Compositions and methods for stabilizing ribonucleic acid in the dry state

Abstract

Embodiments described herein generally relate to compositions and methods for stabilizing RNA in the dry state. In an embodiment, a composition for stabilizing an RNA is provided. The composition includes an air-dried ribonucleic acid. The composition further includes a sugar, a polyol, or combination thereof. The composition further includes 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

Description

PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCTITLE: Compositions and Methods for Stabilizing Ribonucleic Acid in the Dry StateINVENTORS: Thomas C. Boothby; Tyler GonzalezCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 733,970, filed on December 13, 2024, which is incorporated herein by reference in its entirety.GOVERNMENT RIGHTS

[0002] This invention was made with government support pursuant to Grant No. P20GM103432 from the Wyoming IDeA Networks of Biomedical Research Excellence (INBRE) Program of the National Institute of General Medical Sciences (NIGMS). The United States Government has certain rights in the invention.FIELD

[0003] Embodiments described herein generally relate to compositions and methods for stabilizing a ribonucleic acid (RNA) in the dry state.BACKGROUND

[0004] Many organisms have evolved to survive near complete water loss, or desiccation. These organisms are capable of preventing and / or recovering from the severe cellular damages associated with the loss of intracellular water and resuming life upon rehydration. Surviving desiccation has been linked to the accumulation of protectant molecules that protect and stabilize sensitive cellular components. Much of the research into desiccation tolerance has revolved around understanding the stabilization of proteins and membranes. However, there are other biomolecules needed for desiccation recovery, such as RNA, that have been relatively understudied in this context.

[0005] There is a need for new compositions and methods for stabilizing an RNA in the dry state.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCSUMMARY

[0006] Embodiments described herein generally relate to compositions and methods for stabilizing an RNA in the dry state. In some examples, the inventors found that air-dried RNA may be stabilized by disaccharides such as trehalose and sucrose in a concentration dependent manner. These two sugars show significant protection to both non-coding ribosomal RNA (rRNA) as well as coding RNA, but in a non-monotonic fashion. Surprisingly, the inventors also found that too much of a protective sugar may cause deleterious effects on the RNA. The increasing degradation observed as concentration increases may be correlated to the water content of a sample, suggesting that excess water retained by the presence of protective sugars may play a role in RNA degradation at higher concentrations. Selected results presented herein demonstrate that, while these sugars provide protection to RNA, there is a particular amount of sugar for protection and water retention may be detrimental to RNA stability even in the dry state. To date, RNA has been relatively understudied in the context of desiccation tolerance despite its clear importance in recovery from the dry state. The studies presented herein, for example, highlights the effectiveness in stabilizing RNA with mediators of natural desiccation tolerance, and provides avenues for the development of novel storage methodologies for mRNA based vaccines and therapies.

[0007] In an embodiment, a composition for stabilizing an RNA is provided. The composition includes an air-dried ribonucleic acid. The composition further includes a sugar, a polyol, or combination thereof. The composition further includes 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0008] In another embodiment, a composition for stabilizing an air-dried RNA is provided. The composition has a heat of melting that is from about 300 J / g to about 450 J / g.

[0009] In another embodiment, a method for stabilizing an RNA is provided. The method includes introducing a ribonucleic acid with a polyol, sugar, or combination thereof to form a composition, wherein the composition is characterized as stabilizing the ribonucleic acid of the composition in a dry state. The method further includes air dryingPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC the composition at a temperature that is about ambient temperature or higher, with or without vacuumBRIEF DESCRIPTION OF THE DRAWINGS

[0010] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, and may admit to other equally effective embodiments.

[0011] FIGS. 1A-1D: Generalized schematic of degradation analysis assays of hydrated total RNA and coding RNA utilizing the RNA integrity number (FIG. 1A) and in vitro translation (FIG. 1C) assays. Degradation over a 7 day time course at 23 °C, 37°C, and 60°C for hydrated total RNA (FIG. IB) and hydrated coding RNA (FIG. ID). In FIG. IB, the bar without hatching at -80°C is fully intact RNA stored in the hydrated state at -80°C. In FIG. ID, the bar without hatching at -80°C is fully intact coding RNA, stored in the hydrated state at -80°C. Degradation may be observed for total RNA, increasing with both temperature and time (P<0.0001 for both interaction and main effects) as well as for coding RNA (P < 0.0001 for interaction and main effects). **** = P < 0.0001 analyzed using Tukey’s multiple comparisons test following two-way ANOVA.

[0012] FIGS. 2A-2F: Generalized schematic of the experimental design showing degradation of total RNA and coding RNA post drying regime (FIG. 2A). A 7 day time course of dry total RNA (FIG. 2B, P<0.0001 for both interaction and main effects) and dry coding RNA (FIG. 2D, P<0.0001 for both interaction and main effects) at 23°C, 37°C, and 60°C. Comparison between hydrated 7 day time courses (FIG. 1) and desiccated 7 day time course (FIG. 2C, FIG. 2E) for total RNA and coding RNA. In FIG. 2B, the bar without hatching at -80°C is fully intact RNA stored in the hydrated state at -80°C. In FIG. 2D, the bar without hatching at -80°C is fully intact coding RNA, stored in the hydrated state at -80°C. **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed usingPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCTukey’s multiple comparisons test following two-way ANOVA (FIG. 2C, FIG. 2D) or Welch’s unpaired t-test with correction for multiple comparisons (FIG. 2E, FIG. 2F).

[0013] FIGS. 3 A and 3B: Desiccated and hydrated total RNA degradation after 24 hours at 60°C with varying concentrations of sucrose (FIG. 3 A) and trehalose (FIG. 3B). In FIGS. 3A and 3B, the bar without hatching at -80°C is fully intact total RNA, stored in the hydrated state at -80°C. ANOVA P < 0.0001 for both sucrose and trehalose. **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA.

[0014] FIGS. 4A and 4B: Desiccated and hydrated coding RNA degradation after 24 hours at 60°C with varying concentrations of sucrose (FIG. 4A) and trehalose (FIG. 4B). In FIGS. 4A and 4B, the bar without hatching at -80°C is fully intact coding RNA, stored in the hydrated state at -80°C. ANOVA P < 0.0001 for both sugars. **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA.

[0015] FIGS. 5A-5D: The effect of varying experimental parameters on RNA desiccation. Three week 37°C degradation of dry total RNA with varying concentrations of sucrose (FIG. 5A) or trehalose (FIG. 5B). Initial decrease in degradation follows a similar pattern to 60°C trials, but higher sugar concentrations show less degradation (ANOVA P < 0.0001 for both sugars). Comparisons between the degradation of dry total RNA at 1,000 ng / pL RNA or 100 ng / pL RNA with varying concentrations of sucrose (FIG. 5C) or trehalose (FIG. 5D). In FIGS. 5A-5D, the bar without hatching at -80°C is fully intact total RNA, stored in the hydrated state at -80°C. For both sugars, a change in RNA concentration shifts protection but has no effect on degradation. **** = P < 0.0001, *** = P < 0.001, ** = P < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following oneway ANOVA (FIG. 5 A, FIG. 5B) or Welch’s unpaired t-test with correction for multiple comparisons (FIG. 5C, FIG. 5D).

[0016] FIGS. 6A-6D: RNA degradation with extended sugar concentrations. Extended protection of total RNA with sucrose (FIG. 6A) and trehalose (FIG. 6B). Extended protection of coding RNA with sucrose (FIG. 6C) and trehalose (FIG. 6D). The x-axis isPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC sugar concentrations in mg / mL (0-293 mg / mL). In FIGS. 6A and 6B, the bar without hatching at -80°C is fully intact total RNA, stored in the hydrated state at -80°C. In FIGS. 6C and 6D, the bar without hatching at -80°C is fully intact coding RNA, stored in the hydrated state at -80°C. **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA. ANOVA P < 0.0001 for all trials.

[0017] FIGS. 7A-7F: Relationship between heat of melting (FIG. 7A, FIG. 7B) and RNA protection. Total RNA with varying concentrations of sucrose (FIG. 7C) and trehalose (FIG. 7D). Coding RNA with varying concentrations of sucrose (FIG. 7E) and trehalose (FIG. 7F). For all figures, left panels contain all sugar concentrations, top right contains no sugar (0 g / L) to 1 g / L of sugar, and bottom right contains 1 g / L to 293 g / L of sugar. R2and P values are Pearson correlations, and lines of best fit are simple linear regressions with 95% confidence intervals.

