CN116829574A

CN116829574A - Ambient temperature lipid particle storage systems and methods

Info

Publication number: CN116829574A
Application number: CN202180090350.XA
Authority: CN
Inventors: P·莫汉蒂; S·达斯; Y·泰维纳; S·雷努; J·夏普; L·布龙萨特
Original assignee: Upkala Co ltd
Current assignee: Upkala Co ltd
Priority date: 2020-11-19
Filing date: 2021-11-19
Publication date: 2023-09-29

Abstract

Disclosed are methods of non-cryogenic vitrification of particles, lipid particle compositions, and mRNA vaccine compositions comprising lipid particles, the methods comprising the steps of providing lipid particles in a vitrification medium on a capillary network in a drying chamber and providing thermal energy and reduced atmospheric pressure to provide rapid vitrification without the vitrification medium or lipid particles experiencing cryogenic temperatures or boiling due to reduced atmospheric pressure. The lipid particles may be subsequently reconstituted after prolonged storage at ambient temperature or higher and still maintain structural integrity and activity.

Description

Ambient temperature lipid particle storage systems and methods

RELATED APPLICATIONS

The present application claims priority from U.S. provisional application 63/115,943 filed 11/19 in 2020 and U.S. provisional patent application 63/122,792 filed 12/8 in 2020, the contents of which are incorporated herein by reference in their entirety.

FIELD

The present disclosure relates to methods of preparing and storing lipid particles, optionally including one or more ribonucleic acids, without the need for cold chain storage temperatures.

Background

Ribonucleic acids or RNAs play a key core role in biology, providing a tool for enabling genes encoded in chromosomes to be driven to express proteins. The most important ribonucleic acids are probably messenger RNAs (mrnas), which are assembled in one copy from the parent DNA chromosome, cut exons, and transferred to a translation machine for reading and output as expressed proteins.

This key role in controlling protein output makes mRNA an interesting and attractive point for manipulating cells in an organism or indeed even the whole system. Of particular interest are manipulation of cells to express exogenous genes by providing RNA or producing mutant and/or overexpressed forms of endogenous proteins to affect signaling pathways within the cell and ultimately within specific tissues or organs.

Providing cells with mRNA of exogenous genes or parts or fragments thereof is also a powerful means to initiate the immune system of an organism. Forcing the cells to translate foreign mRNA in vivo can result in recognition as foreign or antigen and processing by cells of the immune system to produce antibodies and memory cells. If the foreign gene or fragment thereof is functionally inert at translation but provides recognition of the pathogen at a later time when introduced into the system, the mRNA is already effectively inoculated into the organism without or without the need for attenuated or live inoculants. mRNA is also safer because it is a non-infectious, non-integrated platform and is degraded by normal cellular processes. In a broad sense, mRNA is at the front of vaccine development, gene therapy, and protein replacement therapy.

While the role and attractiveness of mRNA is evident, providing mRNA to a subject is challenging. Initially, attention was focused on efficiently delivering mRNA to cells in vivo. To a large extent, this disorder has been addressed, although further progress is expected, but the challenges of delivery to cells in vivo are smaller (see, e.g., pardi et al Nat. Rev. Drug discovery.17:261-279 (2018)). One key development is the protection of mRNA prior to transfection into target cells. This has been solved in large part by complexing the mRNA with one or more transfection agents, typically in the form of lipid particles that encapsulate the mRNA and protect it from degradation.

Now, a practical barrier arises to be able to supply mRNA to a large population due to rapid overall degradation and loss of activity of mRNA or mRNA in a delivery vehicle at temperatures above freezing. From the point of manufacture to the point of administration, current technology requires that mRNA vaccines (including those packaged in lipid particles) be maintained at refrigeration temperatures, and typically well below zero degrees.

Existing methods of storing such systems rely on freeze-drying to dry the lipid particles so that content degradation is reduced. This entails significant costs and demands which prevent any rapid or massive application, in particular because of the long time scale required for lyophilizing these particles. Thus, there is a need in the art to identify methods of storing lipid particles that are effective but less demanding.

Brief description of the drawings

The figures are not necessarily to scale; some features may be exaggerated or minimized to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present disclosure. Exemplary aspects will become more fully understood from the detailed description and the accompanying drawings, wherein:

Fig. 1A shows a hydrophilic bed 10 with a thin film of liquid 20 placed over it, wherein the capillary force is significantly higher than the viscous force. This limits the amount of liquid that can be dried 21.

Fig. 1B shows a corrugated capillary bed, where drying may occur preferentially at the peaks of the corrugated profile 30, where capillary phenomenon from trough to crest during drying may increase the overall vitrification rate and allow vitrification of large sample volumes relative to fig. 1A.

Fig. 1C shows the surface pattern of liquid filling when there is excess fluid in the corrugated capillary 40, resulting in bubble nucleation and boiling becoming dominant under reduced pressure, which can lead to damage of sensitive molecules.

Fig. 2A shows a general schematic of vitrification in accordance with some aspects provided herein. Deep cryogenic (cryogenic) vitrification has historically been accomplished by rapidly cooling (paths 1-2-3) a liquid (containing biological or other material) below the glass transition temperature, thereby bypassing the freezing zone. The total mass of the material is conserved throughout the process. Similarly, vitrification of the material can be achieved by rapid drying (paths 1-5-6) that bypasses the crystallization process. In this case, a significant mass loss (mainly water) occurs. Deep cryogenic vitrification of large amounts of materials can be challenging due to heat transfer limitations, and is therefore typically performed in vials that provide significant surface area to volume ratios. Similarly, rapid drying is promoted by large surface/volume ratios and in particular under reduced pressure. The reduction in pressure also reduces the boiling point of the liquid, which has the risk of undesirable boiling of the liquid when vitrification of sensitive biomolecules or materials occurs. 1-4-6 show schematic diagrams of some vitrification aspects of the present disclosure, wherein the application of heat and low atmospheric pressure allows for rapid vitrification, thereby avoiding freezing temperature exposure.

FIG. 2B shows a three-phase schematic of water in which the target sample temperatureT ₁ Avoiding triple points and thus avoiding freezing during vitrification.

Fig. 3 shows an exemplary capillary (membrane) to facilitate rapid drying of large volumes of liquid under vacuum to form a vitrified glassy material.

Fig. 4 (a) shows that when excess liquid accumulates on the surface of the capillary membrane, capillary effect is not achieved, so that the accumulated liquid can still boil under vacuum, which is undesirable. To achieve the capillary effect, the liquid may be contained within the pores of the film forming the meniscus. This positioning on the undulating surface is similar and is formed by the peaks and valleys of the material. The fraction of liquid phase at the capillary interface (ζ), i.e. the area occupied by the liquid (in the two-dimensional schematic), is a parameter allowing to optimize the capillary evaporation. Capillary driven evaporation occurs when the viscous pressure drop in the liquid exceeds the maximum capillary pressure at the liquid-vapor interface. The liquid fraction ζ is related to the total pressure drop from the body to the liquid-vapor interface. In the case of atmospheric pressure and without the application of a heat flux (B), the liquid covers a large area, resulting in a liquid phase fraction ζ→1. Under these conditions, the capillary driven evaporation rate is minimal. Decreasing the ambient pressure as shown in (C) decreases ζ and thus increases the evaporation rate. However, above a certain threshold pressure drop, undesired nucleate boiling may occur. The applied heat flux Q as shown in (D) can also increase the evaporation rate, but there is a risk of undesired film boiling when heat is applied to the capillary channel from the supply side of the liquid. The application of heat flux from the surface of the capillary meniscus as shown in (E) significantly reduces the risk of film boiling. Applying large deltap and Q in an inverse gradient as shown in (F) results in a liquid meniscus confined in the aperture, i.e. a liquid phase fraction ζ < <1 (e.g. to 25), resulting in a highest evaporation rate while avoiding boiling. Thus, a temperature gradient between the holding surface and the bulk liquid causes capillary evaporation, as shown in (F), and rapid evaporation can be achieved. As the liquid level drops into the capillary membrane, capillary evaporation can still be achieved as long as the pressure gradient and the temperature gradient are maintained.

FIG. 5 shows the load containsVitrification results of different sized glass films for liquids of 4% BSA, 15% trehalose dihydrate, 0.75% glycerol, 2% Tween-20 and water. For all cases per mm ² The membrane-loaded liquid was kept at 0.316ml. For case 1, the film was cut into 0.25 inch diameter circles, each circle loaded with 10 μl of liquid. A total of 48 samples containing 480 μl were loaded onto a heated (37 ℃) wire mesh in a vacuum chamber. For case 2, three long film strips (240 mmx6.23 mm) each containing 470 μl of liquid were loaded onto a heated wire mesh. For case 3, a single strip (240 mmx22 mm) containing 1700 μl of liquid was used. The chamber was evacuated to 29.5mmHg. The temperature-time diagram indicates the various stages of the vitrification process. At the beginning of the evacuation process, the pressure drops rapidly, while the membrane holder contains mainly liquid, and as expected, the temperature drops with the pressure drop. The heat flux provided from the wire mesh/bed prevents the stent temperature from dropping further to the frozen state. It should be noted that depending on the formulation of the vitrification vehicle/liquid, the freezing point may extend to sub-zero temperatures. In addition to preventing freezing, the heat flux provided from the bed also assists capillary evaporation while preventing the liquid from boiling under reduced pressure, as shown above (fig. 4F). As the moisture evaporates from the support, the temperature rises until it reaches the bed temperature. The heat flux is controlled so that the stent temperature does not exceed a set temperature, typically the bed temperature. As shown in fig. 5, the time required for the membrane support temperature to reach bed temperature varies with the support configuration and the amount of liquid loaded onto it. The time required to reach bed temperature is a measure of the main glass transition time, which means that most of the liquid evaporates during this time. However, the drying process may still extend beyond this time to remove some residual moisture, which may be referred to as secondary drying, which does not rely on capillary phenomenon. The process parameters and scaffold geometry are selected to optimize the volume of liquid that can undergo the primary drying process in a given time. Generally, faster drying rates are required to bypass the crystal precipitated phase boundaries shown in fig. 2A and ensure glass formation. However, there is a threshold rate above which vitrification is ensured, which depends on the chemical nature of the liquid, membrane properties such as hydrophilicity, porosity and size.

