0006] -

[0015] above, a plurality of ALD coated microparticles disclosed herein can be assembled in a single composition and further include at least one excipient. In accordance with these embodiments, the at least one excipient can include any excipient known in the art. In some embodiments, the plurality of ALD-coated microparticles are part of a pharmaceutical composition of use to treat, prevent, reduce onset of, or augment health conditions in a subject.

[0017] In other embodiments and further to paragraphs

[0006] -

[0016] above, methods for treating, preventing, or reducing the risk of onset of a health condition in a subject are disclosed. In accordance with these embodiments, a method can include administering at least one pharmaceutical composition including a plurality of ALD-coated microparticles disclosedherein to a subject. In certain embodiments, the health condition includes a subject having or suspected of developing an infection from a pathogenic organism. In other embodiments, the health condition includes a subject having or suspected of developing a genetic disorder. In yet other embodiments, the health condition includes a subject having or suspected of developing cancer or an inflammatory condition or autoimmune condition. In other embodiments, preventing or reducing the risk of onset of a health condition is prophylactic and / or therapeutic.

[0018] In other embodiments and further to paragraphs

[0006] -

[0017] above, kits are contemplated for generating, storing, transporting and / or administering ALD-coated microparticles disclosed herein. In one embodiment, a kit can include ALD-coated microparticles, at least one pharmaceutical composition and / or a plurality of the same or different ALD-coated microparticles disclosed herein, and at least one container. In other embodiments, a kit can include at least one sample of spray dried lipid assembly-free or naked therapeutic polynucleotide-containing microparticles and at least one container.BRIEF DESCRIPTION OF THE FIGURES

[0019] The accompanying drawings are incorporated into and form a non-limiting part of the specification to illustrate several examples of the present disclosure.

[0020] FIG. 1 represents an exemplary image of scanning electron micrographs (SEMs) of exemplary spray dried and ALD coated lipid assembly-free or naked therapeutic polynucleotide containing microparticles according to some embodiments of the present disclosure.

[0021] FIG. 2 is a graph illustrating size distribution of exemplary lipid assembly-free of naked spray dried and ALD-coated polynucleotide-containing microparticles as measured by well-known techniques in the art according to some embodiments of the present disclosure.

[0022] FIG. 3 represents size distribution of various samples compared to exemplary lipid assembly-free or naked spray dried, ALD-coated polynucleotide-containing microparticles as measured by well-known techniques in the art according to some embodiments of the present disclosure.

[0023] FIG. 4 illustrates antibody titers over time after administration of polynucleotides (e.g., mRNA) samples to an animal model with and without complexing lipids (LNP), with and without ALD coating where the samples were incubated at different temperatures for a pre-determined period prior to administration according to some embodiments of the present disclosure.

[0024] FIG. 5 illustrates an exemplary plot depicting effects on antibody titer produced in an animal study where lipid assembly-free or naked ALD-coated polynucleotide-containing microparticles are compared to a control after incubation at various temperatures for various lengths of time according to some embodiments of the present disclosure.

[0025] FIG. 6 represents an exemplary graph of antibody titer produced in an animal model over time post administration of an exemplary polynucleotide lipid complexed (LNP) (liquid, spray dried or spray dried and ALD coated) compared to naked, lipid-decomplexed or lipid assembly-free ALD-coated polynucleotide-containing microparticles according to some embodiments of the present disclosure.

[0026] FIGS. 7A and 7B illustrate immune response ratios at two time periods after administration of ALD coated lipid assembly-free or naked polynucleotide-containing microparticles compared to lipid complexed polynucleotides (lipid nanoparticles, LNP) of a first exemplary polynucleotide (e.g., OVA) (7 A) and a second exemplary polynucleotide (7B) according to some embodiments of the present disclosure.

[0027] FIGS. 8A and 8B represent an exemplary image in 3D reconstruction of a scanning confocal fluorescence microscopy image of a representative cell (e.g., RAW264.7 cell) following phagocytosis of an exemplary fluorescently-labelled naked, or lipid assembly-free ALD-coated polynucleotide-containing microparticle (8A) according to some embodiments of the present disclosure. FIG. 8B illustrates a percent of phagocytic cells expressing an encoded polypeptide upon lipid assembly-free or naked ALD-coated polynucleotide- containing microparticle uptake versus control samples and release of the sequestered polynucleotide or control over time post phagocytosis according to some embodiments of the present disclosure.DETAILED DESCRIPTION

[0028] In the following sections, various exemplary compositions and methods are described to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentration, times, and other specific details can be modified through routine experimentation. In some embodiments, well-known methods or components have not been included in the description.

[0029] To date, lipid nanoparticles (LNPs) are the most common delivery vector used for mRNA-based vaccines. All currently marketed mRNA vaccines utilize LNPs to protect the mRNA, enhance its ability to transfect cells and generate reliable, reproducible immune responses. A variety of LNPs have been used to create vaccine formulations. These LNPformulations typically include an ionizable cationic lipid, a phospholipid, cholesterol, and a lipid modified by attachment of polyethylene glycol. LNPs offer numerous advantages for mRNA-based vaccines, serving to protect the mRNA from enzymatic degradation, encouraging cellular uptake of the mRNA by endocytic pathways, aiding in endosomal escape to the cytosol and acting as an adjuvant to increase the immune response to proteins expressed after translation of the mRNA within the target cell. However, current mRNA vaccines and other polynucleic acid constructs that rely on LNPs as a delivery vehicle have several disadvantages. For example, the complex mixtures of lipids typically used in LNP- based mRNA vaccines can be expensive, difficult to manufacture, and contribute to the instability of the resulting vaccines and other formulation. To alleviate some of the instability problems with mRNA LNP vaccines, the vaccines typically must be transported and stored under low and sometimes extremely low temperature conditions requiring not just refrigeration but freeze-transport. Most LNP -based mRNA vaccines are maintained at -20 to -70 °C. Naked mRNA formulations have been tested in clinical applications without much success due in part to low transfectivity, and increased degradation of the naked mRNAs. Other disadvantages of these complexes are that some LNP -based mRNA vaccines have additional challenges with unwanted immunogenicity and toxicity due to the particular LNP.

[0030] Therefore, it is desirable to find improved formulations and protections for storage and delivery of mRNAs and other therapeutic polynucleic acid molecules in therapeutic settings. For example, improved compositions and methods to directly administer to a subject in need thereof, lipid assembly-free or naked mRNA as a vaccine or other therapeutic polynucleic acid constructs without the interference of lipid or lipid-like agents. Naked mRNA formulations have been tested in clinical applications; however, the literature demonstrates that naked mRNA preparations tested to date exhibited limited efficacy compared to LNP formulations, and LNP -based vaccine formulations have become so dominant that a recent US FDA publication noted “an mRNA vaccine contains lipid nanoparticles and nucleic acids” because of the general nature of acceptance of these formulated mRNAs in current therapy. It is known that naked mRNA or other polynucleotides can be rapidly degraded compared to LNP formulations.

[0031] One challenge is that vaccine formulations including nanoemulsions and / or nanoparticles of lipids can be thermal instability and typically must be kept refrigerated or in many instances frozen, to reduce degradation, to maintain immunogenic activity or integrity of the polynucleotide(s) and maintain particle structures and compositions that allow for transport of the intact polynucleotide(s) across cell membranes, to escape from endosomes,and for release into cytosolic compartments of cells. These issues can complicate vaccine and therapeutic distribution under any circumstance at any time, but it is most problematic in times of extreme catastrophic events such as pandemics, where for example, vulnerable, underserved and remote areas may have unreliable electricity to keep thermally unstable agents, antigens and / or vaccines under continuously cold or under cool temperatures. Embodiments disclosed herein can solve these issues with respect to polynucleotide delivery by spray-drying lipid assembly-free or naked polynucleic acid sequences or polynucleotides in formulations disclosed herein to imbed these lipid assembly-free, or naked polynucleic acid sequences or polynucleotides within glassy matrices. These lipid assembly-free or naked polynucleic acid sequences or polynucleotides glassy particles or microparticles can be created and used herein for example, to limit molecular mobility and further permit coating of these microparticles using atomic layer deposition (ALD) of coating agents such as metal oxides or alkoxides or the like to apply and completely encapsulate the glassy particles in one or more coating layers of defined thickness (e.g., layers of a thickness formed by at least 1 or about 20 to 500 or more molecular layers). Discoveries disclosed herein go against what has been identified in the past regarding stabilizing naked therapeutic polynucleotide or polynucleic acid sequences within a lipid assembly-free formulation in combination with spray-drying. To date, no formulations without lipid assembly or complex inclusion have been identified to stabilize therapeutic polynucleotides (e.g., mRNAs) but the instantly disclosed compositions and methods enable lipid assembly-free or naked therapeutic polynucleotides to not only remain thermostable, but to delay release of, and improve bioavailability by, for example, increasing transfectivity and preserving or enhancing therapeutic responses (e.g., increased titer) potentially reducing cost of manufacturing, while improving storage, and delivery of lipid assembly-free or naked polynucleotides (e.g., mRNA) formulations.

[0032] Embodiments disclosed herein and further to paragraphs

[0029] -

[0031] above, provide for thermostable metal -oxide, metal alkoxide and / or aluminum-based coated therapeutic polynucleic acid or polynucleotide-containing microparticle formulations. In certain embodiments, compositions and methods are disclosed for creating and using lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing coated particles having improved stability, improved transfection and / or retention or enhancement of immunogenicity or therapeutic effect thereof. Some embodiments provide for lipid assembly- free or naked thermostable metal -oxide, metal alkoxide and / or aluminum-based coated lipid assembly-free or naked therapeutic polynucleotide-containing microparticles. In certainembodiments, compositions and methods are disclosed for using lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations having improved stability, improved transfection, and / or retaining or enhancing immunogenicity (e.g., enhanced titers) or therapeutic effect thereof. In some embodiments, compositions and methods are disclosed herein for ALD-coated lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing particles having improved stability, improved transfection, and / or retaining or enhancing immunogenicity or therapeutic effect thereof compared to LNP -formulated polynucleotides known and currently accepted in the art.

[0033] In certain embodiments and further to paragraphs

[0029] -

[0032] above, polynucleotide molecules of use in compositions and methods disclosed herein are not found as a lipid-polynucleotide complex or assembly (e.g., lipid-mRNA complex or assembly) and are active as a transfective agent when coated by ALD processes. In certain embodiments, powders or microparticles after spray drying disclosed herein can be nearly completely dried or essentially dried lipid assembly-free or naked therapeutic polynucleotides. In other embodiments, therapeutic polynucleotides disclosed herein prior to spray-drying can be decomplexed from a lipid-assembly or lipid nanoparticle or other lipid complex and residual lipids may remain, but the therapeutic polynucleotides are no longer complexed in a lipid assembly. In some embodiments, residual lipids can be present in pre- spray -dried and / or post spray-dried therapeutic polynucleotide formulations, but the therapeutic polynucleotides are un-complexed with lipids or are not formulated with an LNP. In accordance with these embodiments, de-complexed or uncomplexed preparations disclosed herein can contain residual lipids (alternatively, separate lipid-containing agents can supplement the formulations), but the therapeutic polynucleotide has been released from a lipid nanoparticle complex or lipid assembly and is lipid assembly-free or naked polynucleotide.

[0034] As disclosed herein and further to the previous paragraphs, the term lipid assembly- free can mean in absence of a lipid assembly, lipid nanoparticle or other lipid complexed- polynucleotide where therapeutic polynucleotides of use or interest herein are not complexed to a lipid or have been released or disrupted from a lipid assembly for example, prior to spray-drying or at least prior to preparation and coating using ALD. In accordance with these embodiments, certain polynucleotide-containing formulations disclosed herein avoid use of lipid assemblies to produce ALD coated particles disclosed herein.

[0035] In certain embodiments and further to paragraphs

[0029] -

[0034] above, lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations can be spray-dried, further embedded within glassy matrices, microparticles, nanoparticles, orparticles for coating by ALD coating compositions and methods to improve polynucleotide delivery and outcome. In other embodiments, these microparticles harboring essentially dried and / or stabilized lipid assembly-free, or naked therapeutic polynucleic acid sequencecontaining formulations can be coated by one or more coating layer to produce thermal stable, transportable, storage-able, lipid assembly-free, or naked therapeutic polynucleic acid sequences or polynucleotides for therapeutic treatment. In certain embodiments, compositions and methods are disclosed for adapting lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations for ALD-coating resulting in improved stability for prolonged storage, transport, transfectivity, and / or retaining efficacy of the lipid assembly-free or naked therapeutic polynucleic acid sequence or polynucleotides, and / or maintained or enhanced immunogenicity thereof. In other embodiments, lipid assembly-free raw, or naked therapeutic polynucleic acid sequence-containing formulations can be spray-dried to embed the polynucleotides within particles to improve stability alone or with other agents or with multiple different therapeutic polynucleotides by limiting molecular mobility further facilitating efficacy and compatibility. In other embodiments, these ALD- coated essentially dried stabilized lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations can be stored for later use at room temperature or up to 60 °C for prolonged periods with reduced need or without the need for refrigeration and / or freezing. In some embodiments, these spray dried and / or spray dried and ALD-coated lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations can be stored for later use at room temperature or up to 15 °C, up to 20 °C, up to 25 °C or up to about 40 °C to less than 50 °C for prolonged periods with reduced need or without the need for refrigeration and / or freezing. In certain embodiments, these spray-dried and / or spray-dried and ALD-coated lipid assembly-free, raw, or naked therapeutic polynucleic acid sequencecontaining formulations can be stored for later use at room temperature or up to 25 °C for prolonged periods with reduced need or without the need for refrigeration and / or freezing.

[0036] In certain embodiments and further to paragraphs

[0029] -

[0035] above, spray dried lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations disclosed herein can form essentially dried microparticulate powders where these microparticles include essentially dried lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations that can be introduced to a fluidized bed atomic layer deposition (ALD) reactor to apply one or more coating layers. In accordance with these embodiments, each layer of the one or more coating layers can include one or more of metal oxides, metal alkoxides, and / or aluminum-based coating layers alone, in a mixture or in apattern layering of more than one type of coating material (e.g., alternating layers). In certain embodiments, an ALD coating layer can include, but is not limited to, the metal oxide, metal alkoxide, and / or the aluminum-based coating material and can include, but is not limited to, one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCh), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCL), zirconium silicate / zircon (ZrSiCU) and hafnium oxide (HfCL) and combinations thereof. In certain embodiments, the ALD coating layer can include, but is not limited to, aluminum oxide (A12O3), an aluminum alkoxide, and / or silicon dioxide (SiO2), as a mixture of agents, as alternating layers, or other ALD-coating combination. In certain embodiments, the coating layer can include, but is not limited to, aluminum oxide (A12O3), and aluminum alkoxide. It is noted herein that these ALD processes can be performed without liquid solvents and are designed to apply metal-containing formulations to coat thermostabilized lipid assembly-free, or naked therapeutic polynucleotides with precision, in consistent and / or uniform layers for timed-release of a target lipid assembly-free, or naked therapeutic polynucleotides to treat or prevent a condition in a subject. In accordance with these embodiments, combinations of spray-drying of the lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing formulations and ALD-coating, increase the thermostability of the lipid assembly-free, or naked therapeutic nucleic acid sequencecontaining formulations while creating a timed-release of the lipid assembly-free, or naked therapeutic nucleic acid sequence (e.g., mRNA or other therapeutic polynucleotide) once administered to a subject. For example, after in vivo administration, these ALD-coated spray dried lipid assembly-free, or naked therapeutic polynucleotides can release pharmaceutically active polynucleotides as naked therapeutic polynucleotides after extended times, up to several months to over a year, depending on the number of ALD coating layers applied.

[0037] In some embodiments and further to paragraphs

[0029] -

[0036] above, an ALD coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticle can be created starting with a central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle formulation including, but not limited to, at least one lipid assembly-free, or naked therapeutic polynucleotide, and at least one glassforming agent and optionally, at least one buffer salt; and applying one or more ALD coating layers of one or more of metal oxide, metal alkoxide, and an aluminum-based coating layer and combinations thereof covering the central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle. In some embodiments, the central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassymicroparticle includes completely encasing the central or innermost lipid assembly-free, naked, or raw therapeutic polynucleotide containing glassy microparticle with each applied ALD coating layer. In accordance with these embodiments, the ALD applied material layers uniformly, without pinholes or cracks. In some embodiments, the ALD applied material can be layered uniformly and purposely introduce inconsistencies in the surface of outer layers to improve immunogenicity when introduced to a subject for reducing onset of, preventing and / or treating a health condition. In accordance with these embodiments, the lipid assembly- free, or naked therapeutic polynucleotide is not part of a lipid nanoparticle (LNP). In other embodiments, the lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticles before, during, or after spray-drying can be absent or devoid of at least one of cholesterol, phospholipid, polyethylene glycol-modified lipids, and ionizable lipids. In certain embodiments, the lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticles before, during, or after spray-drying can be absent or devoid of all of cholesterol, phospholipid, polyethylene glycol-modified lipids, and ionizable lipids.