[0018] FIGS. 8A-8F: Effect of humidity on RNA degradation and retained water of RNA:sugars mixtures. 24 hour 60°C trials of total RNA (FIG. 8A) and coding RNA (FIG. 8B) degradation at varying humidities. Retained water of 7 pL total RNA with varying concentrations of sucrose (FIG. 8C) or trehalose (FIG. 8D). Retained water of 7 pL coding RNA with varying concentrations of sucrose (FIG. 8E) or trehalose (FIG. 8F). All sugar concentrations from left to right, are 50 g / L, 100 g / L, and 293 g / L. In FIGS. 8A and 8B, the bar without hatching at -80°C is fully intact total RNA, stored in the hydrated state at -80°C. **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA (FIG. 8 A, FIG. 8B). R2and P values are Pearson correlations, and lines of best fit are simple linear regressions with 95% confidence intervals (FIGS. 8C-8F). One-way ANOVA P < 0.0001 for both sugars.

[0019] FIGS. 9A-9D. Retained water of 70 pL of sugar-buffer mixtures vs RNA protection. Retained water of 70 pL. The RNA Storage Solution (TRss, AM7001) with varying concentrations of sucrose (FIG. 9A, FIG. 9C) or trehalose (FIG. 9B, FIG. 9D) compared to total RNA (FIG. 9A, FIG. 9B) or coding RNA (FIG. 9C, FIG. 9D) protection of that sugar concentration. R2and P values are Pearson correlations, and lines of best fit are simple linear regressions with 95% confidence intervals.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0020] FIGS. 10A and 10B: RNA concentration has no effect on hydrated degradation. Hydrated total RNA degradation with varying RNA concentrations (FIG. 10A). Hydrated coding RNA degradation with varying RNA concentrations (FIG. 10B). In FIG. 10 A, the bars without hatching at -80°C is fully intact total RNA, stored in the hydrated state at -80°C. In FIG. 10A, the bars without hatching at -80°C is fully intact coding RNA, stored in the hydrated state at -80°C. **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following two-way ANOVA. ANOVA P < 0.001 for interaction and main effects of all trials.

[0021] FIGS. 11A-11D: pH and Mg2+concentrations changes degrade total RNA and coding RNA. Degradation of total RNA (FIG. 11 A, P<0.05 for main effects, ns for interaction) and coding RNA (FIG. 1 IB, P<0.01 for both interaction and main effects) with increasing concentrations of Mg2+. Degradation of total RNA (FIG. 11C, P<0.0001 for both interaction and main effects) and coding RNA (FIG. 1 ID, P<0.0001 for both interaction and main effects) from pH 4 to pH 10. All experiments were performed at 37°C. In FIGS. 11 A and 11C, the bar without hatching at -80°C is fully intact total RNA, stored in the hydrated state at -80°C. In FIGS. 11B and 11D, the bar without hatching at -80°C is fully intact coding RNA, stored in the hydrated state at -80°C. **** = P < 0.0001, *** = P < 0.001, ** = P < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following two- way ANOVA.

[0022] FIGS. 12A-12D: Sugars have no effect on RIN score analysis but affect coding RNA translation. Effect of sucrose (FIG. 12A) and trehalose (FIG. 12B) on the analysis of frozen total RNA. Effect of sucrose (FIG. 12C) and trehalose (FIG. 12D) on the analysis of frozen coding RNA. **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA. No significance found for FIG. 12A and FIG. 12C; P < 0.0001 for FIG. 12B and FIG. 12D.

[0023] FIGS. 13A-D: Heat of melting of hydrated RNA with varying sugar concentrations. Heat of melting of total RNA with sucrose (FIG. 13 A) or trehalose (FIG. 13B). Heat of melting of coding RNA with sucrose (FIG. 13C) or trehalose (FIG. 13D). **** = p < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA. ANOVA P < 0.0001 for all trials.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0024] FIGS. 14A-14D: Percent water of 7 pL desiccated RNA-sugar samples. Percent water in sucrose with total RNA (FIG. 14 A) and coding RNA (FIG. 14B). Percent water in trehalose with total RNA (FIG. 14C) and coding RNA (FIG. 14D). **** = P < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA. No significance found for FIG. 14B and FIG. 14D; P < 0.01 for FIG. 14A and 14C.

[0025] FIGS. 15A-15D: Water mass increases as sugar concentration increases. Water mass in sucrose with total RNA (FIG. 15 A) and coding RNA (FIG. 15B). Water mass in trehalose with total RNA (FIG. 15C) and coding RNA (FIG. 15D). **** = p < 0.0001, *** = P < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA. All samples had ANOVA P < 0.0001.

[0026] FIGS. 16A-16D: Percent water of 70 pL desiccated buffer-sugar samples. Percent water in sucrose (FIG. 16A) and trehalose (FIG. 16B). **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = P < 0.05 analyzed using Tukey’s multiple comparisons test following one-way ANOVA. No significance found with ANOVA.

[0027] FIGS. 17A-17B: 7 pL and 70 pL desiccated samples contain similar amounts of water. Comparisons between 7 pL and 70 pL desiccations of sucrose (FIG. 17A) and trehalose (FIG. 17B). **** = P < 0.0001, *** = P < 0.001, ** = P < 0.01, * = P < 0.05 analyzed using Welch’s unpaired t-test with correction for multiple comparisons.

[0028] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0029] Embodiments described herein generally relate to compositions and methods for stabilizing an RNA. As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0030] Compositions described herein may include an RNA, an additive, and optionally water. These compositions may be characterized as protecting and / or stabilizing the RNA of the composition in a dry state. The term “dry state”, when referring to a composition described herein, refers to a composition having an amount of water that is 15 wt% or less water based on the total wt% of the composition, such as from 0 wt% to about 15 wt%, such as from greater than 0 wt% to about 15 wt%, such as from about 2 wt% to about 12 wt%, or from about 5 wt% to about 15 wt%, such as from about 6 wt% to about 14 wt%, such as from about 7 wt% to about 13 wt%, such as from about 8 wt% to about 12 wt%, such as from about 9 wt% to about 11 wt%, or about 10 wt% based on a total wt% of the composition. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0031] These compositions may be characterized as protecting and / or stabilizing the RNA of the composition in a dry state at a temperature up to about the glass transition temperature of the composition, such as from about ambient temperature to the glass transition temperature of the composition.

[0032] The glass transition temperature of the composition is determined by differential scanning calorimetry as shown in the Examples. Ambient temperature refers to a temperature of about 20°C.

[0033] The RNA (or RNA molecule) may be in its natural state, in a modified state, derived from a living organism, and / or synthesized. The RNA may be a natural product, a derivative thereof, a component thereof, or combinations thereof. The RNA may be an airdried RNA. Air drying of an RNA may be accomplished by removing at least a portion of the water from the composition at about ambient temperature (or other temperature), with or without vacuum and with or without spinning. Conventional technologies for storing RNA utilize the industry standard of lyophilization. Lyophilization is freezing then removing water.

[0034] The RNA may include total RNA, ribosomal RNA (rRNA), coding RNA, or combinations thereof. “Total RNA” includes all types of RNA such as: rRNA, transfer RNAPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC(tRNA), messenger RNA (mRNA), pre-mRNA, (long and small) non-coding RNA, microRNA, and double stranded RNA.

[0035] “Coding RNA” (also known as messenger RNA or mRNA) includes RNA that encodes a protein product produced through the process of protein translation. Coding RNA also includes precursors to coding RNA known as pre-mRNAs (or pre-messenger RNAs).

[0036] rRNA is RNA that is integral to the structure and function of the ribosome and includes precursors of rRNA.

[0037] Other RNAs that are neither rRNA nor coding RNAs include non-coding RNAs, such as tRNA, long-non-coding RNAs, and small non-coding RNAs.

[0038] The additive of the composition may include a sugar, a polyol, or combinations thereof. A polyol is a compound having more than one hydroxyl group ( OH). Polyols may include sugar alcohols.

[0039] Illustrative, but non-limiting, examples of sugars and polyols may include a disaccharide, a polysaccharide, a polymeric sucrose, glycerol, or combinations thereof. Suitable disaccharides may include trehalose, sucrose, maltose, lactose, or combinations thereof, such as trehalose, sucrose, or combinations thereof.

[0040] Polymeric sucrose (also referred to as polysucroses) are sucrose polymers formed from polymerization of sucrose monomers. In contrast to sucrose monomers, polysucroses are not found as naturally occurring excipients. Instead, polysucroses are commonly used in the biological field for purposes such as separation technology and to study glomerular physiology.