Fig. 6A illustrates an exemplary aspect of the present disclosure, wherein the drying apparatus itself features a corrugated wall. The drying means may be formed of a hydrophilic capillary membrane rolled into a cylindrical shape. Similar to fig. 4, the cylinder may contain a vitrification medium within the film, thereby promoting improved vitrification.

Fig. 6B shows another aspect of the present disclosure, wherein a porous material membrane substrate is placed within a cylinder that can be operably connected to a vacuum and sealed for vitrification of a sample placed on the membrane, wherein the membrane provides a capillary substrate for vitrification.

Fig. 7A illustrates an exemplary aspect of the present disclosure, wherein a cylindrical drying device is placed in a heating block to provide directed heat flux to promote capillary evaporation and prevent the scaffold temperature from falling into a frozen state. The heating method may be conductive or radiant in nature.

Fig. 7B illustrates an exemplary aspect of the present disclosure, wherein additional heat sources are provided from inside the cylinder. The heating method may be conductive or radiant in nature. The heat flux may be provided from only one surface of the membrane or from both surfaces of the membrane.

Fig. 8 shows the improved vitrification produced using a film made of hydrophilic material. The originally hydrophobic film is treated with cold plasma to make it hydrophilic. When the drug formulation is suspended on the membrane, the liquid forms nearly spherical droplets (upper left), while the hydrophilic membrane allows the liquid to flow into the capillary channel. In the vitrification process, the droplets on the hydrophobic membrane are boiled and then frozen, and the liquid on the hydrophilic membrane is rapidly vitrified to form a glassy monolith. Upon release of the vacuum, the frozen droplets again become liquid, but decrease in size due to partial moisture loss. The efficacy of capillary-assisted evaporation for vitrification is evident with hydrophilic membranes.

Fig. 9 shows an overview of the evaluation of the vitrification of mRNA samples for an exemplary two week study procedure. mRNA was vitrified or not and stored as indicated, and assessed as described herein after days 0, 1, 3, 7 and 14, and then normalized and transduced into cells. At each time point, fresh mRNA constructed according to manufacturer's instructions was also assessed as a control point for comparison (IAWMS = according to manufacturer's instructions).

Fig. 10 shows that the amount of antigen-encoded mRNA remaining after vitrification and storage is nearly the same as fresh, indicating nearly 100% mass recovery. Each vitrified sample was loaded with 60 ng/. Mu.L (3. Mu.g in 50. Mu.L) of mRNA. Storage for up to 3 days at various ambient temperatures ranging from-20 ℃ to 55 ℃ resulted in mRNA yields of over 85%.

FIG. 11 shows that the mRNA function encoded by green fluorescent protein was protected from degradation during storage for 3 days at various ambient temperatures ranging from-20℃to 55 ℃. The data presented compares the relative fluorescence units of green fluorescent protein expression after transfection with vitrified and non-vitrified mRNA samples stored at the indicated temperatures. With increasing storage temperature, the non-vitrified samples showed a significant fluorescence loss, while the vitrified samples remained well active even after storage at 55 ℃.

Fig. 12 shows representative fluorescent cell images under specified conditions photographed 3 days after the start of storage as shown in fig. 11.

FIG. 13 shows day 7 fluorescence data for vitrified and non-vitrified samples and fresh mRNA. Although the storage conditions were different, vitrification again retained good activity, whereas the non-vitrified samples showed significant loss of expression activity.

Fig. 14 shows a representative image of the fluorescence presented in fig. 13, arranged in the same manner as provided in fig. 12.

Fig. 15 shows day 14 fluorescence data for both vitrified and non-vitrified samples. Also, although the storage conditions were different, vitrification remained good, while the non-vitrified samples showed significant loss of expression activity at all storage temperatures studied.

Fig. 16 shows a representative image of the fluorescence presented in fig. 15, with control samples on the left, top row vitrified samples, and non-vitrified samples stored as shown in the bottom row.

FIG. 17 shows day 3 fluorescence data for vitrified and non-vitrified samples containing Lipofectamine Messenger MAX (Invitrogen) and fresh mRNA. Also, vitrification allows to retain excellent activity despite different storage conditions, whereas non-vitrified samples show a significant loss of expression activity.

Fig. 18 shows a representative image of the fluorescence presented in fig. 17, left side is a control sample, top row is vitrified sample, and non-vitrified sample is stored as shown in bottom row.

Figure 19 shows the function of successful reconstitution and retention of vitrified mRNA from low starting volumes. (A) An agarose gel is shown depicting liquid mRNA (lanes 2 and 3), reconstituted mRNA (lanes 4 and 5) and non-vitrified mRNA (lanes 5 and 6) subjected to the same storage conditions. (B) Green Fluorescent Protein (GFP) expressed after transfection with fresh mRNA (up), non-vitrified mRNA (in) and reconstituted vitrified mRNA (down) is shown. (C) The percent GFP fluorescence relative to the positive control is shown. The vitrification process does not negatively affect the amount of mRNA recovered or its function.

FIG. 20 shows that lentiviral and fresh liquid lentiviral samples vitrified on water washed PES membrane or PBS-T washed bare filter (bound filter) were transduced and incubated on HEK293 cells for 72 hours. (A) Images taken using a fluorescence microscope after transduction are displayed. (B) The percent transduction efficiency based on fluorescence intensity measured using a fluorescence plate reader is shown and represents the percent transduction relative to a liquid lentivirus positive control. When cells were transduced immediately after vitrification, the performance of vitrification lentiviruses was as good as that of liquid lentiviruses stored at-80 ℃ regardless of the scaffold (bare filter or PES) used, indicating that the vitrification process did not damage the particles.

FIG. 21 shows lentiviruses vitrified on water-washed PES films or PBS-T washed bare filters, and negative controls (not vitrified) were stored at 24℃for 1, 2 or 3 weeks. Fresh liquid lentivirus, vitrified and non-vitrified negative control samples were transduced onto HEK293 cells and incubated for 72 hours. After capturing the transduced images using a fluorescence microscope. Liquid lentiviruses stored at-80℃are indicated as "positive controls". Non-vitrified liquid lentiviruses stored for 1 week at 24 ℃ are indicated as "negative control-I" (virus only) and "negative control-II" (virus and vitrification medium).

Fig. 22A shows the percent transduction efficiency based on fluorescence intensity measured using a fluorescence plate reader for 2 weeks of storage at 24 ℃ and expressed as percent transduction relative to a liquid lentivirus positive control.

Fig. 22B shows the same contents as fig. 22A after 3 weeks of storage at 24 ℃.

FIG. 23 shows lentiviruses vitrified on water-washed PES films or PBS-T washed bare filters, and negative controls (not vitrified) were stored at 37℃for 1 week, 2 weeks and 3 weeks. Fresh liquid lentivirus, vitrified and non-vitrified negative control samples were transduced onto HEK293 cells and incubated for 72 hours. After capturing the transduced images using a fluorescence microscope. Liquid lentiviruses stored at-80℃are indicated as "positive controls". Non-vitrified liquid lentiviruses stored for 1 week at 24 ℃ are indicated as "negative control-I" (virus only) and "negative control-II" (virus and vitrification medium).

Fig. 24A shows the percent transduction efficiency based on fluorescence intensity measured using a fluorescence plate reader for 2 weeks of storage at 37 ℃ and expressed as percent transduction relative to a liquid lentivirus positive control.

Fig. 24B shows the same contents as fig. 24A after 3 weeks of storage at 37 ℃.

Detailed description of the preferred embodiments

The present disclosure relates to methods of preparing lipid particles alone or lipid particles comprising cargo (cargo) molecules (e.g., nucleic acids, proteins, or others) alone or packaged in a deliverable vaccine composition, which allow for storage above cryogenic temperatures while maintaining their activity and/or avoiding their degradation. The method also involves stabilizing the mRNA vaccine composition without the need for freezing or other crystal formation in the sample. The present description generally refers to mRNA such as those contained in lipid nanoparticle or viral structures, but this is for illustration purposes only and is not meant to be limiting. The present invention is generally applicable to protecting structures and stabilizing any cargo within or on lipid particles.

In some aspects, the present disclosure relates to methods and compositions for preparing and/or storing particles. In some aspects, the particles may be or include lipids, proteins, carbohydrates, or any combination thereof. In some aspects, the particles may encapsulate or surround the polynucleotide. In some aspects, the particles may include a membrane of lipids, proteins, and/or carbohydrates that encapsulate the polynucleotide. In some aspects, the particles may include cells encapsulating the polynucleotide, viral particles encapsulating the polynucleotide, and/or lipid nanoparticles, lipid-like nanoparticles, or liposomes encapsulating the polynucleotide. In some aspects, it should be understood that in general, the membrane may include lipids along with proteins and/or carbohydrates dispersed therein.

In some aspects, the particles may be or include a membrane of lipids, proteins, and/or carbohydrates that form the outer shell. In some aspects, a polynucleotide may be present within the housing. In a further aspect, the particles themselves may be polynucleotides. In some aspects, the film may be a single layer or a double layer. In some aspects, the membrane may be a synthetic membrane of lipids, proteins, and/or carbohydrates. In some aspects, the particle is a cell or cell-derived membrane, such as a plant, bacterial, or animal cell, or a viral particle or viral particle-derived membrane. It should be appreciated that in certain aspects, when the membrane is a cell or viral particle, the cell or viral particle may be attenuated.

In some aspects, the methods and compositions provided herein include ionic lipids. In some aspects, the composition may include Lipid Nanoparticles (LNPs) or lipid-like nanoparticles (LLNs) comprising at least one nucleic acid molecule or chain therein. The term "particle" or "lipid particle" as used herein relates to a monolayer or bilayer particle comprising one or more ionic lipids, optionally but not limited to Phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol, polysaccharides, polymers, protamine, and the like. In some aspects, the nucleic acid, protein, or other molecule may be encapsulated within an LNP or LLN of two or more lipids (e.g., three, four, five, or more lipids).