[0038] In certain embodiments and further to paragraphs

[0029] -

[0037] above, ALD coated lipid assembly-free, or naked therapeutic polynucleic acid sequence and / or polynucleotide (e.g., ALD-coated lipid assembly-free therapeutic polynucleotide nanoparticles) can result in increased stability and / or compatibility of active agents of a lipid assembly-free, or naked therapeutic polynucleotide of the coated and stabilized lipid assembly-free, or naked therapeutic polynucleic acid sequence providing for timed-release delivery of incompatible agents encased in the ALD-coated particles. In accordance with these embodiments, incompatible agents once spray-dried have increased compatibility between agents (if normally incompatible to combine) if more than one lipid assembly-free, or naked therapeutic polynucleotide is to be contained within ALD coated particles. Further, these combination particles can contain reduced concentrations of the incompatible lipid assembly-free, or naked therapeutic polynucleotides compared to uncoated or raw polynucleotides (e.g., mRNA) which may be needed to treat, reduce onset of, or prevent a health condition compared to known polynucleotide formulations (e.g. mRNA LNPs). In other embodiments, incompatible polynucleotides can be separately formulated and coated where these coated incompatible polynucleotides can then be mixed together for coadministration of two or more therapeutic polynucleotides prepared by compositions and methods disclosed herein.

[0039] In certain embodiments and further to paragraphs

[0029] -

[0038] above, the lipid assembly-free, or naked therapeutic polynucleotide can include, but is not limited to, singlestranded (ss) or double-stranded (ds) DNA such as linear or circular DNA, RNA, mRNA, siRNA, saRNA or a chimeric molecule thereof (e.g., mixture of polynucleotides or multiple RNAs or DNAs etc. forming a chimera), or other polynucleotide thereof. In some embodiments, the polynucleotide can be mRNA encoding full length or encoding a fragment of a polypeptide or protein thereof, or a mixture of full length and fragments of an encoded polypeptide or protein thereof, for example, of a targeted agent, targeted polypeptide, or antibody or receptor molecule thereof (e.g., virus or bacteria or other pathogen or other therapeutically relevant polynucleotide (e.g., to treat cancer, inflammation or a genetic condition)). In some embodiments, a lipid assembly-free, or naked therapeutic polynucleotide can include, but is not limited to, an mRNA encoding a full-length protein or fragment of a polypeptide or protein thereof, or mixtures of full-length protein and fragments of a polypeptide or protein thereof. In certain embodiments, the encoded full-length protein or fragment of a polypeptide or protein thereof can be from the same pathogenic organism or the same serotype (e.g., virus or bacteria or other pathogen or other therapeutically relevant polynucleotide (e.g., to treat cancer, inflammation or a genetic condition)). In certain embodiments, the encoded full-length protein or fragment of a polypeptide or protein thereof can be from different pathogenic organisms or different serotypes of the same pathogenic organism (e.g., virus or bacteria or other pathogen or other therapeutically relevant polynucleotide (e.g., to treat cancer, inflammation or a genetic condition)).

[0040] In some embodiments and further to paragraphs

[0029] -

[0039] above, a prime and at least one boost dose of ALD coated lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing glassy microparticles (e.g., coated raw, or naked lipid assembly-free therapeutic polynucleic acid sequence) can be administered to a subject in a single administration of these prime and at least one boost coated particles. In other embodiments, ALD coated lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing microparticles (e.g., coated polynucleotide particles) disclosed herein can encode antigens (e.g., immunogenic antigens) or agents against two or more pathogens in the same or in separate coated microparticles.

[0041] In certain embodiments and further to paragraphs

[0029] -

[0040] above, compositions and methods disclosed herein describe critical formulation parameters and methods for spray-drying of lipid assembly-free, or naked therapeutic polynucleic acid sequence for developing thermostable lipid assembly-free, or naked therapeutic polynucleic acid sequence formulations for further coating by metal oxide or metal alkoxide agents. In certain embodiments, lipid assembly-free polynucleotides (e.g., lipid assembly-free mRNAtherapeutic molecules) can be created by formulating naked polynucleotide or lipiddi sassembled molecules that encode a protein antigen or fragment thereof in aqueous solutions including, at least one glass-forming polysaccharide (e.g., trehalose, hydroxy ethyl starch) and optionally, at least one buffer salt (e.g., histidine, phosphate). In certain embodiments, formulations of use herein prior to spray-drying can include the glass-forming agent, trehalose. In certain embodiments, formulations of use herein prior to spray-drying can include the glass-forming agent, trehalose and further include at least one other polysaccharide and / or surfactant. In certain embodiments, formulations of use herein prior to spray-drying can include trehalose at a concentration of about 1.0% to about 20.0% or about 8.0% to about 15.0%. In accordance with these embodiments, these formulations, prior to spray-drying, can be in the absence of at least one of cholesterol, phospholipids, polyethylene glycol-modified lipids, or ionizable lipids typically present to preserve and / or stabilize polynucleotides (e.g., mRNAs). In accordance with these embodiments, these formulations, prior to spray-drying, can be in the absence of cholesterol, phospholipids, polyethylene glycol-modified lipids, or ionizable lipids typically present to preserve and / or stabilize polynucleotides (e.g., mRNAs). In some embodiments, these formulations, prior to spraydrying, can be in the absence of, cholesterol, phospholipids, polyethylene glycol-modified lipids, or ionizable lipids typically present to preserve and / or stabilize polynucleotides (e.g., mRNAs). In certain embodiments, optional agents can be included in these pre-spray-drying formulations. In accordance with these embodiments, optional agents can include other stabilizing additives including, but not limited to, one or more amino acids (e.g., methionine, alanine), one or more non-ionic surfactants (e.g., polysorbates, pluronics) and combinations thereof incorporated into the formulations prior to spray-drying. In certain embodiments, polynucleotides such as mRNA or other polynucleotides can be prepared and purified using techniques well-known in the art. In some embodiments, chemical modifications known in the art to stabilize mRNA or other polynucleotides can be used to further stabilize a polynucleotide contemplated of use herein.

[0042] In other embodiments and further to paragraphs

[0029] -

[0041] above, these formulations can be spray dried, a process wherein a liquid formulation is nebulized to form a mist of microdroplets. These suspended mists of microdroplets can then be contacted with dry, warm air, causing them to dry and form solid microparticles with embedded polynucleotides (e.g., therapeutic mRNA or other therapeutically relevant polypeptide) having reduced mobility or very limited mobility. In accordance with these embodiments, as droplets dry, glass transition temperature of the drying mixture is increased until it exceedsthe temperature in the spray dryer; then excipients disclosed herein surrounding the lipid assembly-free raw or naked mRNA or other lipid assembly-free, or naked therapeutic polynucleotides form a glass or glass-like matrices encasing and protecting the lipid assembly-free, or naked mRNA or other lipid assembly-free, or naked therapeutic polynucleotides. In certain embodiments, the lipid assembly-free polynucleotides such as a lipid assembly-free mRNA is then stabilized (e.g., thermostabilized) by this embedding within the glassy polysaccharide matrix of the microparticles. Because molecular motions are greatly hindered by the high viscosity within the glassy molecules, the normally thermally labile lipid assembly-free mRNA or other lipid assembly-free polynucleotide embedded in the glassy microparticle core can be protected from thermal damage, and no longer require storage at reduced temperatures below refrigeration or below 25 °C. In certain embodiments, no refrigeration conditions are needed for glassy particles disclosed herein.

[0043] In certain embodiments and further to paragraphs

[0029] -

[0042] above, nanoscopic coatings of metal layers such as metal alkoxides and metal oxides e.g., alumina) or other ALD-appropriately applied metal agent, can then be applied to the surface of the thermostabilized lipid assembly-free polynucleotide (e.g., mRNA or other polynucleotide)- containing microparticles or glassy microparticles using atomic layer deposition (ALD) techniques. In accordance with these embodiments, the ALD process involves repeatedly conducting two sequential reactions where gaseous reagents react with a surface of the thermostabilized lipid assembly-free polynucleotide (e.g., mRNA)-containing microparticles or glassy microparticles. ALD chemistries for application of various metal oxides or metal alkoxides to microparticle surfaces have been described and are known in the art. Any ALD method for applying a metal agent, metal -oxide or metal -alkoxide coating is contemplated of use herein to coat the thermostabilized lipid assembly-free polynucleotide (e.g., mRNA or other therapeutically relevant polynucleotide)-containing microparticles or glassy microparticles. For example, in one method, thermostabilized lipid assembly-free polynucleotide (e.g., mRNA)-containing microparticles or glassy microparticles formed by spray drying can be introduced into an ALD fluidized bed reactor. Under vacuum, precise quantities of trimethylaluminum gas can be first introduced to the ALD reactor.Trimethylaluminum reacts quantitatively with hydroxyl groups present on the surface of the spray-dried thermostabilized lipid assembly-free polynucleotide (e.g., mRNA)-containing microparticles or glassy microparticles (e.g., hydroxyls on the polysaccharides that form the particle surface). Subsequent addition of a second reactive gas, e.g., water vapor, which releases methane and leaves the surface of the particles or microparticles coated with amolecular layer of a metal agent (e.g., metal oxide, metal alkoxide etc.), which creates a new layer of exposed hydroxyl groups for repeating the cycle of layering to a desired number of these ALD coatings. These cycles can be repeated as often as desired to produce microparticles with nanoscopic metal agent (e.g., metal oxide and / or metal alkoxide or other metal agent) layers up to any arbitrary or precise / predetermined thickness (e.g., at least one layer, 20-500 coating layers, up to 1000 coating layers or more), as desired and provide accurate, timed-release of the encapsulated raw and / or lipid assembly-free polynucleotide(s) (e.g., lipid assembly-free mRNA).

[0044] In certain embodiments and further to paragraphs

[0029] -

[0043] above, these nanoscopic metal oxide layers on the particle surface protect the interior core from water sorption and exposure of the lipid assembly-free, or naked therapeutic polynucleic acid sequence to RNases, DNases, and / or other hydrolytic enzymes and more importantly, without the need for protection by lipids. In other embodiments, when the ALD-coated microparticles are administered in vivo, the nanoscopic metal -oxide coating erodes over the course of days to months depending on the number of coating layers, releasing the content of microparticle core, including any lipid assembly-free or naked therapeutic polynucleotide such as RNA or any other cargo contained within the microparticle. In accordance with these embodiments, a release profile (e.g., timing of release of a target, therapeutic polynucleotide) can be tailored by modifying the number of molecular layers of metal agent (e.g., metal oxide and / or metal alkoxide or other appropriate metal agent) applied to the microparticles, providing delayed release that can be near zero order, sustained release, delayed, pulsatile release, multi-dose release or the like at predetermined periods, or combinations thereof. In certain embodiments, ALD coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles can contain 1, 2, 3 or more of the same or different therapeutic polynucleotides in the same or different region within the microparticle (e.g., core, middle, or outer layer, etc.) directed to treat a health condition.

[0045] In other embodiments and further to paragraphs

[0029] -

[0044] above, ALD coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles can be administered to a subject and readily phagocytosed by antigen-presenting cells for example, macrophages and dendritic cells. After phagocytosis, erosion of the metal layering (e.g., metal oxide and / or metal alkoxide or other metal agent) commences to expose an outermost lipid assembly-free, or naked therapeutic polynucleotide or polynucleic acid sequence releasing a now uncoated microparticle's outermost lipid assembly-free, or naked therapeutic polynucleotide or polynucleic acid sequence directly into the cells’ cytosol. In accordancewith these embodiments, sequential uncoating or dissolution over a pre-determined or random number of coating layers covering one or more lipid assembly-free, or naked therapeutic polynucleotide(s) until a single lipid assembly-free, or naked therapeutic polynucleotide or multiple lipid assembly-free, or naked therapeutic polynucleotides are exposed to a subject receiving such a formulation. In some embodiments, where for example, the raw polynucleotide is an mRNA, the mRNA can be translated into proteins and / or peptide fragments or a combination thereof. In accordance with these embodiments, expressed proteins and / or peptide fragments formed within the macrophages and dendritic cells can then be presented on a representative cells' surface, eliciting desired antibody responses (e.g., reducing risk of, treating or preventing an infection) or treating a targeted condition. Alternatively, exposure of lipid assembly-free, or naked therapeutic polynucleic acid sequences within cells creates a desired therapeutic response (e.g., targeting tumor cells, alleviating a genetic condition, alleviating an inflammatory condition, or other targeting other cells for therapeutic purposes)

[0046] In some embodiments and further to paragraphs

[0029] -

[0045] above, it is understood that mRNA-based constructs (e.g., vaccines) have until this disclosure, presumed to require complex assembly of mRNA with lipids. These resulting lipid nanoparticles (LNPs) of these formulations are designed to help promote endocytosis of the mRNA into endosomal compartments within the cell followed by fusion of the lipids with endosomal membranes to allow the mRNA to escape from the endosomes into the cytosol and translate to protein. Embodiments disclosed herein skip these lipid-dependent and / or lipid-interference steps, allowing direct delivery of therapeutic polynucleotides (e.g., mRNA or other polynucleotide) to cytosolic compartments of immune-active cells or other targeted cells avoiding lipid and lipid-assembly known delivery and treatment interferences and / or reducing production, reducing therapeutic polynucleotide concentrations for a given dose and / or reducing storage costs in time and money.

[0047] In some embodiments and further to paragraphs

[0029] -

[0046] above, methods for making metal agent, metal oxide, metal alkoxide, aluminum-based or combinations thereof, ALD coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticle are disclosed. In accordance with these embodiments, a method can include combining to make a combination: at least one lipid assembly-free, or naked therapeutic polynucleotide with at least one glass-forming agent (optionally, at least one buffered salt). Drying the combination by any means known in the art. In certain embodiments, drying the combination can include spray-drying or spray-freeze drying the combination to make driedlipid assembly-free, or naked therapeutic polynucleotide-containing microparticles or glassy microparticles. In some embodiments, these dried or essentially dried lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles or glassy microparticles can be used as a central or innermost lipid assembly-free, or naked therapeutic polynucleotide; for coating by ALD, the dried lipid assembly-free, or naked therapeutic polynucleotide microparticles with one or more coating layers of one or more of metal agents, metal oxide, metal alkoxide, aluminum-based or combination thereof or mixture thereof or alternating pattern of coating layers thereof, and encasing the central or innermost essentially dried lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles with one or more coating layers. In accordance with these embodiments, each layer fully encases the microparticle for timed-release or pre-determined timed-release. In other embodiments, the at least one lipid assembly-free, or naked therapeutic polynucleotide or polynucleic acid sequence can further include at least one surfactant before, during or after spray-drying. In some embodiments, the at least one glass-forming agent of a pre-spray dried composition can include at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, glycine, cyclodextrin, and povidone and the like. In other embodiments, the glass-forming agent includes at least trehalose.

[0048] In certain embodiments disclosed herein and further to the preceding paragraphs, a formulation (e.g., vaccine) including an aqueous suspension of LNPs containing an exemplary mRNA encoding for human immunodeficiency virus envelope trimer (HIV Env trimer) protein was reformulated to form a glassy spray dried powder (See the examples). It is understood that this mRNA is an example, and any mRNA or other therapeutic polynucleotide contemplated herein could substitute for the presence of an HIV Env trimer- encoding mRNA in this exemplary experiment or others. The spray-dried powder in these experiments was divided into fractions. One fraction labeled was not subjected to further processing, and for another fraction, 50 ALD cycles were used to apply a roughly 10 nm thick layer of metal oxide (e.g., alumina, 50 coatings) to the surface of a spray-dried powder of the mRNA. Upon reconstitution of spray dried powder to re-form an aqueous suspension, it was observed that the LNPs had released free mRNA from the LNPs (data not shown). These released polynucleotides within the spray dried particles could be further coated using ALD coating. It was observed that, upon suspending the spray dried and ALD-coated powder (e.g., ALD coated spray-dried) microparticles to form an aqueous suspension, it was observed that the mRNA within the ALD-coated spray dried powder was preserved and intact. In lipid- containing polynucleotide constructs, released lipids could cause interferences which may bean issue. It was observed herein that in these experiments, these free therapeutic polynucleotides did not need lipid agents or lipid assemblies to protect the therapeutic polynucleotide and these molecules could be spray dried and ALD coated for storage and / or later use.