[0041] The polymeric sucrose may have any suitable molecular weight, such as from about 10 kiloDalton (kDa) to about 400 kDa, such as from about 70 kDa to about 400 kDa, or from about 20 kDa to about 150 kDa, such as from about 30 kDa to about 125 kDa, such as from about 40 kDa to about 100 kDa, such as from about 50 kDa to about 90 kDa, such as from about 60 kDa to about 80 kDa, such as about 70 kDa. Examples of polymeric sucrose may include, but are not limited to, polysucrose 70 and polysucrose 400. Polysucrose 400 (molecular weight of 400 kDa) is the largest commonly available polymer size of sucrose.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCPolysucrose 70 (molecular weight of 70 kDa) is in intermediate size between the sucrose monomer and polysucrose 400.

[0042] The composition may be glassy and without crystallinity upon air drying, indicating vitrification.

[0043] When a combination of sugars and / or polyols (additive mixture) is used, each of the sugars and / or polyols may be in any suitable amount. As a non-limiting example, an additive mixture may include (i) from about 85 wt% to less than 100 wt% of a disaccharide (e.g., sucrose monomer), based on a total wt% of the additive mixture; and (ii) from greater than 0 wt% to about 15 wt% of glycerol, based on a total wt% of the additive mixture. The total wt% of the additive mixture (the disaccharide plus the glycerol) not to exceed 100 wt%.

[0044] Compositions described herein may further include water. For example, the composition may include 15 wt% or less of water based on a total wt% of the composition. The composition may have an amount of water that is from 0 wt% or more, about 15 wt% or less, or combinations thereof, such as from greater than 0 wt% to about 15 wt%, or from about 2 wt% to about 12 wt%, or from about 5 wt% to about 15 wt%, or from about 6 wt% to about 14 wt%, or from about 7 wt% to about 13 wt%, or from about 8 wt% to about 12 wt%, or from about 9 wt% to about 11 wt%, or about 10 wt% based on a total wt% of the composition. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range. The total wt% of the composition is based on the amount of the RNA, plus the amount of the additive (or mixture of additives), plus the amount of the optional water. The total wt% of the composition is 100 wt%.

[0045] The amount of water in the composition may include water present in starting components (RNA and / or additive) and any added water.

[0046] Compositions described herein may include any suitable amount of RNA and any suitable amount of polyol and / or sugar. In some implementations, which may be combined with other implementations, a number of nucleotide molecules in the composition to a total number of polyol and sugar molecules in the composition (a ratio of nucleotide molecules to polyol and sugar molecules) may be in a range from about 1 :73 to about 1 :0.73, such as from about 1 :66 to about 1 :0.80, such as from about 1 :61 to about 1 :0.88, such asPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC from about 1 :56 to about 1 :0.95, such as from about 1 :53 to about 1 : 1, such as from about 1 :48 to about 1 : 1.1, such as from about 1 :43 to about 1 : 1.25, such as from about 1 :37 to about 1 : 1.5, such as from about 1 :26 to about 1 :2, 1 :18 to about 1 :3, such as from about 1 : 13 to about 1 :4, such as from about 1 : 11 to about 1 :5, such as from about 1 :9 to about 1 :6, such as from about 1 :7.6 to about 1 :7, such as about 1 :7.3. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range. The total number of polyol and sugar molecules in the composition refers to the number of polyol molecules (if any) plus the number of sugar molecules (if any) in the composition.

[0047] As described herein, the composition may be characterized as preserving or stabilizing the RNA of the composition at a temperature up to about the glass transition temperature of the composition, such as about ambient temperature or higher. Such temperatures may include about ambient temperature (about 20°C) to about 100°C, such as from 20°C to about 80°C, such as from about 20°C to about 60°C, such as from about 20°C to about 40°C, such as from about 22°C to about 37°C, such as from about 25°C to about 35°C.

[0048] As used herein, a “stabilized RNA” and “stabilizing” an RNA refers to maintaining the structure and / or the function of the RNA under either aqueous conditions or dried conditions, or after being dried and then rehydrated. The RNA may be stable at a temperature from about -80°C to about 100°C once the RNA is introduced or contacted with the additive, such as those temperatures at which the composition preserves or stabilizes the RNA. By use of embodiments described herein, a percentage (%) of the structure and function of the RNA that is maintained after 7 days at 23°C may be from about 10% to about 100%, from about 10% to about 95%, from about 10% to about 90%, from about 10 to about 85%, from about 10% to about 80%, from about 10% to about 75%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 20% to about 100%, from about 20% to about 95%, from about 20% to about 90%, from about 20% to about 85%, from about 20% to about 80%, from about 20% to about 75%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 30% to about 100%, from about 30% to about 95%, from about 30% to aboutPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC90%, from about 30 to about 85%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, from about 40% to about 100%, from about 40% to about 95%, from about 40% to about 90%, from about 40 to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 60%, from about 40% to about 50%, from about 50% to about 100%, from about 50% to about 95%, from about 50% to about 90%, from about 50 to about 85%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 100%, from about 60% to about 95%, from about 60% to about 90%, from about 60 to about 85%, from about 60% to about 80%, from about 60% to about 75%, from about 60% to about 70%, from about 70% to about 100%, from about 70% to about 95%, from about 70% to about 90%, from about 70 to about 85%, from about 70% to about 80%, from about 70% to about 75%, from about 80% to about 100%, from about 80% to about 95%, from about 80% to about 90%, from about 80 to about 85%, from about 90% to about 100%, from about 90% to about 95%, and the like, of the structure and function of the RNA is maintained. Any of the foregoing numbers can be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0049] Depending on the RNA sample, the percentage (%) of the structure and function of the RNA that is maintained may be determined by assessment of total RNA integrity (RNA Integrity Number) or quantification of coding RNA translatability (fluorescence) as described in the Examples.

[0050] Compositions described herein may be characterized as having a heat of melting. The composition may have any suitable heat of melting such as from about 300 J / g to about 450 J / g, such as from about 325 J / g to about 375 J / g, such as from about 340 J / g to about 360 J / g. Any of the foregoing numbers can be used singly to describe an open-ended range or in combination to describe a close-ended range. Heat of melting is determined by differential scanning calorimetry as described in the Examples.

[0051] Embodiments described herein also generally relate to pharmaceutical compositions. In some examples, pharmaceutical compositions may be used to treat a disease or condition in a patient. For example, the pharmaceutical composition may be aPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC mRNA-based vaccine. For example, the pharmaceutical composition may be an mRNA- based therapy.

[0052] Pharmaceutical compositions described herein may include a composition of the present disclosure (e.g., a composition that includes an air-dried ribonucleic acid). The pharmaceutical composition may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with suitable techniques.

[0053] In at least one embodiment is provided an mRNA-based vaccine. The mRNA- based vaccine may include any suitable composition described herein.

[0054] In some embodiments is provided an mRNA-based therapy. The mRNA-based therapy may include any suitable composition described herein.

[0055] Embodiments of the present disclosure also generally relate to methods of forming the compositions, to methods of stabilizing RNA, and to methods of forming a stabilized RNA. Embodiments described herein may be utilized to stabilize the RNA at temperatures that may be, for example, up to the glass transition temperature of the composition. Compositions formed by methods of the present disclosure include those compositions described herein.

[0056] Methods described herein may generally include introducing or contacting the RNA with the additive to form the composition comprising the RNA and the additive, thereby, for example, stabilizing the RNA. Introducing may be performed under conditions that include suitable temperatures, pressures, and rates of introduction of the RNA with the additive. The conditions may also include mixing the components of the composition by any suitable mixing process.

[0057] In some embodiments, the method further includes removing at least a portion of the liquid content of the composition. The liquid content may include aqueous material. Removing at least a portion of the liquid content may include air drying, or at least partially air drying, the composition that includes the RNA and the additive. Any suitable method of air drying may be utilized. Air drying may be performed with or without vacuum. Air dyingPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC may be performed at a temperature that is up to the glass transition temperature of the composition, such as about ambient temperature or higher, such as from about 20°C to about 60°C, such as from about 20°C to about 40°C. Air drying may be performed for a period of about 10 minutes to about 10 hours, such as from about 30 minutes to about 5 hours, such as from about 1 hour to about 4 hours, such as about 3 hours. Air drying may optionally include spinning the composition such as with use of a centrifuge or other rotating device.

[0058] Prior to introducing the RNA with the additive, the RNA (in the form of a solution or suspension) may be air dried. Alternatively, the RNA (in the form of a solution or suspension) may be introduced with the additive (which may be in the form of a solution or suspension) and the combined RNA and additive may be subjecting to air drying.