In some aspects, the LNP may include an ionic lipid (typically labeled with three moieties, an amine head, a linker, and a hydrophobic tail, e.g., thirty-seven carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA or MC 3), DLinDMA, and DLin-KC 2-DMA). In some aspects, the LNP can include an ionic lipid, polyethylene glycol, and cholesterol. In other aspects, LNPs may include combinations of ionic lipids with polyethylene glycol (PEG), cholesterol, and/or distearylphosphocholine (see, e.g., sabnis et al mol. Ther.26:1509-1519 (2018), pardi et al J. Exp. Med.215:1571-1588 (2018), and Pardi et al J. Control. Release,217:345-351 (2015)). In some aspects, the LNP does not include cholesterol.

In some aspects, the LNP may further comprise a "helper" lipid. The helper lipids may include 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) and/or dioleoyl phosphatidylethanolamine (DOPE) and/or lipofectamine and/or dioleoyl phosphatidylcholine (DOPC) and/or phosphatidylethanolamine (dioleoyl PE) and/or 3β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] -cholesterol (DC-Chol) (see, e.g., du et al Scientific Reports 4:7107 (2014) and Cheng et al Advanced Drug Delivery Reviews (A): 129-137 (2016)).

Storage of the particles by a process involving vitrification presents particular challenges due to the nature of the particles themselves. First, the particles typically encapsulate an aqueous environment, which in some aspects includes one or more functional molecules, such as mRNA, protein, and the like. The purpose of the particles is to protect the cargo and, in some aspects, facilitate downstream delivery, targeting, or other functions of the cargo molecules. Typical existing dry storage methods involve lyophilization, which requires a time scale of several hours to achieve complete drying, and often reduces the functional properties of the reconstituted product. This is typically the result of low temperatures causing crystallization of the lipid bilayer, which prevents water transport from the interior to the exterior of the particle during drying.

The methods provided herein are capable of achieving complete drying in minutes, optionally less than 10 minutes, while significantly improving the functionality of the reconstituted product and without the need for cold chain storage conditions. This process does not result in crystallization of the bilayer of lipid particles, allowing water molecules and stabilins to transport through the membrane much faster by keeping the membrane in a liquid/gel state that facilitates convective transport through the porous layer.

In some aspects, the methods provided herein maintain the temperature of the particles near the phase transition temperature (Tc) of the encapsulation layer or particle film. The permeability of liposomes increases when the bilayer transitions from an ordered gel phase to a disordered fluid phase at Tc. At sufficiently below Tc, the bilayer forms a stiffer gel phase, resulting in reduced flowability and reduced permeability at Tc relative to temperature. Similarly, when the temperature is sufficiently above Tc, the fluidity of the membrane increases, but the permeability also decreases. Thus, by increasing the temperature of the lipid particles to around Tc during drying, the delivery of stabilizing ingredients (e.g., disaccharides) into the particles to stabilize the load and the removal of water from the interior of the particles both maximize the drying time required and significantly improve the storage results by more quickly and efficiently stabilizing the load and lipid bilayer to achieve subsequent functions.

While much of the disclosure relates to protecting and storing mRNA in lipid particles, this is provided as an example only. The processes provided herein are equally applicable to particles comprising other cargo molecules or combinations of cargo molecules, or may simply be empty particles (no particular cargo molecule). Similarly, these processes are equally applicable to particles or empty particles of carbohydrates or proteins, other cargo molecules, or combinations of cargo molecules. Thus, the following description of mRNA and lipid particles applies equally to other cargo molecules or empty lipid particles or other particles. Thus, the expression "lipid particle" can equally be interpreted to include particles of carbohydrates, proteins, carbohydrates and lipids, carbohydrates and proteins, lipids and proteins, and lipid/protein/carbohydrate combinations.

A "polynucleotide" as provided herein may be used synonymously with a nucleic acid and is two or more linked nucleotides (e.g., adenine, guanine, cytosine, thymine, uracil, or any derivative thereof, whether naturally occurring or artificial). The polynucleotide may be DNA, RNA or other.

As used herein, the term "messenger RNA" or "mRNA" may include single-stranded ribonucleic acid copies of a geneShellfish, including pre-and mature mrnas, spliced mrnas, 5' capped mrnas, edited mrnas, and polyadenylation mrnas. mRNA may include gene transcripts having introns and exons or complete gene transcripts or mRNA with introns removed or spliced. mRNA can include single-stranded RNA gene transcripts labeled with a 5' cap, such as RNA 7-methylguanosine caps or RNA m ⁷ G caps. The mRNA may include a start codon of the trimeric ATG base sequence towards the 5' end of the molecule to indicate that the mRNA segment of interest begins translation into a protein, and may also include a stop codon of UAA, UAG or UGA in frame with the start codon to indicate the end of the coding region or the point at which translation ceases. The mRNA may further include an untranslated region (UTR) following the stop codon and may further include a polyadenylation (poly a) tail following the 3' untranslated region (UTR) of the single stranded molecule. The poly-a tail may be provided by template DNA or by using a poly-a polymerase. Those skilled in the art will appreciate that the exact length of the adenosine in the poly-a tail need not be exact, but can generally fall within the range of about 100 to about 200 adenosine residues. In some aspects, the mRNA may be optimized to avoid double-stranded secondary or tertiary structure and/or purified to remove any double-stranded variants (see, e.g., kariko et al Nucleic Acids Res.39:e142 (2011)).

As used herein, a "segment of interest" may refer to a span or sequence of nucleic acids within an mRNA that is to be translated or that is capable of being translated in a cell. The segment of interest may start with a start codon and may terminate with a stop codon, wherein the stop codons are in the same reading frame (i.e., three nucleic acids per codon and/or amino acids added in the translated protein or peptide). In some aspects, the segment of interest can further be characterized by a sequence mutation to replace rare codons with synonymous codons with more abundant cognate tRNA to increase protein yield. The segment of interest may be further adapted to enrich for G: C content to increase steady state mRNA levels (see, e.g., kudla et al PLoS biol.4: e180 (2006)).

As used herein, "capped" or "5 'cap" may refer to a structure or modification at the 5' end of an mRNA. In some cases, the cap may be an N7-methylated guanosine attached to the first nucleotide of the mRNA through a reverse 5'-5' triphosphate linkage or through the binding of N7-methylated GTP. In some cases, the first nucleotide is 2' o methylated. In some aspects, the 5' cap may comprise a synthetic cap or similar cap, such as an anti-reverse cap analogue or a GpppG analogue, see, e.g., muttach et al Bellstein J Org Chem 13:2819-2832 (2017); stepinki et al RNA 7:1486-1495 (2001); schalke et al RNA biol.9:1319-1330 (2012); and Malone et al Proc. Natl. Acad. USA 86:6077-6081 (1989)). The 5' cap may also include cap, cap1, and/or cap2 structures known in the art. The 5' cap may include commercially available modifications, such as clearcap. In some aspects, the 5' cap may be applied post-transcriptionally by using vaccinia virus capping enzymes.

As used herein, "vitrification" is the process of converting a material into an amorphous material. The amorphous solid may be free of any crystalline structure.

As used herein, "vitrification mixture" refers to a heterogeneous mixture of biological materials and/or lipid particles (optionally the lipid particles comprise one or more biological materials encapsulated within the lipid particles) and a vitrification medium comprising a vitrification agent and optionally other materials.

As used herein, a "vitrification agent" is a material that forms an amorphous structure or inhibits the formation of crystals in other materials when a mixture of the vitrification agent and other materials cools or dries. Vitrification agents may also provide osmotically protected or otherwise survive the cell or lipid particle during dehydration. In some aspects, the vitrification agent may be any water-soluble solution that produces an amorphous structure suitable for storage of biological materials. In other aspects, the vitrification agent may be inhaled within a lipid particle, cell, tissue or organ.

As used herein, "storable or storable" refers to the ability of a biological material to be preserved and remain viable for subsequent use.

As used herein, "hydrophilic" refers to attracting or preferentially associating with water molecules. Hydrophilic materials having a specific affinity for water maximize contact with water and have a smaller contact angle with water than hydrophobic materials.

As used herein, "hydrophobic" refers to lack of affinity for water. Hydrophobic materials naturally repel water, resulting in droplet formation and a large contact angle with water.

As used herein, "cryogenic" temperature or a temperature for "cryogenic generation" or similar expressions refer to a temperature at which a biological sample is exposed to freezing conditions. It should be appreciated that in some aspects, the cryogenic temperatures may include the freezing temperatures of the biological sample and/or the vitrification media. It should also be appreciated that the cryogenic temperatures are not limited by a particular threshold or range of temperature values in degrees Fahrenheit or Celsius, but rather may be determined by the relationship between the temperature, pressure, and molecular energy of the vitrified mixture of interest. It should also be understood that, as used herein, although certainly within the definition set forth, "cryogenic generation" and its similar derivatives are not limited to temperatures associated with liquid nitrogen at 1atm or temperatures of about-80 ℃.

As used herein, "above a cryogenic temperature" correspondingly refers to a temperature above the freezing point of the vitrified mixture. Points "above deep cryogenic" may further include temperature values where no freezing conditions exist relative to the surrounding atmosphere and molecular energy. As used herein, room temperature refers to a temperature of about 25 ℃.

"cryopreservation" generally refers to rapid cooling of biological samples, typically by using liquid nitrogen, which can rapidly cool liquid materials because of its low temperature, or small volumes of biological materials by direct immersion. The cooling rate reduces the mobility of the material molecules before they can be stacked into a thermodynamically more favorable crystalline state. Over a longer period of time, the molecules may align to crystallize, which may produce damaging results, particularly in biological samples. Water is an important issue in biological samples because it can crystallize rapidly and its abundance in living tissue can prove to be significantly destructive, allowing it to crystallize more and more destructive. Protective additives (commonly referred to as cryoprotectants) that interfere with the crystallization ability of the primary component can produce amorphous/vitrified materials.

As used herein, "boiling" may refer to the point at which a substance converts to vapor, typically marked by the formation of vapor bubbles within the substance that can escape into the surrounding atmosphere and dissipate therein.

By "glass transition temperature" is meant the temperature above which the material behaves in a liquid-like manner and below which the material behaves in a solid-like manner and enters an amorphous/glassy state. This is not a fixed temperature point, but may vary depending on the properties of the vitrification mixture of interest. In some aspects, the glassy state may refer to a state that a vitrified mixture enters when it falls below its glass transition temperature.