[0049] In some embodiments and further to paragraphs

[0029] -

[0048] above, compositions of use herein are disclosed for creating lipid assembly-free, or naked therapeutic polynucleic acid sequence formulation in a stabilized core by use of spray-drying to form thermostable glassy particles. In accordance with these embodiments, these compositions can contain, but are not limited to, one or more glass-forming agents such as one or more polysaccharide or disaccharide (e.g., trehalose, sucrose, glycine, or the like) and a high molecular weight glassforming agent or surfactant for example, hydroxy ethyl starch, polyvinylpyrrolidone, dextran, carboxymethyl cellulose, human serum albumin, bovine serum albumin, other serum albumin, or the like. In certain embodiments, the at least one glass-forming agent includes, but is not limited to, at least one polysaccharide. In other embodiments, a central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle formulation can include further include at least one additional glass-forming agent or polysaccharide. In accordance with these embodiments, the at least one glass-forming agent includes, but is not limited to, at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, cyclodextrin, and povidone and the like. In some embodiments, the at least one glass-forming agent is trehalose. In some embodiments, the at least one glass-forming agent includes trehalose and the at least one additional high molecular weight glass-forming agent or surfactant includes, but is not limited to, hydroxyethyl starch (HES), polyvinylpyrrolidone, dextran, carboxymethyl cellulose, human serum albumin, bovine serum albumin, other serum albumin, or the like. In certain embodiments, the at least one glass-forming agent includes trehalose and the at least one additional high molecular weight glass-forming agent or surfactant includes, but is not limited to, hydroxyethyl starch. In certain embodiments, in solutions that can be spray dried to form glassy microparticulate powders, the at least one glass-forming agent concentration can be a weight-to-volume (w / v) concentration of about 0.1% to about 40.0%; or about 0.5% to about 30.0%; or about 5.0% to about 15.0%; or about 8.0% to about 15.0%, or about 8.0% to about 12.0%. In accordance with these embodiments, in liquid formulations prior to spray-drying, salts of buffered salt can be included if desired and can be about 0.1 mM to about 250.0 mM (or about 10.0 mM to about 200.0 mM); glass-forming agents / polysaccharide concentrations can be about 1.0% to about 25.0% (w / v) (or about 5.0% to about 20.0%, or about 5.0% toabout 15.0%, or about 8.0% to about 12.0%) and a high molecular weight agent such as a surfactant concentration can be about 0.1% to about 10.0%, or about 1.0 % to about 5.0% (w / v). In certain embodiments a lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle formulation prior to spray or spray-freeze drying can further include at least one surfactant and / or smoothing agent. In accordance with these embodiments, the at least one smoothing agent includes, but is not limited to, hydroxy ethyl starch (HES), polyvinylpyrrolidone, serum albumin, dextran, hetastarch, plasma protein factor and the like. In certain embodiments, the smoothing excipient can also be a primary glass-forming agent for formulating a target therapeutic polynucleotide contemplated herein. In some embodiments, a smoothing agent disclosed herein can be used to create coated particles having a more uniform, or smooth surface, if desired. In accordance with these embodiments, the smoothing excipient can be present in the primary formulation prior to spray drying at a weight-to-volume (w / v) concentration from about 0.1% to about 40%, from about 1% to about 30%, from about 5% to about 20%, or from about 8% to about 15%. In certain embodiments, the smoothing excipient can be different than the primary glass-forming agent, and the smoothing excipient can be present in the primary formulation prior to spray drying in a weight-to-volume (w / v) concentration from about 0.1% to about 10%, from about 0.1% to about 5%, from about 0.1% to about 2.5%, from about 0.1% to about 0.5%. In certain embodiments, the glass-forming agent present in the composition is trehalose and the smoothing excipient for the particles is hydroxyethyl starch. In certain embodiments, a formulation prior to spray-drying smoothing agents disclosed herein can be about 0.1% to about 10.0% (w / v) or about 1.0 % to about 5.0% smoothing agent (e.g., HES), and about 2.5% to about 30.0% (or about 7.50% to about 25.0% or about 7.50% to about 20.0%) glassforming agent (e.g., trehalose). In some embodiments, the at least one surfactant includes a high molecular weight surfactant. In other embodiments, the at least one surfactant includes, but is not limited to, at least one non-ionic surfactant. In some embodiments, the at least one non-ionic surfactant includes, but is not limited to, at least one of polysorbate 80, polysorbate 20, poloxamer 188, poloxamer 403, poloxamer 68, poloxamer 407, other pluronic, Tween 20, Tween 80, or the like. In certain embodiments, surfactants can be included in formulations or compositions prior to spray or spray-freeze drying at concentrations near a critical micelle concentration (CMC) of the given surfactant. In accordance with these embodiments, concentrations can include 0.01 to 100 times the CMC; 0.1 to 10 times the CMC, 0.5 to 2 times the CMC, for example. In accordance with these embodiments, one purpose for these agents is to reduce accumulation of mRNA on a surface of spray dried microparticles toreduce or avoid potential surface-mediated damage to therapeutic polynucleotides (e.g., mRNA).

[0050] In some embodiments and further to paragraphs

[0029] -

[0049] above, an inorganic or organic buffer or buffered salt can be included and can include, but is not limited to, succinate, citrate, acetate, prolamine, arginine, glycine, histidine, borate, carbonate, tromethamine (Tris), glutamate and phosphate buffer systems. In accordance with these embodiments, formulations disclosed herein can include a buffer concentration of about 100.0 mM or less, or about 50.0 mM or less, or about 25.0 mM or less or about 10.0 mM or less or about 5.0 mM or less or about 1.0 mM or less. In some embodiments, the buffer includes a non-chelating organic buffer. In certain embodiments, the non-chelating organic buffer includes, but is not limited to, histidine or similar non-chelating organic buffer. In other embodiments, a buffer or aqueous media can include a phosphate or citrate buffer or a phosphate-citrate combination buffer, a solution containing chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)) or other agent. In certain embodiments, a formulation for generating glassy particles disclosed herein can include histidine and / or phosphate (e.g., sodium phosphate) and / or histidine, and / or tris buffer or similar buffer suitable for inclusion. In accordance with these embodiments, the histidine and / or phosphate concentration can be about 40 mM or less; or about 30 mM or less; about 20 mM or less; about 10 mM or less; or about 5 mM or less. In some embodiments, pH of a pre-spray or spray freeze-dried formulation including inorganic or organic buffer or buffered salt (e.g., histidine and / or phosphate), is about 4.0 to about 9.0 when the concentration of the inorganic or organic buffer or buffered salt (e.g., histidine and / or phosphate) is about 40.0 mM or less; 30 mM or less; 20 mM or less, or 10 mM or less. In some embodiments, pH of a pre-spray or spray freeze-dried formulation including inorganic or organic buffer or buffered salt (e.g., histidine and / or phosphate) is about 5.0 to about 8.0 or other suitable pH depending on a lipid assembly-free therapeutic naked or free polynucleotide to be formulated. In some embodiments, pH of a pre-spray or spray freeze-dried formulation including histidine and / or phosphate is about 4.0 to about 9.0 when the concentration of the histidine and / or phosphate is about 40.0 mM or less; 30 mM or less; 20 mM or less, or 10 mM or less. In some embodiments, pH of a pre-spray or spray freeze-dried formulation including histidine and / or phosphate is about 5.0 to about 8.0 or other suitable pH depending on a lipid assembly-free therapeutic naked or free polynucleotide to be formulated.

[0051] In certain embodiments and further to paragraphs

[0029] -

[0050] above, ALD coated particles disclosed herein can be resuspended in a formulation such as apharmaceutical formulation for delivery to a subject. In some embodiments, the pharmaceutical composition can be formulated into an injectable formulation. In other embodiments, the pharmaceutical composition can be formulated for direct application to a condition (e.g., tumor site, infection site). In accordance with these embodiments, coatings of the ALD coated particles or microparticles harboring or stabilizing one or more lipid assembly-free, or naked therapeutic polynucleotide can be systematically dissolved, layer by layer to expose the one or more lipid assembly-free, naked, or raw therapeutic polynucleotide to the subject (e.g., targeted cells such as immune cells, macrophages, dendritic cells and the like) after administration to the subject in need thereof. In accordance with these embodiments, a suspension formulation for resuspending coated particles disclosed herein can include aqueous solutions buffered with at least one of phosphate, histidine, imidazole, glycine, bis tris methane, tris, bicine, glycylglycine or similar buffer or other similar agent known in the art. In certain embodiments, concentrations of buffers or agents used as coated microparticle suspension formulations disclosed herein can be at a reduced concentration (e.g., 20 mM or less) and can further include, but is not limited to, phosphate (e.g., sodium phosphate). In accordance with these embodiments, the histidine and / or phosphate concentration can be about 40 mM or less; or about 30 mM or less; about 20 mM or less; about 10 mM or less; or about 5 mM or less. In some embodiments, a suspension formulation of sodium phosphate can be a concentration of about 40 mM or less; or about 30 mM or less; about 20 mM or less; about 10 mM or less; or about 5 mM or less.

[0052] In certain embodiments and further to paragraphs

[0029] to

[0051] above, methods of making stable administration-ready ALD-coated thermostable lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles are disclosed. In certain embodiments, a single lipid assembly-free, naked, or free therapeutic polynucleotide is present in the ALD-coated microparticles. In other embodiments, two or more, or three or more lipid assembly-free, or naked therapeutic polynucleotide are present in individual ALD- coated microparticles. In other embodiments, two or more, or three or more lipid assembly- free, or naked therapeutic polynucleotide are present in separate ALD-coated microparticles and mixed together prior to administration. In accordance with these embodiments, these methods can include combining ALD-coated thermostable at least one lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles with at least one nonaqueous suspension agent; and forming a suspension composition. In other embodiments, the ALD- coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles are in a powder form prior to suspension. In other embodiments, the at least one nonaqueoussuspension agent for suspending the ALD coated microparticles disclosed herein can include, but is not limited to, benzyl benzoate, 2-hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures or triglyceride oils and the like. In other embodiments, the at least one refined triglyceride mixture or oil includes, but is not limited to, at least one of refined sesame oil, refined soybean oil, refined palm kernel oil, refined corn oil, refined cottonseed oil, refined peanut oil or other refined triglyceride mixture or refined oil or medium chain triglyceride, or other acceptable refined oil. As disclosed herein, a refined triglyceride mixture can include, but is not limited to, removing impurities from a triglyceride oil mixture for example, to improve purity, reduce contamination and / or reduce presence of interfering agents with a targeted therapy contemplated herein. In certain embodiments, these oils can be used interchangeably (e.g., due to their inert nature).

[0053] In certain embodiment and further to paragraphs

[0029] -

[0052] above, while not wishing to be bound by theory, it is thought that a robust immune response to administration of ALD-coated particles containing lipid assembly-free, or naked mRNA can be ascribed to direct delivery of free or naked mRNA into cells by the metal agent (e.g., metal oxide, metal alkoxide or other ALD compatible metal agent) coated particles. It is observed that this enhanced immune response can be augmented by an adjuvant-like effect of the ALD coating itself (e.g., alumina). In other embodiments, creating an uneven outer surface of an ALD fully coated microparticle disclosed herein can also increase immunogenicity in a subject provided such a microparticle. In other embodiments, ALD fully coated microparticles disclosed herein can be smooth and even on the outermost layers.

[0054] In certain embodiments and further to paragraphs

[0029] -

[0053] above, drying rates in spray drying or spray freeze drying techniques disclosed herein can be modulated by the addition of high molecular weight drying agents such as hydroxy ethyl starch (HES), carboxymethyl cellulose (CMC) or similar high molecular weight polysaccharide or surfactant agent. In some embodiments, these high molecular weight drying agents (e.g., hydroxyethyl starch) can precipitate as a film on a surface of droplets of the formulations before or during a spray-drying or spray-freeze drying process disclosed herein. In accordance with these embodiments, modulation such as a decrease in rate of drying in presence of at least one high molecular weight drying agents can provide a smoothing effect on the particles to further facilitate ALD coating applications. In other embodiments, lipid assembly-free, raw, or naked therapeutic polynucleic acid sequence formulations that have been stabilized by spray-drying to form glassy microparticle powders can be further protectedfrom instabilities by coating the powders using ALD of metal agents (e.g., metal oxides and metal alkoxides and other metal agents) disclosed herein. As disclosed herein, spray-dried microparticles can be introduced to a fluidized bed of an ALD reactor system and nanoscopic layers (e.g., molecular layers) of metal agents can be applied to the microparticles for uniformly encasing the microparticles layer by layer. ALD-coating can further increase the thermostability of the lipid assembly-free, or naked therapeutic polynucleic acid sequence glassified formulations for long term storage. As disclosed herein, these ALD coatings can be dissolved either in vivo or in a salt buffer to expose the lipid assembly-free, or naked therapeutic polynucleic acid sequence. It was surprising that a lipid assembly-free, or naked therapeutic polynucleotide or polynucleic acid sequence within a glassy formulation were stabilized and were stabilized and protected enough to permit ALD-coating while preserving immunogenicity and therapeutic effects of the therapeutic polynucleic acid sequences. In addition, it was found and disclosed herein that lipid assembly-free, or naked therapeutic polynucleic acid sequences in polysaccharide-containing formulations that had been spray- dried to form essentially dried glassy particles or microparticles could be further introduced to a fluidized bed atomic layer deposition reactor to coat the particle surfaces without altering size distribution of the embedded therapeutic polynucleotide contained with a powder or glassy matrix. In in accordance with these embodiments, size of the coated particles or microparticles was maintained as well as their syringability preserving their interior droplet size distribution for later delivery when reconstituting coated microparticles disclosed herein for therapeutic application.

[0055] In some embodiments and further to paragraphs

[0029] -

[0054] above, stabilized lipid assembly-free, raw, or naked therapeutic polynucleic acid sequence formulationcontaining powders, microparticles, or glassy matrices disclosed herein can be coated using ALD with one or more coating layers where each layer of the one or more coating layers can include one or more of a metal agent, metal oxides, metal alkoxides, and / or aluminum-based coating layer or mixture thereof or alternating coating combination thereof. In certain embodiments, the coating layer can include, but is not limited to, metal oxide, metal alkoxide, and / or the aluminum-based coating material and can include, but is not limited to, one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCL), titanium dioxide (TiCL), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCL), zirconium silicate / zircon (ZrSiCU), gallium oxide (Ga2Os), and hafnium oxide (HfCh) and combinations thereof or mixtures thereof or alternating coating combinations thereof. It is contemplated herein that alternating combinations of metal agent layers can affect the timed-release of a therapeutic polynucleic acid sequence disclosed herein. For example, dissolution rates of one metal material versus another can vary by reducing time of exposure or increasing time of exposure of a lipid assembly-free, raw, or naked therapeutic polynucleic acid sequence of an ALD coated microparticle disclosed herein. In accordance with these embodiments, combinations of spray-drying of the lipid assembly-free, raw, or naked therapeutic polynucleic acid sequence formulations and ALD-coating increases the thermostability of the lipid assembly-free raw, or naked therapeutic polynucleic acid sequence formulations. For example, these ALD coated lipid assembly-free, or naked therapeutic polynucleic acid sequence or polynucleotide formulations have at least one of improved transfectivity, and increased stability following storage at room temperature or higher temperatures compared to liquid LNP-containing polynucleotide formulations.

[0056] In certain embodiments and further to paragraphs

[0029] -

[0055] above, ALD coated lipid assembly-free, or naked therapeutic polynucleic acid sequence-containing powders, microparticles or glassy matrices can be stored, transported, and reconstituted for use to treat, reduce onset, correct a genetic disorder, or prevent a medical condition. In accordance with these embodiments, reconstituted coated microparticles maintain size distributions that are syringable (e.g., ability to be taken up and delivered accurately by a syringe, reduced loss from agglomeration or large microparticles) and capable of being introduced to a subject by any delivery method known in the art.

[0057] In certain embodiments and further to paragraphs

[0029] -

[0056] above, one or more agent disclosed herein can include at least one therapeutic naked polynucleotide that forms part of a central or innermost microparticle. In accordance with these embodiments, the central or innermost microparticle can include, but is not limited to, at least one lipid assembly-free, or naked therapeutic polynucleotide, at least one glass-forming agent (e.g., polysaccharide, e.g., trehalose), at least one buffer salt, and at least one surfactant. In other embodiments, the central or innermost microparticle can include, but is not limited to, at least one smoothing agent (e.g., hydroxyethyl starch (HES), polyvinylpyrrolidone etc.). In other embodiments, the central or innermost microparticle further includes, one or more outer metal agent, metal oxide or metal alkoxide or alumina-coating layers covering or completely encasing the central or innermost lipid assembly-free, or naked therapeutic polynucleotide- containing glassy microparticle. In accordance with these embodiments, one, two, three, four, five, up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 750, up to 1,000 or more coating layers can encase stabilized lipid assembly-free, or naked therapeutic polynucleotide-containingpowders or glassy matrices where the coating layers are slowly erodible in a subject once administered to the subject, to expose the one or more lipid assembly-free, or naked therapeutic polynucleotide to the subject by immediate, short-term or other timed-release delivery. In other embodiments, another formulation of the same or different, at least one lipid assembly-free, or naked therapeutic polynucleotide can be stabilized in powder form as disclosed herein and encased in another layer or outer layer of coating agent forming a prime dose of the at least one therapeutic lipid assembly-free or naked polynucleotide. An outer layer of coating by the same or different therapeutic lipid assembly-free or naked polynucleotide can further be coated by additional metal oxide or metal alkoxide-containing compositions using ALD in order for this outer shell applied lipid assembly-free, or naked therapeutic polynucleotide formulation to be time-released prior to the coated central or innermost lipid assembly-free, or naked therapeutic polynucleotide of a microparticle.