[0059] Air drying may be performed according to the following non-limiting procedure. About 1 pL (~1 pg / pL) of RNA in RNA storage solution (TRss), pH ~6.5, may be diluted to a total volume of about 7 pL with nuclease-free water before being placed into a vacuum concentrator (for example, a Savant Speedvac SCI 10 concentrator). For samples, instead of pure nuclease-free water, samples may be diluted in nuclease-free water containing varying concentrations of sugar. Vacuum may be achieved using any suitable vacuum pump (for example, a Thermo Scientific OFP400 vacuum pump). Samples may be spun under vacuum without heating for about 3 h at an atmosphere of about 0.3 mbar (about 30 Pa) to achieve desiccation. During drying, samples may be dried with a consistent volume in the vacuum concentrator (for example, 33 tubes with about 7 pL of solution). For samples that utilize less than this number, additional tubes with nuclease-free water may be added for consistency in drying rates.

[0060] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of embodiments of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for.ExamplesI. IntroductionPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0061] Across all kingdoms of life, many organisms have evolved to survive extreme water loss, or desiccation. These desiccation-tolerant organisms are capable of losing the majority of their intracellular water during drying and entering a metabolically inactive state before resuming normal life processes, even after years or decades of quiescence, upon rehydration. Typically, such extreme water loss causes lethal damage to cellular components, but desiccation-tolerant organisms are able to prevent or repair this damage. Understanding how desiccation-tolerant organisms accomplish this feat is one of the enduring mysteries of organismal physiology.

[0062] A universal strategy for mitigating the deleterious effects of desiccation is the accumulation of protectant molecules, which stabilize and prevent damage to sensitive biomolecules during drying. These protectants range from disordered proteins to small compounds such as the non-reducing disaccharides trehalose and sucrose.

[0063] Trehalose, which includes two molecules of glucose joined by an a, a-1, 1- glycosidic bond, is accumulated to high levels in many desiccation-tolerant organisms ranging from fungi to nematode worms. Trehalose has been shown to not only be directly required for desiccation tolerance in yeast and C. elegans, but also to stabilize sensitive proteins and membranes during drying both in vivo and in vitro.

[0064] Sucrose, a plant sugar that includes one glucose and one fructose joined by an u- 1, P-2-glycosidic bond, has similarly been linked to desiccation survival as well as membrane and protein protection. However, while these two sugars stabilize biomolecules during desiccation both in vivo and in vitro, the exact mechanism by which they provide stabilization is still unknown.

[0065] Most of the research in the desiccation tolerance field has assessed organismal survival or the stabilization of membranes and proteins. The stabilization of other biomolecules, such as nucleic acids, have seen comparatively little research. This may be due to the common paradigm that DNA is typically not directly protected from damage, but repaired rapidly. While DNA does incur oxidative damage during drying in vivo, the direct loss of water does not damage it, but rather provides some stabilization. While the removal of water does provide stabilization in terms of the primary structure of nucleic acids,PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC secondary and tertiary structure is typically altered by dehydration. While the effects of desiccation on DNA is generally known, much less is understood about its counterpart RNA.

[0066] RNA is extremely labile due to the ability of the hydroxyl group on the 2’ carbon of ribose to undergo a hydrolysis reaction with a backbone phosphate resulting in cleavage of the RNA strand. However, despite RNA’s labile nature, it has been shown to be required for desiccation survival in some organisms. In many dry seeds, stored mRNA is used to begin development after rehydration, indicating that mRNA is kept intact while the seed is in the dry state for years and even decades. In addition, there is a correlation between RNA Integrity Number (RIN), a measure of how intact an organism's total RNA is, and seed viability. In animals, pre-mRNA is accumulated before drying in the cysts of brine shrimp, and immediately incorporated into polysomes after rehydration. From these studies, intact RNA may be utilized for some desiccation-tolerant organisms to recover from the dry state.

[0067] Despite RNA’s link to recovery and development upon rehydration in some desiccation-tolerant organisms, there has been little research on the effect of the dry state on RNA or the role common desiccation protectants have on RNA stability. Given the nature of the transesterification reaction, it seems likely that the removal of water will slow its degradation similar to DNA. However, past work comparing dried and hydrated RNA shows no significant difference in degradation between hydrated, dried, and frozen RNA after 2 weeks at room temperature. Other studies have reported success with dry state storage of RNA utilizing proprietary storage matrices, such as RNAstable.

[0068] Lastly, two studies have reported that trehalose has a positive effect on RNA in the dry state, but these studies use paper storage matrices or lyophilization to dry RNA, and only test one concentration of trehalose. In the first of these studies, the researchers did not directly measure RNA integrity or function. Neither of these studies utilized air drying of RNA. In contrast, embodiments of the present disclosure are the first to investigate and utilize air drying of RNA. Furthering the understanding of RNA during extreme drying is useful for a more complete understanding of how organisms withstand such extreme conditions.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0069] In this study, total RNA and coding RNA degradation immediately after drying was investigated as well as after aging in the dry state simulated by thermal stress. It was found that, while both kinds of RNA degrade significantly less in the dry state relative to hydrated conditions, the presence of trehalose and sucrose provide further stabilization to both kinds of dry RNA. Surprisingly, it was found that protection varies in a non-monotonic fashion, where addition of these protective sugars typically increases preservation of RNA up to a particular point, after which further addition of the sugars has a significant and detrimental effect on RNA stability / translatability. In the majority of cases, it was found that this degradation correlates to the amount of residual water left in the dry samples, suggesting that RNA degradation may in part be caused by the miniscule amount of retained water. These results demonstrated that beyond their roles in protein and membrane stabilization during drying, trehalose and sucrose are capable of preserving both total RNA and coding RNA that have been subjected to air-drying. Additionally, the present study provides avenues for the development of formulations to aid in the preservation of air-dried RNA- based therapeutics.II. Materials and MethodsILA. Total RNA

[0070] Human total RNA from K-562 leukemia cell lines were purchased from Invitrogen. (AM7832 and AM7836) and used for all total RNA experiments.II.B. Coding RNA production

[0071] pET28a:GFP (Addgene #60733) in DHIOb E. coli was grown for 16 hours at 37°C in Luria-Bertani (LB) broth at 250 revolutions per minute (rpm) in a shaking incubator. After 16 hours of growth, plasmid DNA was extracted using commercial plasmid miniprep kits (Zymo ZR plasmid miniprep classic - D4015, or QIAprep Spin Miniprep Kit - 27104). pET28a:GFP was linearized with an overnight restriction digest utilizing BamHI-HF (NEB) at 37°C. DNA was then cleaned and concentrated using Zymo Oligo Clean and Concentrator (D4060), and eluted in 9 pL nuclease-free water. Linear DNA concentration and purity was quantified on a ThermoScientific NanoDrop One. Linearized plasmid was then transcribed to coding RNA utilizing MEGAscript T7 transcription kit (AM1334), with a reaction timePCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC of 4 hours. After transcription, RNA was purified with a MEGAclear transcription clean-up kit, and eluted in the RNA Storage Solution (TRss, AM7001) utilizing the second elution method described in the kit. RNA quantification and purity was then determined using a ThermoSci entific NanoDrop One. Samples were diluted to 1000 ng / pL (1 mg / mL) with the addition of TRss. After quantification, RNA was stored at -80°C until use.II.C. Assessment of total RNA integrity

[0072] RNA Integrity Number (RIN) was determined through the use of an Agilent 4200 Tapestation, utilizing high sensitivity RNA screentape. Total RNA (at 1 mg / mL) was first diluted in nuclease-free water to reach the required functional range of the instrument (1-25 ng / pL) before analysis. 2 pL of dilute RNA was mixed with 1 pL high sensitivity screentape buffer and prepared according to the manufacturer's protocol. High sensitivity ladder was prepared in an identical manner, with 2 pL of ladder mixed with 1 pL of high sensitivity buffer before analysis. An initial RIN was taken for each total RNA sample before the experiment began, and then compared to the experimental trials. See FIG. 1 A.