"amorphous" or "glass" refers to an amorphous material in which there is no long-range order of atomic positions (involving an order parameter of 0.3 or less). Solidification of the glassy solid occurs at a glass transition temperature T _g . In some aspects, the vitrification medium may be an amorphous material.

"Crystal" refers to a three-dimensional atomic, ionic or molecular structure consisting of a specific ordered geometric array of periodically repeating and referred to as a lattice or unit cell.

"crystallization" refers to a form of matter consisting of components arranged in an ordered structure at the atomic level, as opposed to glassy or amorphous. Solidification of the crystalline solid occurs at a crystallization temperature T _c 。

In some aspects, the present disclosure relates to methods of providing extended stability and/or storage of lipid particles, lipid particles containing one or more biological agents, mRNA compositions, and/or mRNA vaccine compositions. In the present disclosure, mRNA may be used interchangeably with an mRNA composition comprising mRNA and at least one additional molecule or an mRNA vaccine (which is an mRNA or mRNA composition) suitable for administration to an organism or cell to induce an immune response. In certain aspects, the storage may include a temperature of about-80 ℃ to about 60 ℃. In some aspects, the mRNA may be stored at room temperature of about 25 ℃ to about 60 ℃ for extended or indefinite periods of time or briefly. During such storage, the mRNA is able to maintain structural integrity and physical activity or the ability of such. In some aspects, the present disclosure relates to methods of preparing and storing mRNA such that the storage temperature is largely insignificant, particularly with respect to maintaining the activity and integrity of mRNA.

In some aspects, the disclosure relates to methods of stabilizing, storing and/or preserving mRNA or mRNA compositions, e.g., mRNA vaccine compositions, prior to introducing the mRNA or mRNA compositions, e.g., mRNA vaccine compositions, into a cell or organism and/or incubating with a cell. In other aspects, the disclosure relates to methods of storing and/or preserving mRNA or mRNA vaccine compositions prior to administration to a subject, e.g., including mRNA or mRNA vaccine compositions in injectable compositions and/or compositions for systemic administration.

mRNA and mRNA compositions

In some aspects, the methods of the present disclosure relate to stabilizing, storing and/or preserving mRNA or mRNA compositions. In some aspects, the method can be initiated by obtaining mRNA or mRNA composition or by isolating mRNA to be stored and/or preserved. In some aspects, the mRNA or mRNA composition to be stored and/or preserved may initially be in a solution, such as an aqueous solution. In some aspects, the aqueous solution may be water. In other aspects, the aqueous solution may be predominantly water, with salts and/or buffers added to promote stability of the mRNA therein.

In some aspects, the method comprises providing mRNA or an mRNA composition or an mRNA vaccine composition to the capillary surface. In some aspects, the mRNA or mRNA composition or mRNA vaccine composition may include a Synthetic or recombinant mRNA nucleic acid characterized by a segment of interest that is intended to be translated in a cell (see, e.g., rhodes (ed.) Synthetic mRNA: production, introduction Into Cells, and Physiological Consequences, humana Press, 2016). In some aspects, the mRNA molecules can be prepared by In Vitro Transcription (IVT) or by transcription of a plasmid DNA (pDNA) construct.

In some aspects, the mRNA and/or mRNA composition is a purified mRNA molecule or purified mRNA composition. In certain aspects, the mRNA can be purified by chromatographic methods, including reverse phase rapid protein liquid chromatography or high performance liquid chromatography. Further purification methods may include binding and elution by using poly-a tails with immobilized poly-T or poly-U.

In some aspects, the mRNA molecules can be modified by incorporating modified bases such as pseudouridine, 1-methyl pseudouridine, 5-methylcytidine, N6-methyladenosine, 2-thiouridine, and 5-methoxyuridine.

In some aspects, the mRNA is capped. In some cases, the mRNA molecule or single strand is capped with N7-methylated guanosine attached to the first nucleotide of the mRNA either through a reverse 5'-5' triphosphate linkage or by binding N7-methylated GTP. In some cases, the first nucleotide of the mRNA is 2' o methylated. In some aspects, the mRNA is capped with a synthetic or similar cap (e.g., an anti-reverse cap analog or GpppG analog). In a further aspect, the mRNA is capped by cap, cap1, and/or cap2 structures known in the art. In some aspects, mRNA caps are applied post-transcriptionally by using vaccinia virus capping enzymes. In other aspects, the mRNA is characterized by a segment of interest, UTR, and/or poly a tail.

In some aspects, the mRNA composition and/or mRNA vaccine composition can include a packaged and/or encapsulated mRNA molecule or single strand, e.g., a lipid encapsulated mRNA. In some aspects, mRNA may be encapsulated in an ionizable lipid. In some aspects, the mRNA composition can include Lipid Nanoparticles (LNPs) or lipid-like nanoparticles (LLNs) having at least one mRNA molecule or chain therein. In some aspects, the mRNA can be encapsulated in an LNP or LLN of two or more lipids (e.g., three, four, five, or more lipids).

In some aspects, the mRNA is encapsulated in the LNP. LNPs may include ionic lipids (typically marked by three parts of an amine head, a linker and a hydrophobic tail, e.g., thirty-seven carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA or MC 3), DLinDMA and DLin-KC 2-DMA). In some aspects, the LNP can include an ionic lipid, polyethylene glycol, and cholesterol. In other aspects, LNPs may include combinations of ionic lipids with polyethylene glycol (PEG), cholesterol, and/or distearylphosphocholine (see, e.g., sabnis et al mol. Ther.26:1509-1519 (2018), pardi et al J. Exp. Med.215:1571-1588 (2018), and Pardi et al J. Control. Release,217:345-351 (2015)). In some aspects, LNPs include, but are not limited to, (4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, distearoyl-sn-glycero-3-phosphorylcholine (DPSC), and cholesterol. In certain aspects, the LNP comprises (heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate), 1-monomethoxy polyethylene glycol-2, 3-dimyristoyl glycerol having polyethylene glycol with an average molecular weight of 2000, 1, 2-distearoyl-sn-glycero-3 phosphorylcholine, and cholesterol. Optionally, the LNP is as provided in Schoenmaker et al, international Journal of Pharmaceutics, volume 601,2021,120586.

In some aspects, the mRNA composition and/or mRNA vaccine composition may include a vehicle for improving cellular uptake of mRNA therein, such as a polymer or a polymer modified with a fatty chain or a polymethacrylate with amine-containing side chains or a polyasparagine or poly (β -amino) ester (PBAE) with oligoaminoethylene side chains. In some aspects, the vehicle of the mRNA composition can include a dendrimer, such as a polyamidoamine or a polypropylenimine-based dendrimer.

In some aspects, the mRNA composition and/or mRNA vaccine composition may include a cell-penetrating peptide (CPP) or carrier protein as a carrier to aid in delivering mRNA to cells, including CPP or protamine or D isomer xenry-protamine with arginine-rich amphiphilic RALA repeats. In further aspects, the mRNA composition may include a zwitterionic lipid (ZAL) or a combination of cationic and zwitterionic lipids. Kowalski et al (mol. Ther.27 (4): 710-728 (2019)) set forth an overview of current delivery vehicles for mRNA. Examples of carrier proteins include Tetanus Toxoid (TT), diphtheria Toxoid (DT), CRM197 (DT variant from diphtheria bacillus (C. Diptheriae) C7), meningococcal Outer Membrane Protein Complex (OMPC), haemophilus influenzae (h. Influenca) protein D, and keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH).

In some aspects, the disclosure relates to mRNA compositions for use in mRNA vaccine compositions. In some aspects, the mRNA molecules therein comprise segments of interest to express exogenous proteins or fragments thereof or engineered antigens, whereby expression of the segments of interest allows for cellular processing of translating these and/or presentation of the expressed segments of interest or fragments thereof into immune cells and systems of the cellular host organism. In some aspects, the mRNA is a nucleoside modified mRNA such that some nucleosides are replaced with other naturally occurring nucleosides or synthesized nucleoside analogs, optionally to increase immunogenicity relative to the unmodified mRNA. Examples of a COVID-19 vaccine using modRNA include vaccines developed by Biontech/Pfizer/Fosun International (BNT 162b 2) in concert and developed by Moderna (mRNA-1273), such as Kramer F, nature,2020;586 (7830) 516-527 or Dolgin, E.Nature Biotechnology 2020:d41587-020-00022-y.doi:10.1038/d 41587-020-00022-y.

The segment of interest is optionally any segment encoding the desired protein. In some aspects, the segment of interest encodes a portion of a SARS-CoV-2 virus, influenza virus, or other viral or bacterial antigen. Exemplary proteins encoded by the segment of interest include, for example, the SARS-CoV-2 spike (S) protein and the SARS-CoV-2 nucleocapsid (N) protein. The sequences of the N and S proteins of SARS-CoV-2 are known and commercially available from various suppliers, including, for example, rayBiotech (Peachtree Corners, VA). The segment of interest may be part of a viral antigen that is typically exposed to the environment external to the viral capsid. For example, in certain aspects, the segment of interest can encode the S1 or S2 subunit of SARS-CoV-2 spike protein S. However, the skilled artisan will appreciate that other peptides or fragments thereof, optionally any such exposed protein or protein portion outside of the cell of the capsid or membrane of any infectious agent, may be similarly encoded. Ou, et al, characterization of spike glycoprotein of SARS-CoV-2on virus entry and its immune cross-reactivity with SARS-CoV, nature Communications,11, arc 1620 (2020); and Ibrahim, et al, COVID-19spike-host cell receptor GFP, 78 binding site prediction, J.Infect., S0163-4453 (20) (March 10,2020), each of which is incorporated herein by reference in its entirety.

In some aspects, the mRNA composition and/or mRNA vaccine composition can include an mRNA molecule that includes more than one segment of interest. As will be appreciated, mRNA vaccine compositions may provide expressed antigens and viral replication mechanisms to allow for molecular self-amplification or such modifications necessary to ensure inhibition or elimination of viral replication. In certain aspects, the mRNA or mRNA composition may include segments of interest encoding viral replication mechanisms (either as separate mRNA strands or included in a single strand), e.g., utilizing a viral RNA genome with an antigen segment of interest replacing a structural protein to provide additional RNA complexing agents (see, e.g., gel et al proc. Natal. Acad. Sci. USA 109:14606-14609 (2012) and Pardi et al, nat. Rev. Drug discovery.117:261-279 (2018)). In some aspects, the mRNA vaccine composition may be part of a viral vector, wherein the viral vector is a modified viral genome that is designed to be non-pathogenic and allows transcription of the segment of interest and/or mRNA in the host cell. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, vaccinia viruses, adeno-associated viruses, and modified forms of cytomegalovirus (see, e.g., ura et al, vaccines 2:624-641 (2014)).