[0058] Certain embodiments and further to paragraphs

[0029] -

[0057] above, methods for making stabilized therapeutic lipid assembly-free or naked polynucleotide-containing coated microparticles, stabilized powders or glassy matrices, the methods include combining at least one therapeutic lipid assembly-free or naked polynucleotide with at least one glass-forming agent or polysaccharide to form a primary liquid composition, rapidly spray-drying the composition to form therapeutic lipid assembly-free or naked polynucleotide-containing glassy microparticles, and coating the essentially dried lipid assembly-free, or naked therapeutic polynucleotide-containing glassy microparticles with one or more outer coating layers. In some embodiments, a primary liquid composition (prior to spray-drying) can further include rapid introduction of a second glass-forming agent. In accordance with these embodiments, the second glass-forming agent or polysaccharide can include hydroxy ethyl starch or similar agent. Further, in accordance with these embodiments, hydroxyethyl starch can precipitate as a film on the surface of droplets of the composition as they dry during spray drying, decreasing the rate of drying and reducing collision of particles thereby maintaining integrity of the particles as they dry to a more stabilized state in preparation for coating. Rapid spray drying as disclosed herein can mean spray-drying in a few milliseconds up to a few seconds. In accordance with these embodiments, particulates and / or essentially dried microparticles disclosed herein are formulated and spray dried such that lipid assembly-free, raw, or naked therapeutic polynucleotide-containing formulations are not principally located at the surface of the spray dry particle (reduced exposure). It is known that high molecular weight compounds and low solubility compounds have a higher likelihood of ending up at the surface of a spray dried particle, therefore, embodiments disclosed herein address thiscommonly observed issue by specific additions to the formulation to form an external shelllike layer that is not principally the target therapeutic agent and can, for example, shield the polynucleotides making them more stable under certain conditions. In accordance with these embodiments, particulates and / or essentially dried microparticles disclosed herein can be introduced to an ALD reaction chamber where the particulates, glassy microparticles, and / or essentially dried microparticles flow freely within the chamber for reduced agglomeration and / or aggregation of the particulates and / or essentially dry microparticles. It is noted herein that introduction of polynucleotides directly to an ALD reaction chamber without creating thermostable particulates or essentially dry glassy microparticles as disclosed herein would be unsuccessful. For example, the elevated temperature would degrade the polynucleotides and / or be unable to coat them due to incorrect surface chemistries for complete encapsulation. It was not known until the instant disclosure that formulations disclosed herein could create such a barrier or shell able to protect polynucleotides from such harsh conditions and thoroughly encase them within a glassy particle using compositions and methods disclosed herein. It is further understood by one of skill in the art that formulations and processes disclosed herein are scalable and readily available for manufacture for creating bulk coated lipid assembly-free, raw, or naked therapeutic polynucleotide particles for storage and later use such as in a health crisis.

[0059] In accordance with these embodiments and further to paragraphs

[0029] -

[0058] above, the at least one lipid assembly-free, or naked therapeutic polynucleotide can encode one or more polypeptide, protein, or part of a polypeptide, for example a viral antigen, a bacterial antigen, a toxin, a prion, a protozoan, fungi, a fragment or subunit thereof, a small molecule, an anti-cancer agent, an anti-inflammatory agent, an anti-autoimmune agent, any peptide, polynucleotide, or protein thereof or a combination thereof. In some embodiments, the at least one lipid assembly-free, or naked therapeutic polynucleotide (e.g., mRNA) can encode, but is not limited to, encoding a recombinant peptide, a recombinant protein, a peptide derived from a target protein or pathogen, a synthetic peptide or protein, a polynucleotide encoding a polypeptide, a polynucleotide, a virus-like particle, a live virus, a live, attenuated virus, an inactivated virus, or protein expressed on the surface of a virus or a bacteriophage or a combination thereof. In accordance with these embodiments, a polynucleotide can include, but is not limited to, single stranded (ss) or double-stranded (ds) DNA such as linear or circular DNA, RNA, mRNA, siRNA, saRNA, or a chimeric molecule thereof, or other polynucleotide thereof. In some embodiments, the polynucleotide can be mRNA encoding a full length or fragment polypeptide thereof, for example, of a targetedagent or antibody or receptor molecule thereof e.g., virus or bacteria).

[0060] In certain embodiments and further to paragraphs

[0029] -

[0059] above, the at least one lipid assembly-free, or naked therapeutic polynucleotide can encode at least one polypeptide. In accordance with these embodiments, the at least one lipid assembly-free, or naked therapeutic polynucleotide can encode, but not limited to, encoding a polypeptide of at least one pathogen or antigen derived therefrom. In certain embodiments, a polynucleotide or polynucleic acid sequence disclosed herein includes, but is not limited to, encoding at least one antigen derived from human papilloma virus (e.g., HPV16, HPV18) or other mammalian papilloma virus, ricin toxin, Bacillus anthracis, Clostridium botulinum, Ebola virus, influenza virus, Corona virus (Covid-19, SARS-CoV-2, Alpha, Beta, Delta, Epsilon, Gamma, Omicron or other variants, or mutants thereof), poliovirus, norovirus, rotavirus, hepatitis C, varicella, herpes simplex, cytomegalovirus, Japanese encephalitis, dengue virus, West Nile virus, Zika virus or other flaviviruses, an alphavirus such as chikungunya, EEEV, WEEV, VEEV or other alphavirus, pneumonia virus spp., Yersinia, Pneumococcus, Salmonella, Clostridium difficile, or a combination thereof. In certain embodiments, the at least one lipid assembly- free or naked polynucleotide can be a multimeric complex. In other embodiments, the at least one encoded antigen or agent can include a pathogen or antigen derived therefrom capable of infecting a human (e.g., adult, child, toddler, infant or fetus), companion animal, livestock, wild animal, zoo animal, bird, or reptile.

[0061] In some embodiments and further to paragraphs

[0029] -

[0060] above, the at least one glass-forming agent or polysaccharide disclosed herein can include at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, glycine, cyclodextrin, povidone, or the like and combinations thereof. In certain embodiments, the at least one glass-forming agent or polysaccharide disclosed herein can include trehalose or sucrose or combination thereof. In certain embodiments, the at least one glass-forming agent or polysaccharide disclosed herein can include trehalose alone or in combination with a second polysaccharide. In accordance with these embodiments, the at least one glass-forming agent or polysaccharide disclosed herein can be trehalose at a concentration of about 1.0% to about 40.0% or about 5.0% to about 30.0%, or about 10.0% to about 30.0% or about 15.0% to about 30.0% or about 20.0% to about 40.0% or about 20.0%. In certain embodiments, drying of microdroplets of compositions disclosed herein by spray drying can occur over a rapid timescale of about 50 milliseconds to about 3 seconds, or about 100 milliseconds to about 1 second, or about 300 milliseconds. In accordance with these embodiments, spray-drying of compositions and formulations disclosed herein can provide asuperior product to other drying techniques such as lyophilization. While it is contemplated that any rapid drying technique can be used such as spray-freeze drying, lyophilization or the like, spray-drying can provide a more reliable outcome when creating glassy particles, microparticles or nanoparticles from formulations and compositions of therapeutically relevant polynucleotides disclosed herein.

[0062] In other embodiments and further to paragraphs

[0029] -

[0061] above, the glassforming agent or polysaccharide disclosed herein can include at least one additional polysaccharide agent (or disaccharide agent). In accordance with these embodiments, the at least one additional polysaccharide agent can include, but is not limited to, hydroxy ethyl starch (HES), dextran, hetastarch, carboxymethylcellulose, and the like, or a combination thereof. In accordance with these embodiments, the at least one additional polysaccharide can be present in a primary formulation prior to spray-drying at a weight-to-volume (w / v) concentration from about 0.1% to about 40.0%, from about 1.0% to about 30.0%, from about 5.0% to about 20.0%, or from about 8.0% to about 20.0%. In certain embodiments, the at least one additional polysaccharide agent can be different than a primary or first polysaccharide agent, and the additional polysaccharide agent can be present in a liquid or aqueous pre-spray-drying formulation in a weight-to-volume (w / v) concentration from about 0.1% to about 10.0%, from about 0.1% to about 5.0%, from about 0.1% to about 2.5%, from about 0.1% to about 0.5%. In certain embodiments, a first or primary glass-forming agent present in a pre-spray-drying formulation can be sucrose or trehalose and the at least one additional polysaccharide or disaccharide can be hydroxyethyl starch (HES).

[0063] In some embodiments and further to paragraphs

[0029] -

[0062] above, each layer of the one or more ALD coating layers can include one or more of metal oxides, metal alkoxides, and / or aluminum-based coating layer applied to the essentially dried or powder form of lipid assembly-free, or naked polynucleotide-containing microparticles. In certain embodiments, each layer of the one or more coating layers can include, but is not limited to, one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCh), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCh), zirconium silicate / zircon (ZrSiO4),and hafnium oxide (HfCL) and combinations thereof, alone or in a suitable combination or pattern of alternating layering compositions (1 :1, 2: 1, 1 :2, 3: 1, 1 :3, 4: 1, 1 :4 or other alternating pattern). In certain embodiments, the ALD coating layers are made of the same metal agent, metal oxide, metal alkoxide, and / or an aluminum-based coating layer. In other embodiments, the ALD coating layers are made of a mixture of metal agent, metal oxide, metal alkoxide, and an aluminum-based materials. In yet other embodiments, the ALD coating layers are made of alternating metal agent, metal oxide, metal alkoxide, and an aluminum-based coating layers encasing or covering a central or innermost microparticle or over another outer layer of a lipid assembly- free, or naked therapeutic polynucleotide containing glassy microparticle formulation.

[0064] In accordance with these embodiments and further to paragraphs

[0029] -

[0063] above, each coating layer(s) can be about 0.1 nm to about 20.0 nm in thickness. In certain embodiments, the essentially dried (spray dried) microparticles disclosed herein can include a plurality of outer coating layers sufficient to delay release or provide a timed-release of the at least one lipid assembly-free or naked therapeutic polynucleotide contained in one or more layers of a coated microparticle or from the central or innermost core of the coated microparticles. In certain embodiments, one, two, three, four, five, up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, up to 600, up to 700, up to 800, up to 900, u to 1000 or more coating layers can encase at least one lipid assembly-free, or naked therapeutic polynucleotide-containing powders or glassy matrices or microparticles disclosed herein in the central core or outer layer of a particle where the coating layers are readily dissolvable in a subject once administered to the subject by immediate or timed-release, to expose the one or more lipid assembly-free, or naked therapeutic polynucleotide to the subject.

[0065] In certain embodiments and further to paragraphs

[0029] -

[0064] above, the one or more ALD coating layer(s) disclosed herein can serve as an adjuvant to enhance an immune response in a subject against the encoded polypeptide or naked polynucleotide containing ALD coated microparticles. In some embodiments, the one or more coating layer(s) can include a coating layer or coating layers capable of inducing a rapid immune response in the subject upon exposure. In other embodiments, ALD coated essentially dried at least one lipid assembly-free, or naked therapeutic polynucleotide can be contained in a selected layer or layers and / or the core of the coated microparticles such that when the coating layers dissolve and expose the one or more antigen and / or agent a rapid immune response is induced in the subject to the one or more agent or antigen in the subject. In accordance with these embodiments, the immune response is enhanced compared to delivery of the antigen and / or agent without spray-drying and / or without coating of stabilized microparticles disclosed herein.

[0066] In some embodiments and further to paragraphs

[0029] -

[0065] above, ALD-coated microparticles described herein can be stored without refrigeration temperatures at room temperature, up to about 25° C, up to about 30° C, up to about 35° C, up to about 40° C, up toabout 45° C, up to about 50° C, or up to about 60° C, or up to about 70° C or more for extended periods of time. In certain embodiments, ALD coated microparticles disclosed herein can be stored without refrigeration up to room temperature, up to about 25° C, up to about 30° C, up to about 35° C, up to about 40° C, up to about 45° C, up to about 50° C, or up to about 60° C, or up to about 70° C for a few hours, up to half a day, up to about a day, up to two or more days, up to a week, up to a couple of weeks, up to a few weeks, up to several weeks, up to 1 month, up to 2 months, up to 3 months, up to about 4 months, up to about 6 month, up to about 9 months, up to about 12 months, up to about 15 months, up to about 18 months, up to about 24 months or longer without negative effects on the coated microparticles (e.g. degradation, loss of efficacy, loss of transfection, loss of immunogenicity, reduced delivery of the one or more agents or antigens). In certain embodiments, ALD coated microparticles disclosed herein can be stored without the need for freezing temperatures.

[0067] In other embodiments and further to paragraphs

[0029] -

[0066] above, methods for treating, preventing, or reducing the risk of onset of a health condition in a subject are disclosed. In accordance with these embodiments, a method can include administering at least one pharmaceutical composition including a plurality of, or mixtures of ALD coated lipid assembly-free or naked therapeutic polynucleotide-containing microparticles disclosed herein to a subject. In certain embodiments, the health condition includes a subject having or suspected of developing an infection from a pathogenic organism. In other embodiments, health conditions include a subject having or suspected of developing a genetic disorder. In yet other embodiments, health conditions include a subject having or suspected of developing cancer, inflammatory condition, or autoimmune condition. In certain embodiments, health conditions include cancer caused by a pathogenic organism. In other embodiments, preventing or reducing the risk of onset of a health condition is prophylactic and / or therapeutic. In some embodiments, the at least one pharmaceutical composition including a plurality of, or mixtures of ALD-coated microparticles disclosed herein include a vaccine against one or more pathogenic organism.

[0068] Other embodiments and further to paragraphs

[0029] -

[0067] above, provide for combination compositions or formulations including a plurality of coated lipid assembly-free naked, or raw therapeutic polynucleotide-containing microparticles described herein. In accordance with these embodiments, these combination compositions or formulations can include mixtures of different coated microparticles for treating, reducing onset of, or preventing a single health condition or multiple health conditions (e.g., pathogenic agent infections or prevention of pathogenic agent infections, cancer, inflammation, etc.) andfurther include at least one pharmaceutically acceptable excipient to make a pharmaceutically acceptable composition or formulation. In other embodiments, combination compositions can include at least one representative microparticle-containing pharmaceutical composition as disclosed herein mixed with a standard or known composition or formulation to treat, reduce onset of, or prevent health conditions in a subject. In certain embodiments, the standard formulation can include a standard vaccine formulation against at least one pathogen or a standard or known anti-cancer agent or the like. In some embodiments, ALD coated microparticles disclosed herein can be used as one or more boost doses to a standard treatment directed to treat, prevent, or reduce onset of a health condition (e.g., infection).

[0069] In some embodiments and further to paragraphs

[0029] -

[0068] above, formulations disclosed herein are part of a single-administration formulation that can include a prime dose and at least one boost dose of at least one lipid assembly-free naked, or raw therapeutic polynucleotide sequestered within ALD coated microparticles disclosed herein. In accordance with these embodiments, the prime and at least one boost dose of the at least one lipid assembly-free naked, or raw therapeutic polynucleotide can be in the same coated microparticle, or in separate coated microparticles and administered together as a mixture or separately administered. When in separate particles, the priming dose of the at least lipid assembly-free naked, or raw therapeutic polynucleotide can be sequestered in a microparticle while the at least one boost dose can be in separate microparticle. In certain embodiments, release of a priming dose after delivery to a subject can be immediate or delayed depending on the number of coating layers coating the microparticle priming dose (if not on the outermost or near outermost coating layer) and the at least one boost can be exposed to the subject a short time, minutes, hours, days, weeks, or several months later. In certain embodiments, compositions disclosed herein can be mixed where one composition contains ALD coated microparticles alone or in combinations of different ALD-coated lipid assembly- free or naked therapeutic polynucleotide microparticles targeting the same or different pathogens or health condition or mixtures with other known agents or formulations to treat or prevent a health condition.