[0073] As described above, total RNA includes all RNA. Because rRNA makes up -95% of total RNA, the RIN method is used to quantify the quality and amount of rRNA. It is more or less assumed that this is a good proxy reflection of the other, less abundant species of RNA.II.D. Quantification of coding RNA translatability

[0074] In vitro translation was performed utilizing Promega’s Rabbit Reticulocyte Lysate system (L4960). Reaction size was scaled down by 50%, for a total reaction volume of 25 pL. 7 pL of coding RNA at a concentration of 142 ng / pL (1 pg) was combined with 0.5 pL premixed amino acid solution and 17.5 pL rabbit reticulocyte lysate, mixed gently, and incubated at 30°C for 90 minutes. After 90 minutes, 20 pL from each reaction was added to individual wells of a black 384-well microplate, spun at 900 rpm (174 relative centrifugal force (ref)) for 3 minutes before the fluorescence of each well was measured using a Tecan CytoSpark plate reader. Each experiment included a control translation with no RNA, which was then subtracted as background. All translation experimental trials were paired to identical frozen controls. See FIG. 1C.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCII.E. Desiccation of RNA

[0075] For drying experiments, 1 pg ( 1 pL) of RNA was diluted to 7 pL with nuclease- free water before being placed into a Savant Speedvac SCI 10 vacuum concentrator. Vacuum was achieved using a Thermo Scientific OFP400 vacuum pump. Samples were spun under vacuum with no heating for 3 hours to achieve desiccation. For rehydration, samples were either rehydrated in 100 pL nuclease-free water (for total RNA samples) or in 7 pL nuclease-free water (for coding RNA samples) and allowed to sit for 1 hour, before being utilized in their respective assays.ILF. Heating of RNA

[0076] Hydrated samples were stored to minimize evaporation. For 60°C experiments, hydrated samples were stored in a BioRad Cl 000 thermocycler for their respective time points. For the 37°C and 23 °C hydrated samples, samples were stored in a sealed humidified chamber and placed in incubators at 37°C or 23 °C. For dry samples, all samples were stored in a desiccation chamber with Drierite, before being placed in incubators at their respective temperatures for the indicated time.II.G. Disaccharide preparation

[0077] Five sugar stock solutions (0.01167 g / L, 0.1167 g / L, 1.167 g / L, 11.67 g / L, and 116.7 g / L) were made for both trehalose and sucrose utilizing Amsbio IM trehalose solution (AMS.TS1M-100) or Sigma-Aldrich BioUltra sucrose (84097) 1 M stock solutions. 6 pL of each stock solution was mixed with 1 pL of 1 pg / pL RNA, for a total volume of 7 pL, and sugar concentration of 0.01 g / L, 0.1 g / L, 1 g / L, 10 g / L, and 100 g / L, respectively. Concentrations were chosen not only to test a variety of conditions, but to also include concentrations exhibiting high levels of protection for other biomolecules, whole organisms, as well as being physiologically relevant. Samples were then dried as previously described, and placed at 60°C in a sealed glass desiccation chamber for 24 hours. After 24 hours, samples were cooled to room temperature (about 20°C), and then total RNA samples were rehydrated in 100 pL nuclease-free water while coding RNA samples were rehydrated in 7 pL nuclease-free water. Samples were then analyzed in their respective assays.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCII.H. Differential Scanning Calorimetry (DSC)

[0078] All DSC experiments were performed on a TA instruments DSC 2500 with autoloader. For heat of melting experiments, RNA: sugar mixtures were scaled up by a factor of four for a total volume of 28 pL. Hydrated samples were pipetted into pre-weighed aluminum Tzero pans, the samples mass determined, and then hermetically sealed. Samples were cooled at 1°C per minute to -40°C, held for 20 minutes to ensure thermal equilibrium, and then heated at 1°C per minute to 20°C. The curve at 0°C was then integrated to determine the enthalpy of melting in J / g.ILL Control of humidity

[0079] For experiments with varying humidity, dry RNA samples in open tubes were placed in an ESPEC environmental test chamber (Model BTL-433). To prevent condensation forming on samples at higher humidities, the chamber was preheated to 60°C without humidity control (resulting in a chamber humidity of 2-8%), and samples equilibrated for 30 minutes before humidifying at 60°C. Samples were removed from the chamber after 24 hours, and analyzed as described previously.II.J. Karl-Fischer Coulometry

[0080] Karl-Fischer Coulometry was performed using a Metrohm Eco-coulometer without diaphragm utilizing HYDRANAL Coulomat-AG (34836). Generator current was set to 400 mA and Ipoiwas set to 10 pA. Endpoint was set to 50 mV, and stop drift was 5 pg / min. All experiments were carried out at 25°C with no extraction time. All experiments were blanked in triplicate utilizing an equal volume of solvent prior to experimental samples being analyzed.

[0081] 7 pL samples were dried as described previously in preweighed Eppendorf tubes.After drying, dry mass of a sample was determined before 150 pL of dry 50% methanol and 50% formamide was added to the sample. Sample was vortexed until completely dissolved, briefly centrifuged, then drawn into a 250 pL Hamilton gas-tight syringe with a 4-inch 20- gauge needle. The syringe was then weighed, and the sample injected into the vessel of the coulometer. The empty syringe was then weighed again, and injected mass determined. ForPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC70 pL samples, the protocol was identical except samples were resuspended in 200 pL solvent instead of 150 pL.U.K. Mg2+

[0082] 1.11 mM, 11.1 mM, 111 mM, 1.11 M MgCh in nuclease-free water solutions were made by serially diluting a 2M MgCh in H2O solution. Then, 1 pL of 1 pg / pL RNA was added to 9 pL of the respective MgCh solution for final molarities of 1 mM, 10 mM, 100 mM, and 1 M. Samples were then placed at 37°C as previously described. At each time point, samples were removed, and Mg2+removed utilizing a MEGAclear transcription clean-up kit. For total RNA experiments, samples were then analyzed, while for coding RNA samples were concentrated as described previously using an Oligo Clean and Concentrate kit, and then analyzed by translation.ILL. pH

[0083] To determine the amount of acid or base to reach a given pH, varying amounts of 1 mM HC1 or 1 mM NaOH was added to TRss. This was determined on the milliliter scale to achieve an accurate pH measurement, before being reduced by a factor of 1000 for use on the microliter scale. pH setup was identical for both coding and total RNA. pH 4 - 2:7 TRss: 1 mM HCl, pH 5 - 3:5.5 TRss: l mM HCl, pH 6 - 6:4 TRss: l mM HCl, pH 7 - 6:2 TRss: l mM NaOH, pH 8 - 8.2:3.2 TRss: 1 mM NaOH, pH 9 - 7:3.2 TRss: 1 mM NaOH, 4:6 TRss: 1 mM NaOH. At all pHs other than 10, NaCl was added to account for the additional Na+added by NaOH at pH 10. Samples were then placed at 37°C for 1 day, 3 days, and 7 days.

[0084] After each respective time point, samples were removed from heat, and quenched by being brought to 100 pL with Tris-HCl. Then, samples were cleaned utilizing the Zymo Oligo Clean and Concentrate kit. For total RNA, samples were eluted in 10 pL NFW, and then brought to 100 pL with the addition of 90 pL NFW, before analysis. For coding RNA, samples were concentrated after quenching using the Clean and Concentrate kit as described in the concentration protocol.II.M. ConcentrationPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0085] F or total RNA, 1 pg of RNA was diluted to experimental concentration (5 ng / L, 10 ng / pL, 25 ng / pL, 50 ng / pL, 100 ng / pL) through the addition of TRss. After the time course, samples were all brought to 5 ng / pL with the addition of TRss, and then analyzed on an Agilent Tapestation as described in the assay.

[0086] For coding RNA, 1.5 pg of RNA was diluted to their respective concentrations (5 ng / pL, 10 ng / pL, 25 ng / pL, 50 ng / pL, 100 ng / pL) through the addition of the RNA Storage Solution. After the time course, samples were concentrated utilizing the Zymo Oligo Clean and Concentrator kit and eluted in 8 pL nuclease-free water. Sample concentration was then analyzed on a ThermoScientific NanoDrop One and diluted to 142.8 ng / pL with nuclease-free water. Then, 7 pL (1 pg) from each sample was used to assemble in vitro translation reactions as previously described.III. Non-limiting ResultsIII.A. Hydrated RNA degrades in a temperature and time-dependent manner

[0087] To assess the integrity of total RNA or the translatability of coding RNA after desiccation, two different assays were developed. First, RNA Integrity number (RIN), a common assay to determine RNA degradation, was quantified to assess total RNA integrity (FIG. 1 A). Hydrated total RNA shows more degradation as time and temperature increases (FIG. IB). An in vitro translation assay was utilized to determine coding RNA translatability (FIG. 1C). Similar to total RNA, the translation assay showed that coding RNA translation was reduced after heating and / or extended periods of time in the hydrated state (FIG. ID). Additionally, it was determined that RNA concentration has no effect on the rates of degradation under the conditions tested (FIG. 10), and that pH and Mg2+concentrations affect RNA degradation as expected with both assays (FIG. 11).III.B. The dry state provides significant protection to both kinds of RNA

[0088] After determining that the two assays could reliably measure degradation for both types of RNA, the effects of desiccation on total RNA and coding RNA were then determined. Desiccation was achieved through vacuum centrifugation of RNA samples for 3 hours, followed by resuspension in nuclease-free water after experimental trials (FIG. 2A). 1PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCFor samples analyzed immediately after desiccation (23°C Day 0, FIGS. 2B and 2D), neither total nor coding RNA showed significant degradation when compared to fully intact RNA.