In some aspects, the mRNA is an mRNA vaccine composition. In some aspects, the mRNA vaccine composition includes mRNA molecules encapsulated in a lipid or lipid-like nanoparticle. Such nanoparticles may optionally include ionic lipids, cholesterol (or optionally the absence of cholesterol), polyethylene glycol, and/or helper lipids, such as DOTAP, DOPE, DOPC and/or dioleoyl PE.

The mRNA vaccine composition may include naked mRNA molecules, mRNA and protamine, mRNA in cationic nanoemulsions, mRNA in LNP, mRNA in dendrimer nanoparticles, mRNA in liposomes or LNP and protamine, mRNA in cationic polymers (e.g., polyethylenimine), mRNA in cationic polymer liposomes, mRNA and polysaccharide, mRNA in cationic lipid nanoparticles (e.g., 1, 2-dioleoyloxy-3-trimethylammonium propane or dioleoylphosphatidylethanolamine), mRNA in cationic lipid and cholesterol nanoparticles, and mRNA in cationic lipid, cholesterol and polyethylene glycol (PEG) nanoparticles.

In some aspects, the mRNA vaccine composition can include dendritic cells loaded with mRNA ex vivo. In such aspects, dendritic cells from the subject to be immunized are typically obtained and mRNA is introduced therein for subsequent replacement back into the host subject. Since dendritic cells are potent antigen presenting cells, in vitro mRNA loading provides a mechanism to effectively recruit the immune system upon reintroduction. In such aspects, the dendritic cells themselves can be vitrified by the methods disclosed herein either before or after the introduction of the mRNA. In other aspects, dendritic cells can ingest reconstituted mRNA as described herein.

In further aspects, the mRNA, mRNA composition, and/or mRNA vaccine composition may further include an adjuvant. As described herein in some aspects, mRNA may be added to additional compositions, for example, mRNA is added to a lipid mixture to encapsulate the mRNA in an LNP. In other aspects, mRNA can be stored, reconstituted and adjuvanted as provided herein. In a further aspect, an adjuvant may be mixed with or included in the mRNA or mRNA composition prior to vitrification. Adjuvants may include aluminum-based compounds such as aluminum hydroxy phosphate sulfate, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate. Adjuvants may also include AS04 (monophosphoryl lipid a and aluminium salts), MF59 (oil-in-water emulsions including squalene), AS01B (monophosphoryl lipid a and QS-21 (from chile quillaja)) and CpG1018 (DNA synthesized by cytosine-phosphate guanine) in liposome formulations), and TLR agonists. Other adjuvants may include the presence of other mrnas encoding CD70, CD40L and TLR4 (optionally constitutively active) to allow better cellular uptake of the mRNA and/or cellular expression of the fragment of interest.

Vitrification mixture

In some aspects, the disclosure relates to placing a lipid particle, a lipid particle containing one or more cargo molecules, an mRNA composition, or an mRNA vaccine composition within a vitrified mixture on a capillary or capillary bed. The mRNA may be a naked mRNA, an mRNA composition, or an mRNA vaccine composition as described herein. The mRNA may also be part of a vitrification mixture placed on a capillary or capillary bed. The vitrification mixture may comprise lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition, and a vitrification medium. In a further aspect, the vitrification medium may be added to the capillary bed, and then the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition added thereto to provide a vitrified mixture on the capillary bed. It will be understood in the art that all components that may or appear to be in contact with the mRNA molecules described herein are prepared and/or treated to be free or substantially free of degradative enzymes directed against mRNA, including any potential or potential source of ribonucleases.

The vitrification medium may include a glass former. The identification of glass formers opens the opportunity for successful preservation of biomolecules, cells or tissues. In the presence of a suitable glass former, the biological material may be stored in a vitrification matrix at a temperature above the cryogenic temperature, wherein vitrification is achieved by dehydration as provided herein. The ability to survive in the dry state (hypo-wet dormancy) depends on several complex intracellular physiochemical and genetic mechanisms. Among these mechanisms, sugars (e.g., sugars, disaccharides, oligosaccharides) accumulate in the cell, which act as protectants during drying. Trehalose is an example of a disaccharide that is naturally produced in desiccating-resistant organisms. Pullulan is one example of a polysaccharide that is similarly suitable for drying. Saccharides such as trehalose and pullulan can provide protection in a number of different ways. Because of the unique position of the hydroxyl group on the trehalose molecule, the trehalose molecule can effectively displace hydrogen-bound water molecules from the molecular surface without changing its conformational geometry and folding. In addition, many sugars have high glass transition temperatures, allowing them to form glass at temperatures above deep low temperatures or to form room temperature glass at low moisture content. The highly viscous "glassy state" reduces the mobility of the molecules, which in turn prevents degradative biochemical reactions that lead to functional degradation.

The presence of a suitable vitrification agent in the vitrification medium may be necessary because the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition or mRNA vaccine composition are dried under the ambient conditions described herein. Without the other considerations provided herein, the flash drying process itself does not necessarily ensure viability of the cells or other vitrified biological material after drying. It may be desirable to form glass and/or a vitrification medium that inhibits crystal formation in other materials. The vitrification media may also provide osmotically protected or otherwise allow cells to survive the dehydration of the mRNA or a composition thereof. Illustrative examples of reagents included in the vitrification medium may include one or more of the following: dimethyl sulfoxide, glycerol, sugars (e.g., disaccharides such as trehalose), polyols, methylamine, betines, antifreeze proteins, synthetic anti-nucleating agents, polyvinyl alcohol, cyclohexanetriol, cyclohexandiols, inorganic salts, organic salts, ionic liquids, or combinations thereof. In some aspects, the vitrification medium optionally contains 1, 2, 3, 4, or more vitrification agents.

In some aspects, the vitrification medium may include a concentration of the vitrification agent that depends on the characteristics of the vitrification agent. Optionally, the concentration of the vitrification agent is below a concentration at which the vitrification agent would be toxic to the mRNA or composition thereof that is vitrified, wherein toxicity is such that function or biological activity is not achieved upon subsequent sample use. The concentration of the vitrification agent is optionally from about 500 micromoles (μΜ) to about 6 moles (M), or any value or range therebetween, including about 1, 2, 3, 4 or 5M. For the vitrification agent trehalose, the concentration is optionally from about 1M to about 6M, including 2, 3, 4, or 5M. Optionally, the total concentration of all vitrification agents when combined is optionally from about 1M to about 6M, including 2, 3, 4, or 5M.

Trehalose, a glass forming sugar, has been used for anhydrous vitrification and can provide dry tolerance in a variety of ways. However, the glass transition temperature of the vitrified 1.8M trehalose in water was-15.43 ℃. In order to achieve vitrification above 0℃higher concentrations (6-8M) are required, which may cause damage to mRNA or its composition. Alternatively, the vitrification medium may include buffers and/or salts to increase the Tg value of the VM. In some aspects, the vitrification medium may optionally include water or a solvent and/or a buffer and/or one or more salts and/or other components. The buffer may be any reagent having a pKa of about 6 to about 8.5 at 25 ℃. Illustrative examples of buffers may include HEPES, TRIS, PIPES, MOPS and the like. The buffer may be provided in a concentration suitable to stabilize the pH of the vitrification medium to the desired level.

The glass transition medium comprising 1.8M trehalose, 20mM HEPES, 120mM ChCl and 60mM betaine provided a glass transition temperature of +9℃. Exemplary vitrification media for use in the capillary-assisted vitrification methods disclosed herein may comprise trehalose, and one or more buffers containing large organic ions (> 120 kDa) such as choline or betaine or HEPES, and buffers containing small ions such as K or Na or Cl. In some aspects, the vitrification medium can include trehalose, glycerol, and phosphate buffered saline. The vitrification media may be further sterilized, for example by heat treatment or by filtration, for example through a 0.2 μm membrane filter. In a further aspect, the vitrification medium can be mixed with a volume of mRNA, mRNA composition, or mRNA composition. In some aspects, the vitrification medium is mixed with the mRNA, mRNA composition, or mRNA composition in a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

Pressure and heat

In some aspects, the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or vitrified mixture of mRNA vaccine composition and vitrification medium are placed on a capillary network or continuous capillary network to enhance evaporation of any fluids within the vitrification medium and mRNA. In some aspects, the methods of the present disclosure involve applying a low atmospheric pressure to a vitrified mixture on a capillary network. In some aspects, low pressure is applied while further providing heat to avoid crystallization or freezing of the VM. The present disclosure provides a vitrification process that combines low atmospheric pressure and thermal energy (optionally from a specific direction or location relative to the membrane) to achieve rapid vitrification of mRNA in the vitrification mixture. In some aspects, the disclosure relates to applying thermal energy to a vitrification mixture when vitrification occurs at reduced atmospheric pressure. In some aspects, thermal energy is applied to the vitrification mixture to prevent crystallization of the vitrification mixture or contents therein, such as mRNA or mRNA composition.

In some aspects, the disclosure relates to vitrification of lipid particles, lipid particles containing one or more cargo molecules, mRNA compositions, or mRNA vaccine compositions at low atmospheric pressure. In some aspects, drying may occur in a drying chamber, whereby the vitrification mixture may be placed therein for exposure to low atmospheric pressure. Such a drying chamber may be connected to a vacuum source to apply a low atmospheric pressure to the lipid particles, the lipid particles containing one or more cargo molecules, the mRNA composition, or the mRNA vaccine composition. As described herein, the vitrified mixture may be prepared with a vitrification medium or cryoprotectant such as trehalose and subjected to low atmospheric pressure, for example by application of a vacuum. In some aspects, the low atmospheric pressure is from about 0.9 atmospheres (atm) to about 0.005atm, including 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.255, 0.25, 0245, 0.24, 0.235, 0.23, 0.225, 0.22, 0.215, 0.21, 0.205, 0.2, 0.195, 0.19, 0.185, 0.18, 0.175, 0.17, 0.165, 0.16, 0.155, 0.15, 0.145, 0.14, 0.135, 0.13, 0.125, 0.12, 0.115, 0.11, 0.105, 0.1, 0.095, 0.09, 0.085, 0.08, 0.075, 0.07, 0.06, 0.05, 0.04, 0.02, 0.05, 0.0.04, 0.0.02, 0.05, 0.0.05.