[0070] In some embodiments and further to paragraphs

[0029] -

[0069] above, formulations containing the at least one lipid assembly-free or naked therapeutic polynucleotide can encode at least one polypeptide including, but not limited to, a polypeptide encoding at least one antigen or antigens or other polypeptide or fragment thereof and can be part of a single administration formulation encoding at least two different agents or two different antigens or two different polypeptides capable of eliciting an immune response or other therapeuticresponse to two or more different pathogens or targets. In accordance with these embodiments, the two or more encoded different antigens, different polypeptides, or different agents can be included in the same or separate coated microparticles in the same or different ALD coated microparticle layers of the microparticle (e.g., in a central region / core and at least in one outer layer of the same microparticle where ALD-coating layers separate the two or more encoded different antigens, different polypeptides, or different agents). Precision coating layers can be applied by ALD to create timed-release of the one or more lipid assembly-free naked, or raw therapeutic polynucleotide at predetermined periods once administered to a subject for efficient transfection of a target cell for encoding the different antigens, different polypeptides, or different agents.

[0071] Other embodiments and further to paragraphs

[0029] -

[0070] above, provide for methods for eliciting a response in a subject to the lipid assembly-free, or naked therapeutic polynucleotides sequestered in coated particles disclosed herein, where the method can include administering a pharmaceutical composition of ALD-coated at least one lipid assembly-free or naked therapeutic polynucleotide to treat, prevent or ameliorate a health condition. In accordance with these embodiments, the formulation can be administered by any method known in the art. In some embodiments, administration to a subject can include by injections, by infusion, by bolus administration, by direct administration to a targeted region in a subject, a combination thereof or by other known administration method. In some embodiments, a pharmaceutically acceptable formulation disclosed herein can be administered to a subject to induce a response to the pharmaceutical formulation in the subject. For example, a response can include, but is not limited to, an immune response, an anti-pathogenic organism response (e.g., kill the pathogenic organism or reduce or prevent infection thereof), anti-inflammatory response, an anti-cancer response or other response in the subject administered such a composition. In accordance with these embodiments, an immune response induced by the formulation or composition can be prophylactic, ameliorating, and / or therapeutic, depending on the health condition to be treated or prevented.

[0072] In certain embodiments and further to paragraphs

[0029] -

[0071] above, ALD coated lipid assembly-free, raw, or naked therapeutic polynucleotide microparticles and formulations or compositions thereof disclosed herein can use less therapeutic polynucleotide per treatment than used to formulate current vaccines (e.g., LNP formulations, liquid formulations and the like) against an immunogen or other therapeutic agent e.g., cost saving, dose sparing, exposure reducing), and provide enhanced efficacy after a single administration. This is due in part to the increased transfectivity, stabilization and efficacy of the describedformulations; for example, compared to unstable liquid formulations; and temperature sensitive, expensive LNP vaccine formulations known in the art. In other embodiments, coated lipid assembly-free, naked, raw, or free therapeutic polynucleotide-containing microparticle compositions or formulations provide for thermostable formulations that eliminate and / or reduce refrigeration requirements (e.g., cold chain refrigeration requirements), limit the concentration of adverse effects of agents (e.g., aluminum, excess therapeutic agent, side effects of LNPs, etc.) administered to a subject, and create increased lipid assembly-free therapeutic polynucleotide compatibility within a single ALD coated microparticle or mixture of ALD coated microparticles disclosed herein. In certain embodiments, compositions and methods disclosed herein are applicable to a variety of potential agents or antigens, including, but not limited to, polynucleotides, polynucleotide- containing chimeras, encoded polypeptides, encoded recombinant peptides or encoded protein immunogens, encoded virus-like particles (VLPs), and encoded inactivated or attenuated pathogens (e.g., viruses), encoded bacteriophage conjugated agents, or similar agents.

[0073] In certain embodiments and further to paragraphs

[0029] -

[0072] above, a lipid assembly-free therapeutic polynucleotide encoding a pathogenic agent or antigen derived therefrom can be from a pathogenic virus. In certain embodiments, a pathogenic virus or fragment thereof can be, for example, any pathogenic virus known to infect, humans or other mammals, reptiles, amphibians, birds or other animal. In accordance with these embodiments, a pathogenic virus or fragment thereof of use in creating ALD coated microparticles of the instant inventions can include, but is not limited to, a papovavirus (e.g., papillomaviruses, including human papilloma virus (HPV)), a herpesvirus (e.g., herpes simplex virus, varicellazoster virus, bovine herpesvirus- 1, cytomegalovirus), a poxvirus (e.g., smallpox virus), a reovirus (e.g., rotavirus), a parvovirus (e.g., parvovirus Bl 9, canine parvovirus), a picornavirus (e.g., poliovirus, hepatitis A), a togavirus (e.g., rubella virus), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., dengue virus, hepatitis C virus, West Nile virus, yellow fever virus, Zika virus, Japanese encephalitis virus or other flavivirus), an orthomyxovirus (e.g., influenza A virus, influenza B virus, influenza C virus), a paramyxovirus (e.g., measles virus, mumps virus, respiratory syncytial virus, canine distemper virus, parainfluenza viruses), a rhabdovirus (e.g., rabies virus), a filovirus (e.g., Ebola virus (e.g., Zaire ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, Marburg virus, etc.,), an alphavirus (e.g., chikungunya or WEEV, EEEV or VEEV, or other alphavirus), phleboviruses (e.g., Rift Valley Fever virus) or a coronavirus, SARS or the like or mutants orvariants thereof or combinations thereof. In accordance with these embodiments, therapeutic pathogenic virus-derived polynucleotides can be encased in microparticles and then ALD coated as described herein. In some embodiments, these ALD coated microparticles can further be part of a formulation for administration to a subject and include at least one pharmaceutically acceptable agent and / or excipient.

[0074] In other embodiments and further to paragraphs

[0029] -

[0072] above, a lipid assembly-free therapeutic polynucleotide encoding a pathogenic agent or antigen derived from a pathogenic agent can be from a bacterium or a toxin of a bacterium or toxoid agent. In certain embodiments, a pathogenic bacteria or fragment thereof can be, for example, any pathogenic bacteria known to infect, humans or other mammals, reptiles, amphibians, birds or other animal. In accordance with these embodiments, a lipid assembly-free therapeutic polynucleotide encoding a pathogenic agent or antigen derived from a pathogenic bacteria can include, but is not limited to, Pasteur ella haemolytica, Clostridium difficile, Clostridium haemolylicum, Clostridium lelani, Corynebacterium diphtheria, Neorickettsia resticii, Streptococcus equi equi, Streptococcus pneumoniae, Salmonella spp., Chlamydia trachomatis, Bacillus anthracis, Yersinia spp., Clostridium botulinum, Neisseria gonorrhoeae, Borrelia burgdorferi , Burkholderia pseudomallei , Klebsiella pneumoniae, Acinetobacter baumanii, E. coli, enterococcus faecium, enterococcus faecalis, pseudomonas aeruginosa or other pathogenic bacteria or a combination thereof. In accordance with these embodiments, lipid assembly-free therapeutic polynucleotide encoding agents or antigens derived therefrom can be part of an ALD coated microparticle disclosed herein. In certain embodiments, these ALD coated microparticles can be part of a pharmaceutical composition and further include a pharmaceutically acceptable agent.

[0075] In some embodiments and further to paragraphs

[0029] -

[0072] above, a pathogenic agent can be a fungus. In accordance with these embodiments, a pathogenic fungus can include but is not limited to, Cryptococcus spp. (e.g., neoformans and gatti), Aspergillus spp. (e.g. , fumigatus), Blastomyces spp. (e.g., dermatitidis), Candida albicans, Paracoccidioides spp. e.g., brasiliensis), Sporothrix spp. e.g., schenkii and brasiliensis), Histoplasma capsulatum, Pneumocystis jirovecii and Coccidioides immitis, or other pathogenic fungus or combinations thereof. In accordance with these embodiments, lipid assembly-free therapeutic polynucleotide encoding agents or antigens derived therefrom can be part of a microparticle disclosed herein. In certain embodiments, these ALD coated microparticles can be part of a pharmaceutical composition and further include a pharmaceutically acceptable agent.

[0076] In yet other embodiments and further to paragraphs

[0029] -

[0072] above, apathogenic agent can be a toxin. In accordance with these embodiments, a toxin can include but is not limited to, anthracis toxin, ricin toxin or botulinum toxin or other toxin. In accordance with these embodiments, lipid assembly-free naked therapeutic polynucleotide encoding agents or antigens derived therefrom can be part of a microparticle disclosed herein. In certain embodiments, these ALD coated microparticles can be part of a pharmaceutical composition and further include a pharmaceutically acceptable agent.

[0077] In some embodiments and further to paragraphs

[0029] -

[0076] above, the at least one coated lipid assembly-free, or naked therapeutic polynucleotide-containing particles described herein can be used to manufacture one or more microparticle-containing formulation for use as vaccines or other therapeutic formulation for treating any animal. In certain embodiments, the animal can be a household pet or other companion animal. In some embodiments, an animal can include livestock, or other farm animals, wild animals, or zoo animals. In certain embodiments, the animals can include a non-human mammal, reptile, amphibian, fish, or bird. In accordance with these embodiments, the microparticle-containing composition can be administered, including, but not limited to, to a dog (canine), a cat (feline), a horse (equine), cattle (bovine), a goat (hircine), a sheep (caprine), pig (swine), or poultry (e.g., chicken, turkey, duck, goose). In some embodiments, these microparticle containing compositions can be introduced to water, for example to treat fish or administered using mass or systematic vaccination of farm animals, livestock or companion animals. Any known method for administering these compositions to these subjects is contemplated. Any known instrument for administering or distributing a distribution

[0078] In certain embodiments and further to paragraphs

[0029] -

[0077] above, the at least one lipid assembly-free, raw, or naked therapeutic polynucleotide coated microparticles described herein can be used to generate one or more compositions and / or combined with other therapeutic agent-containing compositions contemplated herein for administering to a human to reduce onset of, treat, and / or prevent an infection or reduce onset of or treat another health condition. In some embodiments, other conditions can include cancer, an inflammatory condition, an autoimmune condition, or diabetes or other health condition.

[0079] In certain embodiments and further to paragraphs

[0029] -

[0078] above, the at least one lipid assembly-free, raw, or naked therapeutic polynucleotide-containing coated microparticles described herein can be used to generate coated-microparticle containing formulations of use to treat, reduce the risk of onset or prevent an infection in a human. In certain embodiments, the at least lipid assembly-free, raw, or naked therapeutic polynucleotide-containing coated particles described herein can be used to deliver one ormore formulation disclosed herein to a human such as an infant or child or adolescent, young adult, adult or elderly human subject to treat, reduce the risk of onset or prevent an infection caused by a pathogenic organism. In accordance with these embodiments, infections caused by a pathogenic organism can include, but is not limited to, lipid assembly-free therapeutic polynucleotide encoding antigens or agents derived from or originating from or for targeting varicella-zoster (chicken pox), diphtheria, Haemophilus influenzae type b (Hib), hepatitis A, hepatitis B, influenza, corona virus, SARS, Ebola virus, measles, mumps, pertussis, polio, pneumococcal disease, rotavirus, rubella, and tetanus. In other embodiments, the at least one lipid assembly-free, naked, or raw therapeutic polynucleotide-containing particles described herein can be used to deliver one or more compositions to a human pre-teen or teen, including but not limited to, lipid assembly-free, or naked therapeutic polynucleotides encoding polypeptides derived from or originating from influenza, tetanus, diphtheria, pertussis, human papillomavirus, meningococcal disease, hepatitis B, hepatitis A, polio, measles, mumps, rubella, and varicella-zoster. In yet other embodiments, the at least one lipid assembly-free, or naked therapeutic polynucleotide-containing coated microparticles described herein can be used to deliver one or more therapeutic compositions to a human adult, including but not limited to, at least one lipid assembly-free, or naked therapeutic polynucleotide encoding polypeptides derived from or to target at least one of, but not limited to, influenza (e.g., A, B or C), tetanus, diphtheria, pertussis, zoster, pneumococcal disease, meningococcal disease, measles, mumps, rubella, varicella, hepatitis A, hepatitis B, and Haemophilus influenzae type b or other pathogenic organism.

[0080] In other embodiments and further to paragraphs

[0029] -

[0079] above, the at least one lipid assembly-free, or naked therapeutic polynucleotide-containing coated particles described herein can be used to generate compositions of use for administering to a human. In accordance with these embodiments, treatment of a human subject can include, but not limited to, the at least one lipid assembly-free, naked, raw, or free therapeutic polynucleotide- containing coated particle-containing composition against travel -related infections. Travel- related infections can include, but are not limited to, hepatitis A, hepatitis B, typhoid fever, paratyphoid fever, meningococcal disease, yellow fever, dengue fever, rabies, Zika virus- related conditions, norovirus infection, West Nile virus infection, Chikungunya disease, and Japanese encephalitis, Covid- 19 or other variant or corona virus infection, or other infection or combinations thereof. In certain embodiments, combination compositions are contemplated that encode polypeptides derived from more than one species and / or strain and / or variant thereof for a single administration against more than one pathogen.

[0081] In yet other embodiments and further to paragraphs

[0029] -

[0080] above, the at least one lipid assembly-free or naked therapeutic polynucleotide-containing coated microparticles described herein can be used to generate compositions of use for administering to a human, including but not limited to, encoded agents or antigens. In accordance with these embodiments, encoded agents or antigens can be derived from and include, but not be limited to, polynucleotides encoding whole or therapeutically effective fragments of human papillomavirus (e.g. HPV 16, HPV18, HPV31, HPV45, or HPV 6 or HPV11, or any other HPV type or serotype), herpes simplex virus, smallpox virus, rotavirus, parvovirus B19 vaccine, chikungunya virus, dengue virus (e.g. dengue- 1, dengue-2, dengue-3 or dengue-4 or any new dengue strain or variant), norovirus, hepatitis C virus, West Nile virus, Zika virus, respiratory syncytial virus, rabies virus, and Ebola virus, SARS, COVID-19 or other strain or mutant, or the like to treat, reduce progression or prevent an infection in the human subject or other appropriate subject.

[0082] In certain embodiments and further to paragraphs

[0029] -

[0081] above, the at least one lipid assembly-free, or naked therapeutic polynucleotide-containing coated microparticles described herein can include a single agent dose or two or more doses of a particular lipid- free, or naked polynucleotide (e.g., prime and boost doses or just doses of the lipid assembly- free or naked polynucleotide for prolonged and timed-release). In some embodiments, the at least one lipid assembly-free, or naked therapeutic polynucleotide-containing coated microparticles can include doses to encode for two or more different agents, polypeptides, or fragments thereof (e.g., two or more different pathogenic organisms or strains). In yet other embodiments, at least one lipid assembly-free, or naked therapeutic polynucleotide- containing coated microparticles including doses of different agents within different coated microparticles can be combined into a mixture of microparticles. A mixture of at least one lipid assembly-free, naked or free therapeutic polynucleotide-containing coated microparticles can be combined into a single administration dose to provide to a subject in need thereof. This can result in a reduced number of administrations reducing needs to return to a clinic for subsequent administration. With respect to livestock, these combined, multitargeted or mixed coated microparticles can be administered to livestock in a single dose to treat, reduce or prevent onset of a health condition in the livestock of which the at least one lipid assembly-free, naked or free therapeutic polynucleotide-containing coated microparticles are targeting.

[0083] In other embodiments and further to paragraphs

[0029] -

[0082] above, one or more adjuvants and / or immune-stimulating agents can be incorporated into the microparticles orlayered onto the coated microparticles disclosed herein. In accordance with these embodiments, the adjuvant or immune-stimulating agents can be combined with the at least one lipid assembly-free or naked therapeutic polynucleotide, and at least one glass-forming agent in a formulation prior to spray-drying or dehydration and / or applied to an outer layer of a coated ALD microparticle disclosed herein. In certain embodiments, compositions disclosed herein can include a co-stimulatory agent to further boost immune responses to one or more target pathogen or other antigen or agent used to treat health conditions (e.g., cancer). In accordance with these embodiments, a co-immunostimulatory agents can include, but are not limited to, one or more of lipid A, lipid A derivatives, monophosphoryl lipid A, chemical analogues of monophosphoryl Lipid A, CpG containing oligonucleotides, TLR-4 agonists, flagellin, flagellins derived from gram negative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 receptors, saponins, analogues of saponins, QS-21, purified saponin fractions, ISCOMS, and saponin combinations with sterols and lipids, or the like, or any known co-immunostimulatory agent, or combinations thereof.

[0084] In some embodiments and further to paragraphs

[0029] -

[0083] above, buffers of use to spray-dry or reconstitute microparticles disclosed herein can include, but are not limited to, acetate, succinate, citrate, prolamine, histidine, borate, carbonate or phosphate buffer, or a combination thereof. In certain embodiments, a buffer can include one or more salts of use in forming formulations to create glassy microparticles and can include, but are not limited to, one or more of salt including, but not limited to, sodium acetate, sodium succinate, potassium succinate, sodium citrate, sodium phosphate, potassium phosphate and the like or a combination thereof. In certain embodiments, the buffer can include histidine, for example, histidine-HCl. In other embodiments, one or more volatile salts can be added to the pre-spray dried formulations and can include, but are not limited to, ammonium acetate, ammonium formate, ammonium carbonate, ammonium bicarbonate, triethylammonium acetate, triethylammonium formate, triethylammonium carbonate, trimethylamine acetate trimethylamine formate, trimethylamine carbonate, pyridinal acetate and pyridinal formate, or combinations thereof. In some embodiments, a volatile salt can include ammonium acetate.