[0089] Given the stability of RNA during drying, next investigated was whether the removal of water would be sufficient to keep RNA stable long-term in the dry state. To test this hypothesis, similar time courses to previous experiments (FIG. 1) were performed in order to determine if RNA in the dry state exhibited increased stability. For both total RNA (FIG. 2B) and coding RNA (FIG. 2C), there was no significant degradation at 23 °C even after 7 days. However, at 37°C both total RNA and coding RNA showed significant decreases in integrity and function during the time course. The 60°C time course exhibited the largest amount of degradation, with both types of RNA exhibiting high degradation at each time point.

[0090] When comparing desiccated RNA to hydrated RNA from previous experiments, significant reductions to degradation at several temperatures and time points in the dry state were observed (FIGS. 2C and 2E). Both dry total RNA (FIG. 2C) and dry coding RNA (FIG. 2E) showed lower degradation relative to hydrated RNA at all 60°C time points and some reduction at lower temperatures, although these were not always significant. While dry RNA appeared to degrade slower than hydrated RNA, degradation was still clearly present in dry samples.III.C. Trehalose and sucrose further stabilize RNA in the dry state

[0091] Given that some desiccation-tolerant organisms can resume life processes after decades of drying induced quiescence, and that mRNA in many cases may be critical for emergence from dormancy upon rehydration, it was hypothesized that RNA could be further stabilized in the dry state by protective biomolecules. To test this, the stabilizing effect of two common desiccation protectants, trehalose and sucrose, on RNA in the dry state was examined. In order to achieve sufficient degradation in shorter time periods, a time and temperature point with moderate degradation from previous experiments was chosen (60°C for 24 hours).

[0092] With sucrose, significant increases in total RNA integrity up to a maximum protection at 1 g / L were observed. However, past this concentration, a subsequent decreasePCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC in protective capacity with a large significant drop-off at 100 g / L was observed (FIG. 3 A). Trehalose exhibited a similar trend (FIG. 3B), with increases in protection up to 1 g / L. Protection remained high until 10 g / L, after which a significant decrease in RNA translatability was observed, although not nearly to the degree observed with sucrose. No effect on RNA stability was observed for either sugar in the hydrated state, implying that these sugars may not be protective to RNA at any concentration tested under hydrated conditions, and that both sugars are free of any RNase contamination as neither are seen to be detrimental to RNA stability even at high levels under hydrated conditions.

[0093] Next, the same concentrations of sucrose and trehalose on coding RNA stability were tested. For sucrose, a similar pattern of degradation as observed with total RNA was found, with increases in protection as sucrose concentration increases, followed by a large decrease in stability past 10 g / L (FIG. 4 A). For trehalose, a similar pattern of protection to previous sugar trials was observed (FIG. 4B). Interestingly, a significant decrease in protection from 1 g / L to 10 g / L, followed by a significant increase in protection from 10 g / L to 100 g / L, was observed (FIG. 4B). After this second increase in protection, there was not a significant difference between the degradation seen at 1 g / L and 100 g / L (FIG. 4B). While this initially looked like such a non-monotonic pattern might be due to inconsistency or outliers in the data, these experiments were repeated 3 times with the same reproducible result each time. Similar to total RNA, neither sugar had an effect on stabilization of coding RNA in the hydrated state. Combined, the data demonstrated that trehalose and sucrose confer protection to total RNA and coding RNA in a dry state, but at higher concentrations these protective effects may be overwhelmed by an emergent perturbing effect.III.D. Temperature and concentration both have effects on desired sugar protection

[0094] In order to gain insight into the possible effects that may be causing protection and / or degradation, experimental parameters were varied in order to, for example, determine how total RNA was effected. First, degradation temperatures were lowered from 60°C to 37°C, and experimental time was extended from 24 hours to 3 weeks.

[0095] For trehalose and sucrose (FIGS. 5 A and 5B), no difference in protection for either sugar at 37°C or 60°C was observed. However, after the particular protectivePCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC concentration (1 g / L) was passed, significantly less degradation in 37°C samples than in 60°C samples was observed, despite control RNA with no sugar (0 g / L) degrading to the same degree between the two temperatures. This result may indicate two different effects at play simultaneously, one causing protection and the other accelerating degradation of RNA.

[0096] Next, the concentration of total RNA down by a factor of 10 (from 1000 ng / pL to 100 ng / pL) was examined to determine how protection varies with RNA concentration. If two different processes are resulting in protection and degradation, then changing RNA concentration may result in an observable to change to one but not the other. Indeed, it was found that after reducing the concentration of total RNA in the sugar protection assay, protection likewise shifted down by a factor of 10 (FIGS. 5C and 5D). However, no similar shift was observed for degradation, with the same trend as seen with previous experiments (FIG. 3). Taken together, these experiments indicated that two different mechanisms are occurring in the dry state, one that stabilizes RNA and another that promotes degradation.III.E. Bound and retained water correlate with RNA degradation for most samples

[0097] After determining the processes that govern protection and increase degradation seem to be distinct, the possible mechanisms behind this phenomenon were investigated. The investigation primarily focused on degradation, since the low dry sample masses during protection made analyses difficult. One possibility that would easily result in increased RNA degradation would be the presence of water, since water is a key player in the non-enzymatic breakdown of RNA, and the inventors showed that simply removing water from RNA does reduce RNA degradation (FIG. 2).

[0098] First, the resolution of the protection experiments was increased with intermediate concentrations of sugars to allow for stronger correlations. The trends observed in the previous sugar protection experiments (FIG. 3 and FIG. 4) were still present when repeated with intermediate concentrations for both total RNA (FIGS. 6 A and 6B) and coding RNA (FIGS. 6C and 6D).

[0099] Next, a comparison of the amount of water bound by both sugars in the hydrated state across all concentrations used in protection experiments was performed. Bound water was determined through heat of melting, since the more water is bound, the lower the energyPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC needed to melt ice (FIGS. 7A and 7B). The results aligned well with the previous experiments, where the amount of detectable bound water changed little up to 1 g / L of sugar, and then increased with sugar concentration (FIG. 13). If RNA stability is correlated with heat of melting, no relationship is observed between the two variables until 1 g / L of sugar, the onset of degradation, and then significant negative correlations for most samples are observed (FIGS. 7C-7E). The one exception was with coding RNA and trehalose where a non-significant positive correlation was observed (FIG. 7F), due to the protection seen at high levels of sugar in those samples. While this data fits well with the hypothesis of a process that stabilizes RNA and a process that increases degradation, this data was performed in the hydrated state and may not be pertinent to the dry state.

[0100] In order to better understand the effect of water on dry state RNA, the effect of increasing humidity on dry RNA degradation was determined. In brief, dry RNA was placed at 60°C at relative humidities from 15-75% for 24 h before being resuspended and analyzed as described previously. Both total RNA and coding RNA show significant degradation as relative humidity (RH) increases, and both exhibit levels of degradation close to hydrated RNA at 75% RH (FIGS. 8A and 8B) indicating that water still plays a role in degradation even in the dry state.

[0101] Next, the relationship between the amount of water in a dry sample and the degradation of RNA was investigated. It was found that at all concentrations of sugar tested (50 g / L, 100 g / L, and 293 g / L), each sample averaged ~10 percent water content (FIG. 14). However, the total mass of water in each sample did increase with concentration (FIG. 15). If the molar ratio between ribonucleotides and water for each sugar concentration tested is instead calculated, a negative relationship between protection and water: nucleotide ratio emerged (FIGS. 8C-8E). However, only total RNA and trehalose showed a significant P value for the correlation between protection and water: nucleotide ratio (FIG. 8D) given the small number of data points in the correlation.