In other aspects, the pressure within the drying chamber is reduced to a point above the triple point of the vitrification mixture. In other aspects, the pressure is reduced to a point above the triple point of water, for example greater than 0.006atm. As described herein, the reduced atmospheric pressure reduces the temperature of the vitrification mixture while also reducing its boiling point. In some aspects, the pressure within the drying chamber is reduced to about 0.04atm or about 29mmHg.

In a further aspect, the temperature of the vitrification mixture is controlled during drying and/or vitrification. For example, the vitrification mixture is placed in a drying chamber and thermal energy is applied to the vitrification mixture to limit or prevent the vitrification mixture from experiencing deep cryogenic temperatures. In some aspects, thermal energy is transferred to the vitrification mixture to prevent crystallization therein.

In some aspects, the temperature of the lipid particle, the lipid particle containing one or more cargo molecules, the mRNA composition, or the mRNA vaccine composition is controlled within a vacuum or reduced atmospheric pressure applied around the vitrification mixture, optionally within 30 ℃ from Tc, optionally within 20 ℃ from Tc, optionally within 10 ℃ from Tc, optionally within 5 ℃ from Tc, optionally within 4 ℃ from Tc, optionally within 3 ℃ from Tc, optionally within 2 ℃ from Tc, optionally within less than 1 ℃ from Tc. As discussed herein, the application of low atmospheric pressure can significantly reduce the temperature of the vitrification mixture, thereby causing the vitrification mixture to crystallize. If mRNA or the surrounding medium crystallizes, irreparable damage can occur therein, which can negatively affect any desired activity or use upon reconstitution. As also identified herein, a decrease in the atmospheric pressure surrounding the vitrification mixture can alter the molecular activity within the vitrification mixture, thereby lowering the boiling point. Similar to what happens at deep low temperatures, boiling the mRNA and/or vitrification media or overheating may be detrimental. Boiling of the vitrification mixture can lead to loss of tertiary structure, crosslinking and degradation of mRNA components therein, such that any activity upon reconstitution is compromised. In certain aspects, the methods of the present disclosure involve maintaining the vitrification mixture at a temperature above a cryogenic temperature while in a low atmospheric pressure, such as a vacuum, partial vacuum, or generally a reduced pressure atmosphere.

In certain aspects, the vitrification mixture comprising the lipid particles, the lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition, and the vitrification medium can be directly heated to control the temperature thereof during drying. In other aspects, the temperature of the vitrification mixture comprising the lipid particles, the lipid particles containing one or more cargo molecules, the mRNA composition, or the mRNA vaccine composition, and the vitrification medium can be controlled by conduction, convection, and/or radiation. In other aspects, the temperature of the vitrification mixture comprising the lipid particles, the lipid particles containing one or more cargo molecules, the mRNA composition, or the mRNA vaccine composition, and the vitrification medium can be controlled by controlling the temperature outside the drying chamber and by controlling the temperature of the vitrification mixture by conduction through the drying chamber or a portion thereof. In this case, it should be understood that the physical properties of the drying chamber wall need to be taken into account. For example, a poorly conductive material of the drying chamber may require a temperature to be applied that is different from the temperature required for the vitrification mixture to allow the vitrification mixture to receive the appropriate thermal energy. Such necessary adaptations will be readily appreciated by those skilled in the art. In some aspects, heat may be applied by heating pads, heating baths, flames, heating beds such as glass beads, heating blocks, and the like. In some cases, the thermal energy may be from a power source that generates heat and/or thermal energy released by combustion and/or thermal energy generated by electrical resistance.

In some aspects, thermal energy may be provided to the vitrification mixture through the underlying support substrate. While the porous material of the continuous capillary network may also provide thermal energy to the vitrified mixture, in some cases the porous material is a poorly conductive material, such as glass or a polymer. However, the underlying substrate may be a metal or similar highly efficient conductive material and is easily connected to a heat source or power source external to the drying chamber and provides heat through the electrical resistance generated therein. The application of thermal energy from the solid support may further provide a temperature gradient to assist in capillary evaporation.

The thermal energy may be applied from a desired direction. It has been found that in some aspects, the application of heat from below or within the capillary channel or membrane such that the heat is targeted to the bulk of the liquid itself can be detrimental because film boiling can be induced in the material before the glassy state is achieved. Alternatively, the application of heat from a direction above the meniscus formed by the ends of the capillary channel promotes vitrification without causing boiling of the liquid alone or during exposure to reduced atmospheric pressure. The direction above the meniscus may be at both ends of the capillary channel, for example when the channel or film is loaded with a vitrification mixture and subjected to heat and an atmospheric pressure reduction to promote vitrification of the material. By leaving a liquid-free space (aerated or evacuated) between the heat source and the end of the capillary channel or the surface of the vitrification membrane, improved vitrification is achieved, thereby increasing the stability of the bioactivity of the mRNA.

In some aspects, a vitrification mixture comprising lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition, and a vitrification medium is maintained at a temperature above its cryogenic temperature during vitrification at low atmospheric pressure. In some aspects, the vitrification mixture is preheated at low atmospheric pressure prior to drying. In other aspects, the vitrification mixture is heated at a low atmospheric pressure during the vitrification process. In other aspects, heating is performed at or before the onset of vitrification. It should be appreciated that the amount of thermal energy applied to the vitrification mixture may be constant or may vary during the vitrification process at low atmospheric pressure. In some aspects, introducing a low atmospheric pressure within the drying chamber can result in a rapid drop in the temperature of the vitrification mixture. In such aspects, preparing the vitrified mixture to receive or having received thermal energy can increase the recovery rate from the temperature drop (see, e.g., fig. 5).

In certain aspects, a constant temperature is applied to the vitrified mixture such that the vitrified mixture remains at about T from the vitrified mixture _g (. Degree. C.) to about 40 ℃ (including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, and 39 ℃). In some aspects, can be to The drying chamber or porous material applies a higher temperature to provide the necessary thermal energy to the vitrification mixture. Such applied temperatures may be from about 15 ℃ to about 70 ℃, depending on the size of the drying chamber and the conduction means available for efficient transfer to the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition or mRNA vaccine composition and/or vitrification media.

In some aspects of the disclosure, the vitrification mixture is placed in a vacuum or partial vacuum at an elevated temperature or maintained at a cryogenic temperature above the vitrification mixture at the applied atmospheric pressure such that the vitrification mixture does not experience cryogenic temperatures during rapid decreases in atmospheric pressure. In a further aspect, the temperature of the vitrification mixture will drop below T of the vitrification medium _g To allow vitrification of the mRNA or a combination thereof.

In some cases, maintaining a low atmospheric pressure may require that the vitrification mixture be contained in a sealed enclosure (e.g., a drying chamber). Those skilled in the art will appreciate that providing and/or maintaining a low atmospheric pressure around the vitrification mixture generally requires a drying chamber capable of withstanding the low pressures therein. This may be any suitable or desired shape and/or material, limited by the requirement to maintain a low atmospheric pressure therein, requiring adequate sealing and adequate wall strength. The drying chamber may be operatively connected to a vacuum source to reduce the atmospheric pressure therein while further allowing air to return when vitrification is complete. The drying chamber may be sufficiently sealed or closed to allow a vacuum to be applied to effectively reduce the atmospheric pressure in the drying chamber to a desired range.

Capillary assisted evaporation

In a further aspect, the capillary network may prevent the vitrification mixture from boiling at reduced atmospheric pressure. The principles of capillary assisted evaporation and devices useful for vitrification can be as described in U.S. patent 10,568,318, the entire contents of which are incorporated herein by reference. In some aspects, thermal energy may be applied to the vitrification mixture as the vitrification mixture undergoes drying and vitrification on a capillary network. In some aspects, the underlying capillary network may allow for uniform and complete vitrification and drying of the vitrified mixture receiving thermal energy while protecting the vitrified mixture from boiling. The capillary network may be a continuous capillary network. In some cases, the capillary network may be provided by an underlying porous material, such as a membrane, or by an underlying corrugated or ridged surface, with the valleys and peaks thereof providing a capillary bed.

The presence of the vitrification mixture on the capillary network allows for rapid evaporation by drawing the vitrification mixture out by capillary action. The presence of a continuous capillary network further allows the fluid volume of the vitrification medium to evaporate uniformly and prevents boiling while also preventing excess fluid from accumulating on the mRNA or its composition, which may also undergo destructive boiling. Similarly, a porous material such as a membrane may provide the underlying capillary network. In these aspects, a porous material, such as a membrane, is located directly under the mRNA or a combination thereof, and capillary action therein provides enhanced evaporation. Thus, in some aspects of the present disclosure, the vitrified mixture is placed on a continuous capillary network. In a further aspect, the vitrified mixture is placed on a patterned and/or ridged and/or corrugated porous material of a continuous capillary network. In a further aspect, the continuous capillary network is formed by a pattern and/or ridges and/or wavy contours in the drying chamber or on the wall. In other aspects, the capillary network is provided by a porous material.

Referring to fig. 1A, a continuous hydrophilic bed 10 covered by a thin liquid layer to which a vitrification mixture 20 is applied is depicted. As shown in fig. 1A, the use of extremely thin liquid films on hydrophilic surfaces can avoid and/or reduce the prevention of boiling under reduced atmosphere. However, while boiling may be prevented, the available surface area reduces the amount of liquid that can be vitrified. The presence of a contoured surface (e.g., as shown in fig. 1B) effectively provides a surface upon which the vitrification mixture can undergo capillary action because preferential drying occurs at the peaks, thereby absorbing moisture from the valleys during vitrification, and this can similarly protect the mRNA or combination thereof from boiling. Furthermore, when the sample is vitrified at the peaks of the wavy profile, capillary action draws fluid from the valleys underneath, thereby promoting excellent vitrification of the vitrified mixture. Similarly, if the porous material of the membrane of the capillary supports mRNA or a combination thereof, capillary action will draw fluid from the capillary channel when the vitrification mixture is placed thereon and provide uniform and complete vitrification and drying of the mRNA or a combination thereof. However, as shown in fig. 1C, if capillary action is not successful in drawing fluid, for example, in the event of excessive fluid loading, the liquid may fill the surface pattern or remain in the valleys, where bubble nucleation and boiling becomes dominant under reduced pressure, which may lead to damage to the sensitive molecules contained therein.