[0085] In some embodiments and further to paragraphs

[0029] -

[0084] above, molecular deposition techniques can be used to apply nanometer-thick coatings of inorganic, or metal oxide or metal alkoxide materials on the surface of essentially dried glassy at least one lipid assembly-free, naked or free therapeutic polynucleotide-containing microparticles. In certain embodiments, the coating or sequestering layer can be an aluminum-based material including, for example, aluminum oxide or metal oxide, or metal alkoxide, and / or an aluminum alkoxide(e.g., alucone) or mixture thereof. In accordance with these embodiments, an aluminum- containing material can be deposited on or applied to the surface of the one or more microparticles to coat or sequester the one or more microparticles in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, up to 20, up to 30, up to 50, up to 100, up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, up to 500, up to 600, up to 700, up to 800, up to 900 or more layers of the metal oxide, metal alkoxide, or other metal-based-containing material to form encased or fully coated microparticles.

[0086] In some embodiments and further to paragraphs

[0029] -

[0085] above, metal-based coating layers (e.g. metal oxide, metal alkoxide or ALD-compatible coating agent) can be used as a coating applied to spray dried or essentially dried lipid assembly-free, or naked therapeutic polynucleotide microparticles including at least one glass-forming or polysaccharide agent. In accordance with these embodiments, ALD is used to apply the metal -based coating layer on the microparticles. In certain embodiments, one or more layers of a metal-based film (coating) applied by ALD can be formed by coupling trimethyl groups of the metal-based agent (e.g. metal oxide, metal alkoxide or ALD-compatible coating agent) to hydroxyl groups of the microparticles, a layer of amine groups can be formed by coupling ethanolamine to the layer of metal agent-containing material (e.g. metal oxide, metal alkoxide or ALD-compatible coating agent), and a second layer of hydroxyl groups can be formed by coupling maleic anhydride to the available amine groups. An ABC-type reaction, for example, can be self-limiting, and can be used to deposit molecular layers of alucone or other metal -containing compositions disclosed herein (e.g. metal oxide, metal alkoxide or ALD- compatible coating agent). Further to these embodiments, hydroxyl groups on the substrate (e.g., microparticles) react with trimethyl aluminum, ethanolamine then reacts, leaving terminal amine groups on the surface, and available maleic anhydride reacts with terminal amine groups, regenerating a surface of hydroxyl groups for a repeat of the ALD-applied coating, if desired. This ABC-type molecular deposition process can be repeated to provide additional layers as desired (up to 10, up to 20, up to 100, up to 200, up to 300, up to 400, up to 500, up to 600, up to 700, up to 800, up to 900 or more, or any desired coating number in between), and can be used to deliver, for example, 1, 2, 3, 4, 5 or 6 doses of the at least one lipid assembly-free, or naked therapeutic polynucleotide coated microparticles to a subject in a single administration depending on the composition or make-up of the coated microparticles. In other embodiments, various chemical substitutes can be used in these coating or sequestering processes (e.g., alternative sources for the metal or ALD compatible coating agent, amine, and / or hydroxyl groups), as would be recognized by one of ordinaryskill in the art and based on the present disclosure. One of skill in the relevant art would understand that this disclosure provides support for any ALD coating process to be used to coat lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles disclosed herein.

[0087] In certain embodiments and further to paragraphs

[0029] -

[0086] above, a binary reaction sequence can be used to deposit one or more layers of alumina, metal oxide or metal alkoxide, or ALD-compatible coating agent on spray-dried or an essentially dried microparticles disclosed herein. In accordance with these embodiments, microparticles can be subjected to alternating gas streams containing either trimethyl aluminum or water vapor within an ALD reaction chamber during coating of the microparticles. In certain embodiments, the number of cycles can be varied to control formation of the number of coating or sequestering layers on the at least one lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles. It is further understood that spray dried or essentially dried at least one lipid assembly-free, or naked therapeutic polynucleotide- containing microparticles disclosed herein introduced to an ALD reactor for coating are introduced for a predetermined time to receive a predetermined number of coating layers on the microparticles depending on what is desired. It is understood that coated microparticles disclosed herein removed from the ALD reactor can be reintroduced if additional coating layers are desired and / or further processed for storage, transport, preparation and / or use in treating, reducing onset of, or preventing a health condition in a subject.

[0088] In accordance with these embodiments and further to paragraphs

[0029] -

[0087] above, some advantages of depositing one or more coating layers on the spray dried at least one lipid assembly-free, or naked therapeutic polynucleotide microparticles include, but are not limited to, the coating layers can dissolve slowly or at a pre-determined rate when the microparticles are administered to a subject, thus allowing temporal control of the release of the particle contents (e.g., the one or more lipid assembly-free, or naked therapeutic polynucleotides). Release times can be tailored by adjusting composition(s) of the coating layers and number and / or thickness of molecular layers applied to the agent-containing microparticles. In some embodiments, up to about 10, up to about 20 to about 1000 or more coating or sequestering layers can be used to form the completely coated or sequestered microparticles of the present disclosure. In certain embodiments, coating of these spray dried microparticles cannot be performed by simply mixing metal agent compositions with the spray dried microparticles because for precision and complete nanoscopic coating, ALD application is needed. In some embodiments, release of the antigens or agents from thecoated or sequestered lipid-assembly or naked therapeutic polynucleotide containing microparticle’s core can occur within hours, up to about 1 day, or days, or up to about 7 days or up to about 30 days or up to about 60 days or up to about 90 days or up to about 120 days or up to about 150 days or up to about 180 days, or up to about a year or more after administration to the subject. In some embodiments, release of lipid assembly-free, raw, or naked therapeutic polynucleotide from the coated microparticles can occur from about 10 days to about 90 days or more after administration to the subject. In other embodiments, release of the one or more innermost sequestered or coated lipid assembly-free, or naked therapeutic polynucleotide from the microparticle can occur from about 30 days to about 180 days after administration to the subject. In some embodiments, release of the innermost lipid assembly-free, or naked therapeutic polynucleotide can occur from about 30 days to about 120 days, or about 30 days to about 90 days, or about 30 days to about 90 days, or about 30 days to about 60 days, after administration to the subject. In some embodiments, release of an innermost lipid assembly-free, raw, or naked therapeutic polynucleotide can occur from about 10 days to about 180 days after administration to the subject. In some embodiments, release of the innermost lipid assembly-free, or naked therapeutic polynucleotide can occur from about 14 days to about 120 days after administration to the subject. Further, in some embodiments, release of the innermost at least one lipid assembly-free, or naked therapeutic polynucleotide can occur from about 3 days to about 90 days after administration to the subject.

[0089] In certain embodiments and further to paragraphs

[0029] -

[0088] above, particle size (e.g. microparticle, nanoparticle etc.) of an encased one or more at least one lipid assembly- free, or naked therapeutic polynucleotide-containing microparticles is from about 0.1 pm to about 10 pm, or about 1.0 pm to about 5.0 pm or less than 5.0 pm. In other embodiments, an encased lipid assembly-free, or naked therapeutic polynucleotide-containing microparticle having multiple layers is less than about 5.0 pm in size.

[0090] In certain embodiments and further to paragraphs

[0029] -

[0089] above, one advantage of using one or more aluminum-based materials as coating or sequestering layer(s) of microparticles disclosed herein is that the aluminum-based materials can also act as an adjuvant but can also be used to reduce the need for supplemental adjuvants at reduced concentrations. In accordance with these embodiments, the aluminum-based coating layers sequestering or surrounding the microparticles expose essentially the same surface chemistries to immunoactive cells in a subject being treated as do standard aluminum -based adjuvant particles known in the art. In some embodiments, nanoscopic aluminum-basedcoating layers layered on at least one lipid assembly-free, or naked therapeutic polynucleotide-containing glassy microparticles disclosed herein can be significantly thinner than what is found in particles of conventional vaccines; therefore, total amounts of aluminum per administration can be essentially negligible, enhancing safety of administration and exposure with reduced side effects of these agents. In certain embodiments, an aluminabased coating layer can be sufficiently thin so that the total aluminum concentration per administration of the composition to a subject is less than about 100 pg, less than about 50 pg, less than about 25 pg, less than about 15 pg, less than about 5 pg, or less than about 1 pg in total aluminum per dose, for example.

[0091] In some embodiments and further to paragraphs

[0029] -

[0090] above, coating layers other than aluminum-based coating layers can be used to coat or sequester the at least one lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles. In accordance with these embodiments, non-aluminum coating layers can include, but are not limited to, other ALD-compatible coating agents. In certain embodiments, ALD-compatible coating agents can include, but are not limited to metal oxide, metal alkoxide, and / or the aluminum-based coating material and can include, but are not limited to, one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCL), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCh), zirconium silicate / zircon (ZrSiC ), hafnium oxide (HfCL) and combinations thereof used either in combination with aluminum-based coating layers, or alone to the exclusion of aluminum -based coating layers or as mixtures or alternating patterns of layers. With each type of material having different characteristic dissolution times, layers of these different materials can be deposited on the microparticles disclosed herein to vary temporal release of the lipid assembly-free, naked, or free therapeutic polynucleotide from the microparticles’ core or other outer layer when administered to a subject. In some embodiments, a microparticle disclosed herein can be coated with one or more coating layers of one material, followed by one or more layers of a different material. In accordance with these embodiments, different materials can dissolve more slowly or more rapidly; for example, than an aluminum-based coating layer. These alternative or mixed coatings can be tailored for the desired timed-release. Using selected ALD-compatible materials for coating the microparticles can reduce the number of coating layers necessary to provide for a given release time, minimizing the amount of adverse agent exposure per dose. In some embodiments, using selected ALD-compatible materials for coating the microparticles, can reduce the number of aluminum-based layers necessary to provide for a given release time,minimizing the amount of aluminum or other metal oxide or metal alkoxide per dose and reducing subject exposure to adverse effects of these coating agents.

[0092] In other embodiments and further to paragraphs

[0029] -

[0091] above, one or more coating layers can be deposited on the one or more lipid assembly-free, or naked therapeutic polynucleotide-containing particles by, for example, ALD (e.g., any instrumentation capable of atomic layer deposition can be used). ALD includes a thin film deposition technique that is based on the sequential use of a gas phase chemical process. ALD is considered a type of chemical vapor deposition. In certain methods, a majority of ALD reactions use two chemicals, referred to as precursors. These precursors react with the surface of a material one at a time in a sequential, self-limiting, or directed manner. Through the repeated exposure to separate precursors, a thin film can be precisely deposited. Use of ALD to deposit coating layers on agent-containing microparticles can be based on sequential, self-limiting reactions and provides for layer thickness control at the angstrom level as tunable coating layer compositions as described herein. Examples of ALD procedures of use in methods disclosed herein for depositing coating or sequestering layers on microparticles can be found in the art now that stabilized lipid-free, or naked therapeutic polynucleotide-containing microparticles can be provided as discovered and disclosed herein.

[0093] Embodiments of the present disclosure and further to paragraphs

[0029] -

[0092] above, provide for thermostable at least one lipid assembly-free, naked or free therapeutic polynucleotide-containing microparticle in a composition, where the composition can be produced by formulating the at least one lipid assembly-free, or free therapeutic polynucleotide-containing particles into a composition further comprising a buffer. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient. In certain embodiments, the composition can be used as a vaccine. For example, the at least one lipid assembly-free, or naked therapeutic polynucleotide encodes a polypeptide able to elicit an immune response in a subject.

[0094] In some embodiments and further to paragraphs

[0029] to

[0093] above, compositions disclosed herein that include ALD coated lipid assembly-free, or naked therapeutic polynucleotides can be used to produce a more balanced immune response. In accordance with these embodiments, ALD coated lipid assembly-free, or naked therapeutic polynucleotides can more equally induce a non-specific immune response concerning T cell activation. In some embodiments, ALD coated lipid assembly-free, or naked therapeutic polynucleotides can more equally induce T helper (TH) cell responses (e.g., compared to raw or uncoated therapeutic polynucleotides). In accordance with these embodiments, ALDcoated lipid assembly-free, or naked therapeutic polynucleotides can more equally induce THI and TH2 responses in a subject for a more balanced immunity; for example, when compared to lipid-containing, LNPs, or raw or uncoated therapeutic polynucleotides. In certain embodiments, inductions of THI and TH2 responses in a subject receiving formulations disclosed herein can be equal or nearly equal when the formulation includes ALD-coated lipid assembly-free, or naked therapeutic polynucleotides compared to LNPs and / or free therapeutic polynucleotides. Therefore, coated microparticles of the instantly claimed inventions can create a more balanced immune response in a subject receiving such a composition.

[0095] Certain embodiments and further to paragraphs

[0029] -

[0094] above, include methods to elicit an immune response to at least one lipid assembly-free, or naked therapeutic polynucleotide of coated stabilized microparticle or combination of agents of one or multiple agent-containing microparticles disclosed herein, by administering to the subject a composition including microparticles disclosed herein. The composition including the microparticles can be administered in therapeutically effective amounts. That is, in amounts sufficient to produce a protective immunological response or other therapeutic outcome. Generally, the immunogenic, therapeutic, prophylactic, and / or vaccine compositions can be administered in active agent dosages ranging from about 0.001 mg to about 20.0 mg active agent or about 0.01 mg to about 20.0 mg agent, or about 0.1 mg to about 10.0 mg agent.Single or multiple dosages can be administered in a single administration composition. Where multiple doses of an immunogenic, therapeutic, prophylactic, and / or vaccine composition can be administered in a single administration composition, for example, in prime-boost or prime and more than one boost compositions, these doses can be temporally controlled for release at a pre-selected time after administration.

[0096] Certain embodiments and further to paragraphs

[0029] -

[0095] above, concern compositions and methods for reconstituting ALD coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles disclosed herein in a buffer. In some embodiments, the buffer can include a buffer for delivering the ALD coated lipid assembly- free, or naked therapeutic polynucleotide-containing microparticles to a subject. In certain embodiments, the buffer can be a pharmaceutically acceptable buffer that includes at least one pharmaceutically acceptable carrier or excipient. Any pharmaceutically acceptable buffer is contemplated of use to reconstitute ALD coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles disclosed herein. In other embodiments, at least one nonaqueous suspension agent can be used to reconstitute ALD-coated microparticlesdisclosed herein. In accordance with these embodiments, nonaqueous suspension agents can include, but are not limited to, at least one of benzyl benzoate, 2-hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures or oils and the like. In other embodiments, the at least one refined triglyceride mixture or oil can include, but is not limited to, at least one of refined sesame oil, refined soybean oil, refined corn oil, refined cottonseed oil, refined peanut oil or other triglyceride mixture or oil or other acceptable refined triglyceride mixture or oil. In certain embodiments, suspension formulations disclosed herein combine ALD coated microparticles with nonaqueous solvent agents for allocation and / or distribution and use with reduced loss of product and in ready -to-administer delivery devices. In accordance with the embodiments, these suspension compositions are stable at elevated temperatures (e.g., room temperature or above) for prolonged periods (e.g., one hour to several months or more).

[0097] In certain embodiments and further to paragraphs

[0029] -

[0096] above, ALD- coated therapeutic agent containing microparticles disclosed herein can be suspended in a buffered suspension agent. Buffered suspension agents are well known in the art.

[0098] In certain embodiments and further to paragraphs

[0029] -

[0097] above, administration to a subject of reconstituted ALD coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticle formulations of the present disclosure can be performed using any acceptable means or methods known in the art. In some embodiments, formulations disclosed herein can be administered parenterally, locally or systemically. In certain embodiments, formulations can be administered, including by way of example, orally, intranasally, by inhalation, intravenously, subcutaneously, intradermally, intravaginally, by suppository, intramuscularly, and topically, by bolus administration or any other known mode of administration. In some embodiments, administration of compositions disclosed herein can be affected by factors including a natural route of infection by a particular pathogen. Dosage or dosages administered can depend upon factors including the age, health, weight, kind of concurrent treatment, exposure, if any, to pathogen, for example. Reconstituted ALD coated lipid assembly-free, or naked therapeutic polynucleotide- containing microparticle formulations disclosed herein can be employed in dosage form such as intravenous application, targeted application to a site in a subject, injectable, capsule, liquid solution, suspension, or elixir, for oral administration, or sterile liquid formulations such as solutions or suspensions for parenteral or intranasal use.