[0102] To address this issue, desiccation volumes were scaled up by a factor of 10 and increased drying time to 24 hours, allowing the determination of the water content of samples down to 5 g / L. Due to the increase in sample volume, RNA was excluded from these mixtures that included 10 pL of buffer and 60 pL of sugar. After desiccation, allPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC samples were analyzed via Karl Fischer Coulometry as described previously. Similar to the 7 pL trials (FIG. 8) all samples retained -10% water content (FIG. 16). Both 70 pL trials for trehalose and sucrose exhibited no statistical differences in moisture content between 7 pL trials and 70 pL trials without RNA, indicating that the drying regimes are equivalent (FIG. 17). After converting to nucleotide: water ratio, similar correlations emerged for both trehalose and sucrose for both types of RNA as observed previously with the 7 pL samples (FIGS. 8E-8H, FIGS. 9A-9D), with significant correlations for all samples except for total RNA and sucrose. All correlations observed were negative with the exception of coding RNA and trehalose (FIG. 9D), where a positive correlation between water: nucleotide ratio and water appeared. These results indicate that as the total amount of water in a sample increases, degradation tends to increase. However, while some trials exhibited extremely high R2values, others showed lower R2values and even positive correlations. This indicates that water content may not be the only factor influencing stability in some RNA-sugar mixtures.IV. Non-limiting Discussion

[0103] While the chemistry and molecular behavior of RNA in the hydrated state has been studied, there is less information in regards to how RNA behaves in the dry state. However, the pharmaceutical industry has performed large amounts of research into solid- state reaction kinetics, which has provided insight into the properties of DNA in the dry state. Relevant to RNA is the notion that reactions that typically occur in solution will still occur in the dry state, but at different rates due to the large change in molecular mobility between the hydrated and dry states. It has been shown that residual water may still participate in hydrolysis reactions in the solid-state. This aligns well with the differences in degradation seen between hydrated and dry RNA in the present study (FIGS. 2D and 2E), where the removal of water decreased degradation but did not halt it.

[0104] When looking at protection in the dry state, it was observed that the addition of trehalose or sucrose improves dry state protection significantly, but then typically dropped off past 1 g / L (FIG. 3 and FIG. 4). Interestingly, changing experimental parameters had an influence on the buildup of protection or onset of degradation, but not both (FIG. 5). Lowering temperature resulted in significantly less degradation but had no effect onPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC protection (FIGS. 5A and 5B), while lowering RNA concentration affected protection but had no effect on degradation (FIGS. 5C and 5D). Taken together, the results appear to indicate that the process stabilizing RNA and the process accelerating degradation are separate.IV.A. RNA Protection

[0105] One striking observation was the nearly identical build up in protection up to 1 g / L observed not only for both sugars, but both kinds of RNA tested. This result was unexpected not only due to the differences in size between the coding RNA and ribosomal RNA (which is quantified in the RIN assay) but also due to the homogeneity of the coding RNA solution when compared to the total RNA solution. However, 1 pg of RNA was used for all trials, so the weight of RNA and number of nucleotides comprising all RNA in each sample was identical.

[0106] Given this similarity, an estimate of the number of sugar molecules per nucleotide was calculated, finding a nucleotide to sugar ratio of 1 :7.3 at 1 g / L of sugar. This was surprising, as nucleotides are typically hydrated with a similar number of water molecules in the hydrated state. Given the already established water replacement hypothesis in the desiccation tolerance field, which hypothesizes that biomolecules are stabilized by protectants replacing the hydrogen bonds in order to maintain a hydrated-like state during drying, it may be that trehalose / sucrose are replacing the hydrogen bonds around RNA during drying. Disaccharides may replace hydrating water around nucleotides in solution, destabilizing secondary structure and lowering melting temperatures. That being said, unlike proteins which may unfold upon the loss of stabilizing hydrogen bonds, nucleic acids are typically stabilized upon the loss of water in vitro (FIGS. 2D and 2E). It seems unlikely that maintaining hydrogen bonds would be necessary for RNA stability, but another established hypothesis in desiccation tolerance, the preferential exclusion hypothesis, may have an answer.

[0107] The preferential exclusion hypothesis states that, during drying, water is excluded from sugar matrices. In the context of proteins, this exclusionary action may keep water next to the protein entrapped in the sugar matrix, once again maintaining its hydrogenPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC bond network. For RNA, the opposite may be occurring, with disaccharides replacing water during drying and excluding water from RNA, preventing any interactions that may lead to degradation. At lower concentrations, the number of sugar molecules may be unable to fully replace and exclude water, allowing some to remain bound to RNA and providing an opportunity for hydrolysis reactions. This may explain the gradual increase of protection observed with all sugars and both types of RNA (FIG. 3, FIG. 4, FIG. 6) as sugar concentration increased up to 1 g / L, as well as the apparent insensitivity to RNA type. Reducing total RNA concentration by a factor of 10 (FIGS. 5C and 5D) shifted the onset of protection down by a factor of 10. This indicates that the ratio of RNA: sugar may be important for protection, aligning with the previously mentioned hypothesis of sugars replacing water around a nucleotide.IV.B. RNA Degradation

[0108] A more puzzling observation was the decrease in protection seen in most experimental trials past 1 g / L (FIG. 3, FIG. 4, FIG. 6). Both sugars bound more water (FIGS. 7C-7F) and retained more water after drying (FIG. 7, FIG. 8) as sugar concentration increased. Given that water in the dry state may participate in hydrolysis reactions, it seems possible that this degradation may be caused by the increasing amounts of water in samples. Both bound water and water per RNA in the dry state showed negative correlations with RNA protection for most samples (FIG. 7, FIG. 8, FIG. 9) except for coding RNA and trehalose (FIG. 7F, FIG. 8F, FIG. 9D), which showed a slight positive correlation. The lower R2values observed in sucrose trials were largely caused by the sudden drop in integrity observed past 1 g / L, for example directly after 10 g / L for both total RNA and coding RNA (FIG. 3A, FIG. 4A). Given the differences between RNA degradation with sucrose or trehalose, but the similarities in water content, other factors may be influencing RNA stability other than just water content.

[0109] One explanation for the differences in degradation is the tendency of sucrose to crystallize more readily than trehalose. This is detrimental to protein protection because protection is associated with amorphous, or glassy, solids. If crystallinity is equally detrimental to RNA protection, then sucrose’s tendency to crystallize may explain the large drops in protection (FIG. 3, FIGS. 6A and 6C) seemingly unrelated to water content. It isPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC possible that crystallinity itself is not necessarily detrimental, since growing crystals exclude impurities from crystalline lattices. This could exclude RNA and water from a forming crystal, forcing water-RNA interactions and causing degradation. As to why crystallization appears to be dependent on sucrose concentration, there are several possible explanations. For example, polymers may reduce the nucleation rate of crystallization in other systems. As the constant mass of RNA becomes more and more of the minority in terms of total sample mass, nucleation rates could increase. Another, simpler explanation is that simply having more mass would make crystalline nucleation more common in that sample. Crystallization could also explain the differences observed in degradation between 3 weeks at 37°C and 24 hours at 60°C with sucrose experiments (FIG. 5A, FIG. 3A). Crystalline growth occurs faster at higher temperatures due to increased molecular mobility, so 60°C samples may have more rapid growth rates than 37°C samples.

[0110] The reduction of molecular mobility at lower temperatures may also provide an explanation for the decreased degradation observed at the higher concentrations of trehalose at 37°C when compared to 60°C. At lower temperatures, excess water in higher concentrations of sugar would remain excluded away from RNA and unable to react, due to the amorphous nature of the solid. At higher temperatures, the increase in molecular mobility may allow for water that would normally be excluded away from RNA to move to positions capable of reacting with RNA. This seems even more likely given that 60°C is above the Tg(glass transition temperature) of dry trehalose, a point at which amorphous solids transition to a rubbery phase, accompanied by a large increase in molecular mobility. However, while these hypotheses provide explanations for much of the observations, none sufficiently explain the degradation pattern with increasing trehalose for coding RNA (FIG. 4B, FIG. 6D).[OHl] With the high protection observed with coding RNA and trehalose at even the highest concentration of sugar tested (FIG. 6D), it seemed clear that whatever process is causing this elevated protection would need to be substantial. Different RNA secondary structures are stabilized / destabilized differently with equal concentrations of sucrose which seems reasonable to apply to trehalose given similar results with DNA. Secondary structure has been linked to RNA stability in the hydrated state, so it may be that the secondaryPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC structure of coding RNA is being stabilized with high concentrations of trehalose. However, one would expect similar observations with sucrose if this was the case. RNA sequence may also have a major effect on the crystallization of RNA itself. Given the homogenous nature of not only RNA size, but RNA sequence in the coding RNA trials, it may be possible that RNA crystallization is promoted in coding RNA trehalose samples, resulting in RNA crystals or possibly RNA-trehalose crystals. This would trap RNA away from water and avoid degradation, but also prevent increasing water and temperature from affecting RNA since crystals do not have a Tglike glasses. However, a typical indicator of crystallization is reduced water content and solubility, which was not observed for any coding RNA trehalose samples (FIG. 14D, FIG. 15B). It is possible that such crystallization only emerges at elevated temperatures, or remains local to RNA and forms micro / nanocrystals. The latter is not uncommon in dry disaccharides, with sucrose forming both amorphous and crystalline regions when dry. Unfortunately, the interactions between dry state RNA and disaccharides have seen little research, making conclusions difficult.