The capillary network formed by the underlying patterned ridge support or porous material, such as a membrane, may be made of a material that is non-toxic and non-reactive to mRNA or a combination thereof and does not chemically or physically react with the vitrification medium. The material may be a suitable polymer, metal, ceramic, glass or combinations thereof. In some aspects, the continuous capillary network is formed from a material of Polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulfone (PES), polyester (e.g., polyethylene terephthalate), or the like. Illustrative examples of membranes containing capillary channels suitable as surfaces in the devices and methods provided herein include hydrophilic filtration membranes such as those sold by EMD Millipore, bellerica, MA. In certain aspects, the porous material does not substantially bind, alter, or otherwise chemically or physically associate with the mRNA or a composition thereof and/or a component of the vitrification medium. The porous material is optionally not derivatized. Optionally, the capillary channels may be formed in a substrate (e.g., a dry chamber wall) of the desired material and thickness by PDMS formation techniques, laser drilling, or other drilling formation techniques known in the art.

In some aspects, the capillary network has a sufficient thickness to limit liquid or fluid accumulation on its surface. To achieve the capillary effect, the liquid may be contained within the pores of the film forming the meniscus. The liquid phase fraction (ζ), i.e. the volume occupied by the liquid, at the capillary interface is a parameter considered to provide improved capillary evaporation.Capillary driven evaporation occurs when the viscous pressure drop in the liquid exceeds the maximum capillary pressure at the liquid-vapor interface. The liquid fraction ζ is related to the total pressure drop from the body to the liquid-vapor interface. In the case of atmospheric pressure and no applied heat flux (fig. 4B), the liquid covers a large fraction, resulting in a liquid phase fraction, ζ→1. Under these conditions, the capillary driven evaporation rate is minimal. Decreasing the ambient pressure decreases ζ and thus increases the evaporation rate as shown in fig. 4C. However, above a certain threshold pressure drop, undesired nucleate boiling may occur. The heat flux Q applied as shown in fig. 4D may also increase the evaporation rate, but there is a risk of film boiling, which is also undesirable. Applying a heat flux from the capillary meniscus surface as shown in fig. 4E eliminates or reduces the risk of film boiling. In the case of applying large deltap and Q in an inverse gradient manner as shown in fig. 4F, this results in a confinement of the liquid meniscus in the aperture, i.e. the liquid phase fraction ζ <<1 (-0.25), resulting in the highest evaporation rate while avoiding boiling [ ]Where p is the distance between the ridges or the height of the film and d is the diameter of the circle formed by the shape of the liquid meniscus). Thus, the temperature gradient between the holding surface and the bulk liquid causes capillary evaporation, as shown in fig. 4F, so that rapid evaporation can be achieved. When the liquid surface is withdrawn back into the capillary membrane, capillary evaporation still occurs as long as the pressure gradient and the temperature gradient are maintained. In some aspects, the capillary network under the mRNA or composition thereof can aid in the evaporation process during drying. />

As described herein, capillaries may be provided by patterning or contoured the walls of the drying chamber to effectively provide an underlying capillary bed (see, e.g., fig. 1B and 6A) or by providing a continuous capillary network of porous material (e.g., using a membrane) (see, e.g., fig. 3). In some aspects, the capillary network provided by the porous material and/or patterned and/or contoured surface features pores of about 20 μm or less, such that the pores provide underlying capillaries to aid vitrification. In some aspects, the peak-to-peak distance in the pore or undulating bed may have an average opening of about 20 μm to about 0.1 μm, including about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 μm. The capillary channel may have a length optionally defined by the thickness of the substrate forming the channel or by the one or more individual channels themselves. The capillary channel length is optionally about one millimeter or less, but should not be construed as limited to these dimensions. Optionally, the capillary channel length is from about 0.1 microns to about 1000 microns, or any value or range therebetween. Optionally, the capillary channel length is from about 5 to about 100 microns, optionally from about 1 to about 200 microns, and/or optionally from about 1 to about 100 microns. The capillary channel length is optionally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 microns. In some aspects, the length of the capillary channel varies among the plurality of capillary channels, optionally non-uniformly.

The cross-sectional area of the capillary passage may be about 2000 μm ² Or smaller. Optionally, the cross-sectional area is about 0.01 μm ² To about 2000 μm ² Optionally about 100 μm ² To about 2000 μm ² Or any value or range therebetween. Optionally, the capillary channel has a cross-sectional area of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 μm ² Or smaller.

The capillary assisted evaporation rate may be affected by atmospheric requirements (humidity, temperature and velocity of the air/gas at the evaporation surface) and (i) the nature of the capillary channel that produces the driving capillary force, (ii) the liquid meniscus depth, and (iii) the viscous drag of flow through the capillary. Thus, complex and highly dynamic interactions between capillary properties, transport processes and boundary conditions lead to a broad range of evaporation behaviors. For flash drying, key parameters may include: (1) Supporting the conditions under which a liquid network is formed and maintained at the evaporation surface, and (2) promoting the formation of capillary pressure that creates sufficient flow at the evaporation surface to supply water.

In some aspects, the porous material may be ridged and/or corrugated or placed on an underlying support substrate such that the porous material adopts a similar shape when placed or pressed thereon. The undulating contours and/or ridges of the patterned material may increase surface area to provide increased exposure for evaporation.

In a further aspect, increased surface area of the porous material may be achieved by arranging or shaping the membrane. As shown herein, a drying chamber with corrugated walls can provide increased surface area for a porous material. However, shaping an otherwise flat porous material may further provide improved surface area for efficient capillary-assisted vitrification (see, e.g., fig. 6A and B).

In some aspects, the membrane is hydrophilic. It should be understood that mRNA is soluble in water or water-based solutions. In some cases, the mRNA is in solution within the lipid or LNP or similar vehicle described herein. Thus, it may be beneficial to have a capillary network that does not repel aqueous solutions. It is also beneficial to have a hydrophilic capillary system to separate the expelled water from the mRNA composition as it dries. It is also understood that rapid and/or efficient absorption of the aqueous solution from the mRNA or mRNA composition and/or the vitrification medium will prevent or reduce the chance of re-dissolution and/or re-absorption, thereby improving the overall vitrification process.

In some aspects, the capillary network has a hydrophilic material. In other aspects, the capillary network may have a hydrophobic material and be further treated to be hydrophilic or more hydrophilic in nature, such as by plasma treatment. As shown in fig. 9, the originally hydrophobic film was treated with cold plasma to make it more hydrophilic. When the drug formulation is suspended on the membrane, the liquid forms nearly spherical droplets (upper left), while the hydrophilic membrane allows the liquid to flow into the underlying capillary channel. In the vitrification process, the droplets on the hydrophobic membrane are boiled and then frozen, and the liquid on the hydrophilic membrane is rapidly vitrified to form a glassy monolith. Upon release of the vacuum, the frozen droplets again become liquid, but reduce in size to a point where some of the water is lost. The efficacy of capillary evaporation on vitrification is evident in the uniform vitrification observed with hydrophilic films.

Vitrification method

In some aspects, the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition are coated or submerged in a vitrification medium and placed on a support substrate that provides capillary action to hold these during the vitrification step shown herein. In certain aspects, the capillary network absorbs some of the vitrification mixture while allowing a thin layer of fluid to remain above the membrane. In a further aspect, as the pressure around the vitrification mixture decreases, the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition become vitrified. The application of heat will prevent crystallization of the mRNA or its composition or vitrification media, while the application of a thermal gradient across the capillary network will prevent boiling, all of which together allow uniform and complete vitrification of the mRNA and/or its composition.

In some aspects, the disclosure relates to methods for vitrifying at least one lipid particle, a lipid particle containing one or more cargo molecules, an mRNA composition, or an mRNA vaccine composition. The method comprises preparing a lipid particle, a lipid particle containing one or more cargo molecules, an mRNA composition, or an mRNA vaccine composition. For example, a lipid particle containing one or more cargo molecules, an mRNA composition, or a vitrified mixture of an mRNA vaccine composition and a vitrification medium is placed on or in contact with a solid support matrix. In some aspects, the underlying solid support is corrugated and/or ridged to provide an underlying capillary network. In some aspects, the underlying support is part of a drying chamber, such as a wall thereof. In other aspects, the porous membrane may be disposed between the vitrification mixture and the solid support. In some aspects, a continuous capillary network supports the vitrified mixture and draws fluid from it. The capillary network and/or porous material should be of sufficient thickness or quantity to avoid the presence and/or pooling of liquid above the surface of the capillary network.

The vitrification method of the present disclosure further comprises placing the vitrification mixture comprising lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition in a drying chamber operably connected to a vacuum device or other device for reducing the atmospheric pressure therein. In certain aspects, the vitrification mixture is immobilized on a porous or corrugated material within a drying chamber. In some aspects, the vitrification mixture is placed on a portion of the drying chamber, wherein the portion is patterned and/or contoured to provide an underlying capillary network. In some aspects, a solid support substrate, a porous material such as a membrane, and a vitrification mixture are placed in a drying chamber.

In some cases, the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition may be coated and/or mixed with a vitrification medium in a drying chamber. In other aspects, the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition may be prepared with a vitrification medium prior to placement in the drying chamber.

Once assembled, the methods of the present disclosure may include reducing the atmospheric pressure around the vitrified mixture, providing capillary-assisted evaporation to the vitrified mixture, and/or applying thermal energy to the vitrified mixture without causing boiling or freezing of the vitrified mixture or any components contained therein. As described herein, the application of all three can provide rapid and uniform vitrification and drying of the vitrification mixture while avoiding experiencing cryogenic temperatures and avoiding boiling, thereby significantly reducing any damage to the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition during processing and significantly improving the activity of the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition after reconstitution.