[0099] In other embodiments and further to paragraphs

[0029] -

[0098] above, compositions and methods are contemplated for using raw or naked therapeutic polynucleotides from acomplex. In some embodiments, compositions and methods are contemplated for using raw or naked therapeutic polynucleotides formulated with an LNP or originating from a lipid- containing formulation. In accordance with these embodiments, raw or naked therapeutic polynucleotides can be released from the lipids and these released raw or naked therapeutic polynucleotides can be further formulated into microparticles contemplated herein. In other embodiments, these released naked therapeutic polynucleotides formulated into microparticles as provided herein can be coated by ALD coating procedures disclosed herein. In some embodiments, released naked therapeutic polynucleotide formulations can include lipids or lipid complexes no longer complexed with these released, naked polynucleotides but that remain present in a formulation disclosed herein. It is contemplated herein that these lipids or lipid complexes no longer complexed with polynucleotides disclosed herein do not affect a released naked polynucleotide from being processed to ALD coated microparticles as described herein.

[0100] In certain embodiments and further to paragraphs

[0029] to

[0099] above, methods of making stable administration-ready ALD-coated thermostable at least one lipid assembly- free, or naked therapeutic polynucleotide-containing microparticle are disclosed. In accordance with these embodiments, these methods can include combining spray dried or spray freeze-dried ALD-coated lipid assembly-free, or naked therapeutic polynucleotide- containing microparticles with at least one nonaqueous suspension agent; and forming a suspension composition. In other embodiments, the ALD-coated lipid-free, or naked therapeutic polynucleotide-containing microparticles include a powder form of the ALD- coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles. In other embodiments, the at least one nonaqueous suspension agent can include, but is not limited to, at least one of benzyl benzoate, 2-hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures or oils and the like. In other embodiments, the at least one refined triglyceride mixture or oil can include, but is not limited to, at least one of refined sesame oil, refined soybean oil, refined corn oil, refined cottonseed oil, refined peanut oil or other triglyceride mixture or oil or other acceptable refined triglyceride mixture or oil. In certain embodiments, suspension formulations disclosed herein combining ALD coated microparticles with nonaqueous solvent agents can be readily used to fill any container for allocation and or distribution and use with reduced loss of product and in ready -to-administer delivery devices. In certain embodiments, suspension formulations disclosed herein combining ALD coated microparticles with nonaqueous solvent agents can be readily used tofill containers or delivery devices including, but not limited to, syringes, dermal administrators, inhaler, or the like). In accordance with the embodiments, these suspension compositions are stable at elevated temperatures (e.g., room temperature or above) for prolonged periods (e.g., one hour to several months or more).Kits

[0101] In other embodiments and further to paragraphs

[0029] -

[0100] above, kits are contemplated for storing, transporting and / or administering ALD-coated microparticles disclosed herein. In one embodiment, a kit can include ALD-coated microparticles disclosed herein, at least one pharmaceutical composition or a plurality of ALD-coated microparticles disclosed herein, and at least one container.

[0102] Other embodiments and further to paragraphs

[0029] -

[0101] above, provide kits of use with the methods (e.g., methods to elicit an immune response in a subject or treat a health condition) and compositions described in the present disclosure. In certain embodiments, a kit may contain one more microparticles in a dehydrated form. In certain embodiments, microparticles can also be provided. Different coated and uncoated microparticles can be mixed in a single pharmaceutical composition or separately provided to a subject. In certain embodiments, different microparticles are provided mixed together or in separated containers for storage and transport. Microparticle-containing pharmaceutical compositions can be provided in a known or predetermined quantity and / or at a predetermined ratio so that when the particles are reconstituted, the result is a composition with a known concentration of targeted agent or encoding polynucleotide.

[0103] In other embodiments and further to paragraphs

[0029] -

[0102] above, kits are contemplated of use for compositions, and methods described herein. Kits can be for storage or can be portable. In some embodiments, kits can include administration-ready pre-filled delivery devices containing nonaqueous solvent suspended coated microparticles disclosed herein. In certain embodiments, kits can be used to transport formulations disclosed herein to remote areas such as military installations or remote villages or towns. The thermostability of the ALD coated microparticles disclosed herein allows for transport and storage without the need for a cold chain (e.g., refrigeration, freezing) for a powder form, essentially dried formulation or reconstituted form of coated microparticles disclosed herein. These advantages can also be advantageous in healthcare facilities, as it reduces costs associated with cold storage, loss of product or unreliable delivery.

[0104] In other embodiments and further to paragraphs

[0029] -

[0103] above, kits can include an appropriate carrier or diluent suitable for reconstituting ALD coated particles ormicroparticles disclosed herein. In certain embodiments, the carrier or diluent can be a pharmaceutically acceptable aqueous buffer suitable for injection that includes pyrogen-free water and can resist changes in pH upon addition of an inorganic compound, organic compound, acid, alkali, or dilution with a solvent or diluent.

[0105] In some embodiments and further to paragraphs

[0029] -

[0104] above, kits can include one or more suitable containers, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe, or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the microparticles, pharmaceutically acceptable carrier or diluent and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0106] In yet other embodiments and further to paragraphs

[0029] -

[0105] above, kits can include ALD coated or uncoated lipid assembly-free, naked or free therapeutic polynucleotide-containing microparticles and compositions and optionally, instructions for coating uncoated microparticles. In other embodiments, kits can include instructions for reconstituting and / or administering reconstituted ALD coated lipid assembly-free, naked or free therapeutic polynucleotide-containing microparticle-containing formulations to a subject. In other embodiments, the kit can include an apparatus for performing such ALD coatings on microparticles. In accordance with these embodiments, the kits allow for production coated microparticles disclosed herein. In other embodiments, ALD coating layers disclosed herein can serve to reduce exposure to moisture, reducing degradation of lipid free, raw or naked therapeutic polynucleotide microparticles prior to administration. These coatings can function to protect water-soluble drug formulations or other moisture sensitive agents from degradation or dissolution until desired exposure to a subject after administration. In certain embodiments, compositions, components, formulations, sequestered and coated therapeutic polynucleotides and immunogenic compositions disclosed herein or encapsulated molecules using layering / coating technologies described herein can be transported, stored, and administered directly to a subject or to an affected bodily region of a subject such as the liver, lymph nodes, stomach, eye, kidney, or brain, tumor, or other targeted region in a subject.EXAMPLES

[0107] The materials, methods, and embodiments described herein are further defined in the following Examples. Certain embodiments are defined in the Examples herein. It is understood that these examples, while indicating certain embodiments, are given by way ofillustration only. From the disclosure herein and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.Example 1Atomic layer deposition coatings of aluminum oxide on spray dried microparticles containing mRNA and lipids:

[0108] In one exemplary experiment, a process is demonstrated for depositing layers of agents such as metal agents (e.g., metal oxides (e.g., aluminum oxides)) using repeated two- part, gas-phase reaction cycles that each deposit a molecular layer of the agent (e.g. alumina oxide) on the surface of spray dried microparticles. An exemplary polynucleotide (e.g., mRNA) was formulated at about 25 or 30 pg / mL for spray drying in about 9.5% low- endotoxin trehalose, about 2.5% hydroxyethyl starch, about 0.05% F-68, about 50 mM 1- histidine, about 1 mM 1-methionine in nuclease-free water at about pH 7.2. Formulated mRNA was kept on ice prior to spray drying. Lipid-free (e.g., naked, uncomplexed or lipid- assembly-free) polynucleotide solutions were spray dried to form dry, flowable microparticulate powders containing approximately 200-300 ng mRNA / mg powder. Spray drying resulted in complete incorporation of the polynucleotide (e.g., mRNA) within the glassy microparticles; polynucleotide concentrations were unchanged after spray drying and reconstitution of the spray dried polynucleotide-containing powders to their original volumes. Then, ALD was used to coat the spray dried microparticles. To coat the microparticles, the spray dried microparticles were suspended with an inert gas in a fluidized bed reactor of an ALD assembly. Alternating pulses of trimethyl aluminum (TMA) and water vapors were then added to the fluidized bed reactor with each two-pulse cycle depositing a single molecular layer of alumina approximately 2.3 A thick on the microparticle surface. The microparticles were coated with 30 or 50 molecular layers of the metal agent (e.g., alumina) in the custom- built fluidized bed reactor by these ALD cycles, yielding microparticles with alumina contents as determined by gravimetric analysis after calcination of 3.6 and 5.2%, respectively. It is contemplated that any ALD reactor can be used for these processes.

[0109] In another exemplary method, ALD-coated microparticles were imaged by scanning electron microscopy (SEM) (FIG. 1) demonstrating uniformly coated and distributed microparticles and flow imaging microscopy (FIM) to assess particle size (See for example FIG. 2 and FIG. 3). FIG. 2 represents an exemplary bar graph of particle size distribution for ALD-coated spray dried lipid assembly-free or naked polynucleotide-containingmicroparticle formulation representing average percent of the total ALD-coated microparticles at each given diameter size examined in pM. It is demonstrated that ALD coated microparticles of 2-3 pM had the highest percent of over 40% and about 90% of the microparticles were 8 pM or less. This is an important observation that demonstrates the nanocoating layers do not affect microparticle sizes and there is little to no agglomeration which for example, preserves product and improves syringability. In another exemplary method, FIG. 3 represents a comparison of particle size distribution for different microparticle processing, supporting the observation that lipid assembly-free or naked ALD coated polynucleotide-containing microparticles had the largest percentage of smaller sized particles (particles with a diameter under 7.0 pM with a majority of these 3.0 pM or less) when compared to spray dried naked, uncomplexed or lipid assembly-free mRNA microparticles without ALD coating and with ALD coating indicating greater syringability, reduced agglomeration than observed for larger particles. It is understood by one of skill in the art that microparticles 5.0 pM or less in diameter are known to be more readily phagocytosed than larger particles into macrophages and dendritic cells making the sequestered polynucleotide readily available for translation and / or presentation to the immune system of a subject receiving such a formulation.Example 2Immunogenicity and thermal stability of alumina-ALD coated polynucleotide (mRNA) preparations

[0110] In another exemplary method, immunogenicity of ALD-coated spray dried mRNA- containing microparticle (e.g., alumina oxide) preparations without lipid complexes were compared to a lipid complexed polynucleotide (e.g., mRNA-LNP) at different concentrations of an exemplary polynucleotide. This example used an mRNA encoding ovalbumin (OVA- mRNA) as a model therapeutic polynucleotide contemplated herein. The ALD-coated therapeutic polynucleotide containing microparticle powders were prepared as follows: 9.5% low-endotoxin trehalose, 2.5% hydroxyethyl starch, 0.05% F-68, 50 mM 1-histidine, 1 mM 1- methionine was introduced to the OVA-mRNA in nuclease-free water at pH 7.2 for spray drying. Then the samples were spray dried using standard methods. These spray dried powders were prepared and suspensions of spray-dried OVA-mRNA microparticles were introduced to an ALD reactor and coated with 50 layers of alumina oxide (ALD OVA- mRNA). These ALD coated or uncoated microparticles or powders were then resuspended in a suspension buffer (USP grad Super Refined TM Sesame Oil) for parenteral administrationand injected into mice intramuscularly at either 0.3 pg or 3.0 pg samples. Blood from the mice was collected via the submandibular vein. Serum samples were then used to determine IgG titers by ELISA at various times after injection.[OHl] In these studies, mice receiving OVA-mRNA without ALD coating did not seroconvert. However, seroconversion was observed in all mice immunized with ALD OVA- mRNA at both the high (3.0 pg) and low (0.3 pg) mRNA doses. All mice immunized with 3.0 pg doses of OVA-mRNA LNPs (not coated by ALD) seroconverted whereas only half of the mice immunized with OVA-mRNA LNPs (not coated by ALD) seroconverted at the lower dose of 0.3 pg mRNA.

[0112] FIG. 4 illustrates that anti-OVA IgG titers generated in response to immunization with 3.0 pg mRNA in ALD coated OVA-mRNA were significantly higher (by at least an order of magnitude) than those observed following immunization with uncoated OVA-mRNA LNPs at the same mRNA dose. FIG. 4 further illustrates that for both ALD OVA-mRNA and OVA-mRNA LNP, reducing the mRNA dose by 10-fold (e.g., from 3.0 to 0.3 pg) reduced anti-OVA IgG titers but the reduction in a lipid complexed mRNA sample, (uncoated OVA- mRNA LNP) was significantly reduced compared to the 3.0 pg. sample and significantly (about 2 or more logs) less than that of the average titer for the ALD OVA mRNA samples at either 0.3 pg or 3.0 pg. These data indicate that the ALD OVA-mRNA induced a greater response at both doses tested which were lOx difference in concentration, demonstrating improved immunogenicity when compared to the lipid complexed formulations (LNPs) alone without ALD coating and that the concentration was less important than the ALD-coating preparation. Further, FIG. 4 also illustrates that incubation temperatures prior to administration of the ALD coated OVA-mRNA to the animals did not impact the seroconversion observed in the mice or the resulting anti-OVA titers when incubated for 12 days at temperatures of about -80 °C, 4 °C, 25 °C or 40 °C. These observations demonstrate the stability and increased immunogenicity of these ALD coated OVA-mRNA preparations when prepared by methods disclosed herein (spray dried and ALD coated as uncomplexed mRNAs).Example 3Thermal stability of alumina-coated lipid assembly-free mRNA preparations

[0113] In another exemplary method, the thermostability of ALD-coated OVA-mRNA microparticles was studied by incubating the ALD-coated OVA-mRNA microparticles for up to three months at temperatures of about 4 °C, 25 °C, or 40 °C prior to administration to theanimal model (e.g., intramuscular injection in mice). The OVA-mRNA microparticles were prepared by adding 9.5% low-endotoxin trehalose, 2.5% hydroxyethyl starch, 0.05% F-68, 50 mM 1-histidine, 1 mM 1-methionine in nuclease-free water at pH 7.2 to the OVA-mRNA and then spray dried by methods known in the art. These spray dried OVA-mRNA were then coated with 50 layers of alumina (ALD-coated OVA-mRNA) by processes detailed herein. Then, the ALD-coated OVA-mRNA microparticles (powders) were resuspended in a nonaqueous suspension buffer (USP grade Super Refined ™ Sesame Oil). These oils permit ready administration with reduced product loss. These resuspended samples were incubated at the indicated temperatures for up to about 3 months.

[0114] After 1- or 3 -months incubation at the above referenced temperatures, the resuspended ALD coated OVA-mRNAs in oil were administered (injected) to mice (e.g., intramuscularly), and sera samples were collected and evaluated by ELISA. It was observed that at 3 months post incubation, the 4 °C, 25 °C, and 40 °C samples maintained titer close to the control level at -80 °C, see examples and FIG. 5, supporting the observation that the ALD coated OVA-mRNA are thermostable at increased temperatures for up to three months to at least to 40 °C.Example 4Lipid Free or naked mRNA encoding HIV antigens

[0115] In another exemplary method, mRNA-LNPs encoding for N332-GT2 (“trimer- mRNA” a therapeutic polynucleotide encoding an HIV trimer, a single polypeptide that, once expressed, intrinsically forms a protein trimer) and lipid-assembly-free or naked trimer- mRNA were spray-dried and coated by ALD. The ALD-coated therapeutic polynucleotide containing microparticles were prepared as indicated previously. The mRNAs, either mRNA- LNP or lipid assembly-free or naked mRNA were combined with 9.5% low-endotoxin trehalose, 2.5% hydroxyethyl starch, 0.05% F-68, 50 mM 1-histidine, 1 mM 1-methionine in nuclease-free water at pH 7.2 for spray-drying. Then, these spray dried powders were coated by ALD (e.g., alumina coating) as described above and herein.

[0116] Mice were immunized with about 2.5 pg of: uncoated trimer-mRNA LNPs formulated in a liquid suspension reconstituted from spray-dried powders, or suspensions containing about 2.5 pg of ALD-coated trimer-mRNA LNPs, or suspensions of about 2.5 pg of ALD-coated trimer-mRNA (lipid assembly-free or naked mRNA). Control, spray-dried microparticles, were reconstituted in nuclease-free water immediately prior to immunization. ALD-coated microparticles were suspended in a suspension buffer (e.g., USP Grade Super Refined ™ Sesame Oil) for parenteral administration. Following immunization, sera sampleswere collected, and titers were determined by ELISA after a predetermined period.

[0117] Liquid suspensions of trimer-mRNA LNPs elicited anti-trimer IgG titers following a single intramuscular immunization. FIG. 6 represents a graph demonstrating seroconversion in all immunized mice two weeks post-injection, and anti-trimer antibody titers reached a plateau by four weeks post-injection. Immune responses and seroconversion were present in fewer mice when trimer-mRNA LNP (lipid complexed) were spray-dried and reconstituted. Mice receiving lipid-free trimer-mRNA (uncomplexed, uncoated / without ALD coating) solutions or reconstituted spray-dried lipid-free trimer-mRNA solutions did not seroconvert and thus their titer values are not illustrated in FIG. 6. SD = spray dried microparticles without ALD coating; ALD = atomic layer deposition coated particles; liquid = liquid control of mRNA / LNP formulation.