[0112] The study demonstrates that, while RNA is stabilized by the removal of water, desiccation protectants such as trehalose and sucrose provide further dry-state stabilization. Given the prevalence of these disaccharides in desiccation, it seems reasonable that organisms may rely on them not only to stabilize proteins and membranes, but also RNA. Here, it was shown that degradation is linked to water content at elevated temperatures. Overall, embodiments of the present disclosure show sugar and disaccharide mediated stabilization of RNA in the dry state.Embodiments Listing

[0113] The present disclosure provides, among others, the following embodiments, each of which may be considered as optionally including any alternate embodiments:

[0114] Clause 1. A composition for stabilizing a ribonucleic acid, comprising: an air-dried ribonucleic acid; a sugar, a polyol, or combination thereof; andPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%, wherein the composition is characterized as stabilizing the air-dried ribonucleic acid of the composition in a dry state.

[0115] Clause 2. The composition of Clause 1, wherein the air-dried ribonucleic acid comprises total RNA, coding RNA, ribosomal RNA (rRNA), or combinations thereof.

[0116] Clause 3. The composition of Clause 2, wherein the coding RNA comprises messenger RNA (mRNA).

[0117] Clause 4. The composition of any one of Clauses 1-3, comprising 7 wt% to about 13 wt% of the water based on the total wt% of the composition.

[0118] Clause 5. The composition of any one of Clauses 1-4, wherein a number of nucleotide molecules in the composition to a total number of polyol and sugar molecules in the composition is from about 1 :73 to about 1 :0.73.

[0119] Clause 6. The composition of any one of Clauses 1-5, wherein a number of nucleotide molecules in the composition to a total number of polyol and sugar molecules in the composition is from about 1 :26 to about 1 :2.

[0120] Clause 7. The composition of any one of Clauses 1-6, wherein the polyol, sugar, or combination thereof comprises a disaccharide, a polysaccharide, a polymeric sucrose, glycerol, or combinations thereof.

[0121] Clause 8. The composition of Clause 7, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

[0122] Clause 9. The composition of any one of Clauses 7 or 8, wherein the disaccharide comprises trehalose, sucrose, or combinations thereof.

[0123] Clause 10. The composition of any one of Clauses 7-9, wherein the polymeric sucrose has a molecular weight that is from about 10 kDa to about 400 kDa.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0124] Clause 11. The composition of any one of Clauses 7-10, wherein the polymeric sucrose has a molecular weight that is from about 40 kDa to about 100 kDa.

[0125] Clause 12. The composition any one of Clauses 1-11, wherein the composition is glassy and without crystallinity.

[0126] Clause 13. The composition any one of Clauses 1-12, wherein the composition is characterized as stabilizing the air-dried ribonucleic acid of the composition in the dry state at a temperature up to about a glass transition temperature of the composition as determined by differential scanning calorimetry.

[0127] Clause 14. The composition of any one of Clauses 1-13, wherein the composition has a heat of melting that is from about 300 J / g to about 450 J / g.

[0128] Clause 15. The composition any one of Clauses 1-14, wherein the air-dried ribonucleic acid maintains 50% or more of its structure and function after 7 days at 23°C.

[0129] Clause 16. A composition for stabilizing an air-dried ribonucleic acid, the composition having a heat of melting that is from about 300 J / g to about 450 J / g.

[0130] Clause 17. The composition of Clause 16, comprising: the air-dried ribonucleic acid; a sugar, a polyol, or combination thereof; and15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0131] Clause 18. An mRNA-based vaccine, comprising: the composition of any one of Clauses 1-17.

[0132] Clause 19. An mRNA-based therapy, comprising: the composition of any one of Clauses 1-17.

[0133] Clause 20. A method for stabilizing a ribonucleic acid, the method comprising:PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC introducing a ribonucleic acid with a polyol, sugar, or combination thereof to form a composition, wherein the composition is characterized as stabilizing the ribonucleic acid of the composition in a dry state; and air drying the composition at a temperature that is about ambient temperature or higher, with or without vacuum.

[0134] Clause 21. The method of Clause 20, wherein the composition comprises the composition of any one of Clauses 1-19.

[0135] Clause 22. The method of any one of Clauses 20-21, wherein the composition comprises: an air-dried ribonucleic acid; a sugar, a polyol, or combination thereof; and 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%, wherein the composition is characterized as stabilizing the air-dried ribonucleic acid of the composition in a dry state.

[0136] Clause 23. The method of any one of Clauses 20-22, wherein a number of nucleotide molecules in the composition to a total number of polyol and sugar molecules in the composition is from about 1 :73 to about 1 :0.73.

[0137] As is apparent from the foregoing general description and the specific aspects, while forms of the aspects have been illustrated and described, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, process operation, process operations, an element or a group of elements is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, process operation, process operations, element, or elements and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC

[0138] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0139] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, aspects comprising “a disaccharide” includes aspects comprising one, two, or more disaccharides, unless specified to the contrary or the context clearly indicates only one disaccharide is included.

[0140] While the foregoing is directed to aspects of the present disclosure, other and further aspects of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PCClaimsWhat is claimed is:

1. A composition for stabilizing a ribonucleic acid, comprising: an air-dried ribonucleic acid; a sugar, a polyol, or combination thereof; and15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%, wherein the composition is characterized as stabilizing the air-dried ribonucleic acid of the composition in a dry state.

2. The composition of claim 1, wherein the air-dried ribonucleic acid comprises total RNA, coding RNA, ribosomal RNA (rRNA), or combinations thereof.

3. The composition of claim 2, wherein the air-dried ribonucleic acid comprises the coding RNA.

4. The composition of claim 1, comprising 7 wt% to about 13 wt% of the water based on the total wt% of the composition.

5. The composition of claim 1, wherein a number of nucleotide molecules in the composition to a total number of polyol and sugar molecules in the composition is from about 1 :73 to about 1 :0.73.

6. The composition of claim 1, wherein a number of nucleotide molecules in the composition to a total number of polyol and sugar molecules in the composition is from about 1 :26 to about 1 :2.

7. The composition of claim 1, wherein the polyol, sugar, or combination thereof comprises a disaccharide, a polysaccharide, a polymeric sucrose, glycerol, or combinations thereof.PCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC8. The composition of claim 7, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

9. The composition of claim 7, wherein the disaccharide comprises trehalose, sucrose, or combinations thereof.

10. The composition of claim 7, wherein the polymeric sucrose has a molecular weight that is from about 10 kDa to about 400 kDa.

11. The composition of claim 7, wherein the polymeric sucrose has a molecular weight that is from about 40 kDa to about 100 kDa.

12. The composition of claim 1, wherein the composition is glassy and without crystallinity.

13. The composition of claim 1, wherein the composition is characterized as stabilizing the air-dried ribonucleic acid of the composition in the dry state at a temperature up to about a glass transition temperature of the composition as determined by differential scanning calorimetry.

14. The composition of claim 1, wherein the composition has a heat of melting that is from about 300 J / g to about 450 J / g.

15. The composition of claim 1, wherein the air-dried ribonucleic acid maintains 50% or more of its structure and function after 7 days at 23°C.

16. A composition for stabilizing an air-dried ribonucleic acid, the composition having a heat of melting that is from about 300 J / g to about 450 J / g.

17. The composition of claim 16, comprising: the air-dried ribonucleic acid; andPCT Patent ApplicationAttorney Docket No.: UWYO / 0123PC a sugar, a polyol, or combination thereof.

18. A method for stabilizing a ribonucleic acid, the method comprising: introducing a ribonucleic acid with a polyol, sugar, or combination thereof to form a composition, wherein the composition is characterized as stabilizing the ribonucleic acid of the composition in a dry state; and air drying the composition at a temperature that is about ambient temperature or higher, with or without vacuum.

19. The method of claim 18, wherein the composition comprises: an air-dried ribonucleic acid; a sugar, a polyol, or combination thereof; and15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%, wherein the composition is characterized as stabilizing the air-dried ribonucleic acid of the composition in a dry state.

20. The method of claim 18, wherein a number of nucleotide molecules in the composition to a total number of polyol and sugar molecules in the composition is from about 1 :73 to about 1 :0.73.

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