[0118] Trimer-mRNA LNPs that were spray-dried (SD trimer-LNP, open square) and reconstituted prior to administration yielded decreased anti-trimer IgG titers and reduced seroconversion rates compared to those generated by control, liquid trimer-mRNA LNP formulations. Overall, mice immunized with ALD coated trimer-mRNA (lipid assembly-free or naked mRNA, diamonds) demonstrated a strong and lasting titer response over time, maintained at least to 112 days. In another experiment (data not illustrated), mice were injected subcutaneously rather than intramuscularly, which is known in the art to be less effective than intramuscular immunization. Surprisingly, the ALD coated trimer-mRNA (lipid assembly-free or naked mRNA) demonstrated improved responses when compared to trimer-mRNA LNPs not coated by ALD (data available upon request).Example 5Assessing balanced immunity: TH1 / TH2 polarization in immunized mice resulting from administration of lipid assembly-free mRNA within ALD-coated microspheres

[0119] In one exemplary method, mice were immunized with about a 3.0 pg dose of OVA- mRNA contained within suspensions of ALD-coated OVA-mRNA particles (“ALD”, uncomplexed, lipid assembly-free or naked mRNA) or liquid formulations of OVA-mRNA LNPs (“LNP”) (FIG. 7A). In another method (FIG. 7B), mice were immunized with 2.5 pg of an exemplary therapeutic polynucleotide, trimer-mRNA, in suspensions of ALD-coated trimer-mRNA microparticles (“ALD”) or liquid formulations of trimer-mRNA LNPs (“LNP”). The IgG2c / IgGl ratio which is an indicator of the TH1 / TH2 response in the animal model was calculated using the relative titer of each antibody subclass (see for example FIGS. 7 A and 7B). The microparticles used in both exemplary methods were prepared in 9.5% low-endotoxin trehalose, 2.5% hydroxy ethyl starch, 0.05% F-68, 50 mM 1-histidine, 1mM 1-methionine in nuclease-free water at pH 7.2 for spray-drying, then the spray dried samples were coated by ALD.

[0120] Prior to immunization, ALD-coated mRNA microparticles (alumina coated) and OVA-mRNA LNPs were stored at -80 °C, 4 °C, 25 °C, or 40 °C for 12 days. After immunization, sera samples were collected, and titers were determined by ELISA. The IgG2c / IgGl ratio was calculated using a mass concentration of each antibody subclass.

[0121] For reference, an IgG2c / IgGl ratio greater than one indicates a TH1 -biased response in mice. Mice immunized with mRNA-LNPs exhibited TH2 biased responses with IgG2c / IgGl ratios below one, regardless of the encoded antigen. Surprisingly, median IgG2c / IgGl ratios of approximately one in mice immunized with ALD-coated OVA-mRNA or ALD coated trimer-mRNA indicated a shift to a more balanced TH1 / TH2 polarization. This more balanced TH1 / TH2 polarization indicates that the ALD-coated OVA-mRNA or ALD coated trimer-mRNA likely will have fewer side effects and a longer lasting immune response when compared to the non-ALD coated lipid complexed mRNA formulations (LNPs). In addition, no effect of storage temperature was observed regarding median IgG2c / IgGl ratio for exemplary ALD coated OVA-mRNA preparations incubated for 12 days at -80 °C, 4 °C, 25 °C, or 40 °C. This demonstrates the stability of these molecules even when temperatures of pre-incubation are increased compared to the control.Example 6Uptake of ALD-coated lipid assembly-free or naked mRNA particles by immune cells

[0122] It is understood by one of skill in the art that macrophages and dendritic cells have both been identified as antigen-presenting cells (APCs) for polynucleotides including mRNA- vaccines. In another exemplary method, formulations containing an exemplary polynucleotide, in this case an mRNA-LNP encoding for a fluorescent reporter protein and another formulation containing lipid assembly-free or naked polynucleotide microparticles, in this case lipid assembly-free or naked mRNA encoding for the same fluorescent reporter protein were prepared in 9.5% low-endotoxin trehalose, 2.5% hydroxyethyl starch, 0.05% F- 68, 50 mM 1-histidine, 1 mM 1-methionine in nuclease-free water at pH 7.2 for spray-drying and then the spray dried samples were coated with 50 layers of metal oxide (e.g., alumina) by ALD. Both samples were ALD coated for these experiments. Macrophages and dendritic cells were incubated with the ALD-coated (e.g., alumina-coated) microparticles for up to 1 week. Detection of fluorescent reporter protein by a flow cytometry assay demonstrated that administration of ALD-coated microparticles to macrophage and dendritic cells leads to uptake of the ALD-coated microparticles, release of mRNA-LNP, or lipid assembly-free ornaked mRNA cargo, and expression within the cells. It was demonstrated that the ALD- coated lipid assembly-free or naked mRNA microparticles are capable of transfecting antigen presenting cells (APCs) independent of lipid-mediated mechanisms to promote desired immune responses.

[0123] RAW264.7 cells were transfected with GFP-encoding mRNA (GFP-mRNA) to determine whether phagocytic cells expressed the encoded protein upon particle uptake and release of mRNA from the particles. Spray-dried microparticles containing lipid-assembly- free or naked polynucleotides, GFP-mRNAs, were coated with 30 ALD layers (this is just an example of coating layers) of alumina particles and incubated with cells. As positive and negative controls, cells were incubated with GFP-mRNA using the lipopolyamine RmesFect (RF, a different lipid assembly than an LNP), a lipofection agent to assist transfection of mRNA into cells for in vitro experiments, or were incubated in PBS, respectively. FIG. 7B illustrates that cells expressed GFP by 24 hours after addition of ALD coated GFP-mRNA microparticles. In vitro transfection of RAW264.7 cells with ALD coated GFP-mRNA (open and back slash bars of the bar graph) was higher than with RF-mRNA complexes (solid and forward slash bars of the bar graph, FIG. 8B). It appears again that the different concentrations of the tested mRNA (pg) did not produce a significant difference in the ALD coated microparticles but the different concentrations of the RF samples demonstrated that the higher concentration was required to even see an effect.

[0124] In another exemplary method, to visualize cells following phagocytosis of alumina- coated particles, cells were allowed to adhere overnight prior to the addition of the alumina- coated particles containing AF647-trimer. After three hours, cells were washed, trypsinized and re-seeded in new wells. Cells were again allowed to adhere overnight before they were treated with Dil (Invitrogen) and Hoechst dye (Invitrogen) to visualize cell membranes and nuclei, respectively. After staining, cells were fixed with 4% paraformaldehyde then imaged on a Nikon AXR laser scanning confocal microscope with a 60x water immersion lens. Image stacks encompassing the full height of the cell (20 pm total, 0.5 pm z-steps) were captured then imported into Imaris (Oxford Instruments) for deconvolution and 3D reconstruction. RAW264.7 cells were seeded in 96-well PhenoPlate imaging dishes. To visualize particle uptake by RAW264.7 cells, spray-dried particles containing AlexaFluor-647-labelled trimer protein molecules were coated with 50 ALD-deposited layers of alumina and incubated with cells in culture media for three hours. Scanning confocal microscopy images of these cells demonstrated that the ALD-coated microparticles were present within the cells. A photographic representation of these cells is illustrated in FIG. 8A.All the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of embodiments, it is apparent to those of skill in the art that variations maybe applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.

Claims

What is claimed is:

1. An atomic layer deposition (ALD) coated lipid assembly-free, naked therapeutic polynucleotide-containing microparticle comprising: a central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle comprising at least one lipid assembly-free or naked therapeutic polynucleotide having transfective activity, and at least one glass-forming agent; and one or more ALD coating layers comprising one or more ALD coating agents comprising at least one of a metal agent, a metal oxide, a metal alkoxide, and aluminum-based agent, and mixtures and combinations thereof, covering the central or innermost lipid assembly, naked therapeutic polynucleotide containing glassy microparticle.

2. The microparticle according to claim 1, wherein the lipid assembly-free, or naked therapeutic polynucleotide is not part of a lipid nanoparticle (LNP), and not within a lipid- containing complex or other lipid assembly.

3. The microparticle according to claim 1 or 2, wherein the lipid assembly-free or naked therapeutic polynucleotide containing glassy microparticle is in absence of cholesterol, phospholipid, polyethylene glycol-modified lipids, and ionizable lipids.

4. The microparticle according to any one of claims 1-3, wherein the polynucleotide of the lipid assembly-free, or naked therapeutic polynucleotide comprises one or more of a single stranded (ss) or double-stranded (ds) DNA, RNA, mRNA, siRNA, saRNA, a chimeric polynucleotide molecule thereof, or other polynucleotide thereof.

5. The microparticle according to any one of claims 1-4, wherein the polynucleotide of the lipid assembly-free, or naked therapeutic polynucleotide comprises mRNA encoding a full-length polypeptide or one or more polypeptide fragment(s) thereof.

6. The microparticle according to any one of claims 1-4, wherein the lipid assembly- free, or naked therapeutic polynucleotide containing glassy microparticle further comprises at least one buffer salt.

7. The microparticle according to any one of claims 1-6, wherein the at least one glassforming agent comprises at least one polysaccharide.

8. The microparticle according to any one of claims 1-7, wherein covering the central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle comprises completely encasing the central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle with each ALD coating layer.

9. The microparticle according to any one of claims 1-8, further comprising at least one additional glass-forming agent or polysaccharide.

10. The microparticle according to any one of claims 1-9, wherein the at least one glassforming agent comprises at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, cyclodextrin, and povidone and the like.

11. The microparticle according to any one of claims 1-10, wherein the at least one glassforming agent comprises trehalose.

12. The microparticle according to any one of claims 1-11, wherein the at least one glassforming agent concentration comprises a weight-to-volume (w / v) concentration of about 0.1% to about 40.0%; or about 8.0% to about 15.0%.

13. The microparticle according to any one of claims 1-12, further comprising at least one surfactant or smoothing agent.

14. The microparticle according to claim 13, wherein the at least one smoothing agent comprises at least one of hydroxy ethyl starch (HES), polyvinylpyrrolidone, serum albumin, dextran, hetastarch, plasma protein factor and the like.

15. The microparticle according to claim 13, wherein the at least one surfactant comprises a high molecular weight surfactant.

16. The microparticle according to claim 13, wherein the at least one surfactant comprises at least one non-ionic surfactant; optionally, wherein the at least one non-ionic surfactant comprises at least one of polysorbate 80, polysorbate 20, pol oxamer 188, pol oxamer 403 or poloxamer 407, Tween 20, Tween 80, or the like.

17. The microparticle according to claim 1 or 2, further comprising at least one surfactant; optionally, wherein the at least one surfactant comprises a high molecular weight surfactant; optionally, wherein the at least one surfactant comprises at least one non-ionic surfactant; optionally, wherein the at least one non-ionic surfactant comprises at least one of polysorbate 80, polysorbate 20, poloxamer 188, poloxamer 403 or poloxamer 407, Tween 20, Tween 80, or the like.

18. The microparticle according to any one of claim 1-17, wherein at least one metal agent, metal oxide, metal alkoxide, and an aluminum-based coating layer comprises one or more of aluminum oxide (A12O3), an aluminum alkoxide, silicon dioxide (SiO2), titanium dioxide (TiO2), silicon nitride (Si3N4), zinc oxide (ZnO), zirconium dioxide (ZrCh), zirconium silicate / zircon (ZrSiC ), gallium oxide (Ga2Os), and hafnium oxide (HfCh) and mixtures and alternating layers or combinations thereof.

19. The microparticle according to any one of claims 6-16, or 18, wherein the lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle further comprises at least one buffer salt and the at least one buffer salt comprises at least one of succinate, acetate, citrate, prolamine, arginine, histidine, borate, carbonate, phosphate, and the like.

20. The microparticle according to any one of claims 1-19, wherein the central or innermost lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle formulation further comprises at least one amino acid.

21. The microparticle according to any one of claims 1-20, wherein the naked polynucleotide comprises a naked polynucleotide with increased transfectivity and capable of producing higher antibody titer in a subject when compared to a same polynucleotide complexed in a lipid assembly or lipid nanoparticle.

22. The microparticle according to any one of claims 1-21, wherein the ALD coated microparticles form part of a pharmaceutical composition and further include a pharmaceutically acceptable excipient.

23. The microparticle according to claim 22, wherein the pharmaceutical composition comprises an injectable formulation.

24. The microparticle according to any one of claims 1-23, wherein a therapeutic polynucleotide of the lipid assembly-free, or naked therapeutic polynucleotide comprises at least one polynucleotide: derived from a pathogenic organism, derived from a tumor, derived from a genetic disorder, derived from an anti -cancer agent, derived from an antiinflammatory agent, derived from an anti -autoimmune agent, or fragment thereof, or combination thereof.

25. The microparticle according to claim 24, wherein the pathogenic organism comprises a virus, bacterium, fungi, protozoa, prion, toxin or a fragment or subunit thereof.

26. The microparticle according to claim 1, wherein a therapeutic polynucleotide of the lipid assembly-free, or naked therapeutic polynucleotide encodes at least one of a recombinant peptide, a recombinant protein, a peptide derived from a target protein or pathogen, a synthetic peptide or protein, a virus-like particle, a protein expressed on the surface of a virus or a polypeptide derived from a bacteriophage or a combination thereof.

27. The microparticle according to claim 25, wherein the pathogenic organism comprises a virus or fragment thereof and the virus or fragment thereof comprises a virus-like particle (VLP), a live virus, a live, attenuated virus, an inactivated virus, or protein expressed on the surface of a virus or a combination thereof.

28. The microparticle according to claim 1, wherein a therapeutic polynucleotide of the lipid assembly-free, or naked therapeutic polynucleotide comprises mRNA derived from a pathogenic organism.

29. A method of making a metal oxide or metal alkoxide-coated lipid assembly-free, or naked therapeutic polynucleotide-containing microparticle according to any one of claims 1-28, the method comprising: a) combining at least one lipid assembly-free, or naked therapeutic polynucleotide with at least one glass-forming agent; b) spray-drying or spray freeze drying a) to make dried lipid assembly-free, or naked therapeutic polynucleotide-containing microparticles to generate a central or innermost lipid assembly-free, or naked therapeutic polynucleotide; and c) coating by ALD, the dried lipid assembly-free, or naked therapeutic polynucleotide microparticles of b) with one or more ALD coating layers comprising one or more of a metal agent, metal oxide, metal alkoxide, aluminum- based or combination, mixture, or alternating layers thereof, and encasing the central or innermost lipid assembly-free, or naked therapeutic polynucleotide- containing microparticles with each ALD applied layer.

30. The method according to claim 29, wherein the at least one lipid assembly-free, or naked therapeutic polynucleotide in a) further comprises at least one surfactant.

31. The method according to claim 29 or 30, wherein the at least one glass-forming agent comprises at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, cyclodextrin, and povidone and the like.

32. The method according to any one of claims 29 to 31, wherein the lipid assembly-free, or naked therapeutic polynucleotide containing glassy microparticle further comprises at least one buffer salt.

33. The method according to any one of claims 29-32, wherein at least one buffer salt comprises at least one of succinate, acetate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate, phosphate, and the like.

34. A microparticle-containing composition, comprising a plurality of microparticles according to any one of claims 1-29, or a combination thereof; and at least one excipient or suspension agent.

35. The microparticle-containing composition according to claim 34, wherein the suspension agent comprises a nonaqueous solvent.

36. A method for treating, preventing, or reducing risk of onset of or treating a health condition in a subject, the method comprising administering to the subject a composition according to claim 22-25 to the subject.

37. The method according to claim 36, wherein the health condition comprises having or suspected of developing an infection from a pathogenic organism.

38. The method according to claim 36, wherein the health condition comprises cancer.

39. The method according to claim 36, wherein the health condition comprises at least one of a genetic disorder, an inflammatory disorder or an immune disorder.

40. The method according to claim 36, wherein the preventing or reducing the risk of onset of the health condition comprises prophylactic or therapeutic treatment to prevent or reduce onset of the health condition.

41. The method according to any one of claims 36-40, wherein the subject comprises a human.

42. The method according to any one of claims 36-41, wherein the subject comprises a non-human mammal, pet, livestock, bird, amphibian, reptile, fish or other domesticated animal.

43. The method according to any one of claims 39-40, wherein the subject comprises a human and the human comprises a neonate, an infant, a toddler, a child, an adolescent, a young adult, an adult, or an older adult subject.

44. A kit comprising at least one microparticle according to any one of claims 1-21 or 24- 28, or the pharmaceutical composition according to claim 22 or 23, and at least one container.

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