In some aspects, the methods of the present disclosure involve applying a low atmospheric pressure to a vitrified mixture on a capillary network. In some aspects, low pressure is applied while further providing heat to avoid subjecting the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition to freezing conditions. The present disclosure relates to a vitrification process that combines low atmospheric pressure and thermal energy to achieve uniform and rapid vitrification of a vitrified mixture. In some aspects, the disclosure relates to applying thermal energy to a vitrification mixture, the vitrification occurring at a reduced atmospheric pressure. In some aspects, thermal energy is applied to the vitrification mixture to prevent crystallization of the vitrification mixture.

Once the vitrification mixture is placed in the drying chamber, the atmospheric pressure therein is reduced. In some aspects, the atmospheric pressure is reduced to a point above the triple point of the vitrified mixture or lipid particles therein, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition. In other aspects, the atmospheric pressure is reduced to a point above the triple point of water. In a further aspect, the pressure within the drying chamber is reduced to about 0.04atm.

In some aspects, thermal energy is applied to the vitrification mixture as the vitrification mixture undergoes vitrification on the capillary network. In some aspects, the underlying capillary network may allow for uniform and complete vitrification of the vitrified mixture that receives thermal energy while protecting the vitrified mixture from boiling. The capillary network may be a continuous network of capillaries. In some cases, the capillary network may be provided by an underlying porous material, such as a membrane, or by an underlying corrugated or ridged surface, wherein the valleys and peaks thereof provide a bed sufficient to subject the liquid vitrification mixture to capillary action during vitrification.

Fig. 2A is an overview of one exemplary aspect of the vitrification process of the present disclosure. Conventional vitrification is shown in paths 1-2-3, where the liquid (containing biological or other material) is rapidly cooled below the glass transition to bypass the freezing zone. The total mass of the material is conserved throughout the process. Deep cryogenic vitrification of large amounts of materials can be challenging due to heat transfer limitations, and is therefore typically performed in vials that provide significant surface area to volume ratios. Vitrification of the material can also be achieved by drying (bypassing the crystallization process), as shown in paths 1-5-6. In this respect, significant mass losses (mainly water) occur. Traditional methods of dehydration of biological materials have focused on creating fixed droplets on a substrate and evaporative drying in a low humidity enclosure. The process is characterized by slow speed and uneven drying. When the biological material therein dries, a glassy skin forms at the interface between the liquid and the vapor. The formation of the glassy skin slows down and eventually prevents further drying of the vitrified mixture, limiting the vitrified mixture to only a certain level of drying, wherein there is a spatially significant inhomogeneity of the water content in the sample. As a result, some areas are not vitrified, but are now degraded by the retention of high molecular mobility. The large surface area to volume ratio and especially under reduced pressure can promote drying rates.

In some aspects, the disclosure relates to paths 1-4-6 of fig. 2A, wherein maintaining a desired temperature and low pressure of the vitrification mixture provides near deep temperature and dry mixing. However, as the pressure decreases, the boiling point also decreases. As shown in fig. 2B, maintaining the low pressure above the triple point of water may provide a temperature window between freezing and boiling for vitrification of the vitrification mixture. In some aspects of the disclosure, the applied temperature is maintained at the applied low pressure at a temperature above the deep low temperature point of the vitrification mixture. As further described in fig. 2 and 4, reducing the temperature from the applied low ambient pressure allows the temperature of the vitrification mixture to drop below the glass transition temperature without boiling, thereby providing uniform vitrification of the entire vitrification mixture.

Fig. 4A shows how rapid drying of a large volume of liquid can be conveniently achieved under vacuum by deploying a porous material of a capillary network to facilitate capillary evaporation, for example by introducing a membrane of continuous capillary channels. However, when liquid accumulates on the surface of the capillary membranes, boiling may still occur in the accumulated liquid, which may be an undesirable situation as described herein. As shown in fig. 4E and 4F, the presence of a temperature gradient between the surface and the bulk liquid allows capillary evaporation, wherein rapid evaporation can be achieved.

Thus, in some aspects of the present disclosure, the volume of fluid present in the vitrified mixture may be determined such that the fluid may fill the capillary network without spilling or pooling on the surface.

Fig. 5 depicts the results seen from the application of 37 ℃ heat from a wire mesh as an underlying solid support and a glass film as a porous material thereon. Figure 5 shows a comparison between membrane and volume dimensions and the rate at which the sample temperature recovers after reducing the pressure when the liquid loading is kept constant. In all cases, the application of vacuum resulted in a rapid drop in the temperature of the vitrification mixture, as shown in fig. 5, while smaller films produced a faster complete vitrification, as observed by returning to the starting temperature. With further reference to fig. 5, it can be seen that once vitrification is complete, the temperature of the sample stabilizes.

In some aspects, the methods of the present disclosure include providing capillary-assisted evaporation of the vitrified mixture. In some aspects, the underlying capillary network provided by the corrugated and/or ridged support or by the porous membrane will provide the necessary features to enhance evaporation.

In some aspects, the methods of the present disclosure can be performed for a certain drying time. The drying time is a time sufficient to promote proper drying to vitrify the vitrification media. The drying time is optionally from about 1 second to about 1 hour, including but optionally not more than about 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, and 55 minutes. Optionally, the drying time is from about 1 second to about 30 minutes, optionally from about 5 seconds to about 10 minutes.

Vitrification composition

In some aspects, the disclosure relates to vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA compositions, or mRNA vaccine compositions. The vitrified mRNA vaccine composition may comprise at least one single stranded mRNA molecule encapsulated in a Lipid Nanoparticle (LNP) or a lipid-like nanoparticle. In some aspects, the vitrified mRNA vaccine composition may further include a vitrified vitrification medium, e.g., around or near and/or around the mRNA in the LNP or LLN to provide stability thereto. LNP and/or LLN may include ionizable lipids, optionally using cholesterol, PEG, and/or helper lipids, such as DOTAP, DOPE, DOPC, etc. In some aspects, the vitrified mRNA composition may be immobilized to a capillary network, such as a membrane, by a vitrified or dried vitrification medium.

(Storage)

In certain aspects, the disclosure relates to the handling and storage of vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA compositions, or mRNA vaccine compositions. For all materials used, care should be taken to avoid exposing the vitrification composition to potential sources of degrading enzymes, including rnases, during vitrification and any processing, storage or reconstitution steps taken later.

After the vitrification step, the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition or mRNA vaccine composition will be effectively preserved in a dehydrated state in the vitrification medium on the capillary membrane. The vitrified molecules may remain thereon and move into a sealed environment. In terms of the vitrification mixture within the drying chamber, the capillary network or the drying chamber itself may be moved to a sealed or enclosed environment to protect the lipid particles, the lipid particles containing one or more cargo molecules, the mRNA composition, or the mRNA vaccine composition from humidity and from exposure to degrading enzymes.

In some aspects, the vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition can then be stored at any desired temperature. Since the lipid particle, the lipid particle containing one or more cargo molecules, the mRNA composition or the mRNA vaccine composition is in a dehydrated state, exposure to sub-deep low temperatures at this time does not result in the same crystallization as expected before vitrification, since the ability of the molecules therein to rearrange into a crystalline structure is negated due to the dehydrated, vitrified state of the molecules therein. Thus, the vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition can be stored at about-80 ℃ to about-20 ℃ to about-5 ℃ to about 0 ℃.

The storage of vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA compositions or mRNA vaccine compositions does not require at zero or below zero temperatures to maintain structural integrity and activity. As demonstrated herein, the lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition can be stored at room temperature (e.g., about 20 to about 34 ℃) for extended periods of time for at least several months without significant loss of structural integrity or functional activity (e.g., translation of mRNA). The vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA composition or mRNA vaccine composition may also be stored at higher temperatures (including up to about 50, 55 or 60 ℃) for extended periods of time, including weeks and months.

In other aspects, the vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition need not be stored at a constant or near-constant temperature to maintain functional activity, including withstanding seasonal fluctuations from below freezing point to 40 ℃ or higher, including up to 60 ℃ or higher.

Those skilled in the art will appreciate that storage may be prolonged by improving or deliberately preventing exposure to significant environments with high humidity, particularly at high temperatures. Since the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition or mRNA vaccine composition is in a vitrified state, preventing exposure to moisture can prolong and preserve the ability to be reconstituted without loss of intended functional activity. The more water is allowed to be absorbed from the surrounding atmosphere by the vitrified lipid particles, lipid particles containing one or more cargo molecules, mRNA composition or mRNA vaccine composition, the faster the damage to the active or retained structure can be expected.

In some aspects, the storage of the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition may include sealing and/or inclusion of a desiccant to help prevent any moisture absorption from the surrounding atmosphere. In some aspects, the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition may remain viable for 2-20 days above cryogenic storage. In other aspects, the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition can remain viable when stored for 10 weeks above cryogenic temperatures. In other aspects, the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition may remain viable for up to one year above cryogenic storage. In other aspects, the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition may remain viable for up to 10 years above cryogenic storage.

Reconstruction

In some aspects, the disclosure may include reconstituting and/or purifying lipid particles as disclosed herein, lipid particles containing one or more cargo molecules, mRNA compositions, or mRNA vaccine composition molecules and compositions. In some aspects, the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition can be purified by reconstitution with an aqueous solution such as water or salt and/or buffered aqueous solution or a solution comprising an encapsulating composition such as lipid to form lipid nanoparticles, cholesterol, or the like. Purification of the reconstituted material may include chromatography, if desired, for example using poly (T) or poly (U) coupling resins to bind mRNA, followed by elution with high salt and/or high pH denaturation.

In some aspects, the disclosure relates to washing lipid particles, lipid particles containing one or more cargo molecules, mRNA compositions, or mRNA vaccine compositions from a capillary network or membrane. Elution may be achieved by rehydration with sterile or purified water or sterile/purified saline or buffered solution or aqueous medium so as to allow the vitrification material to reabsorb water and revert to its natural state. In some aspects, reconstitution of the lipid particle, lipid particle containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition will result in their presence in the reconstitution medium and the underlying capillary system can be removed or isolated therefrom.

The present disclosure relates in some aspects to adding a reconstituted lipid particle, a lipid particle containing one or more cargo molecules, an mRNA composition, or an mRNA vaccine composition to another composition, e.g., a vaccine composition, or to a vehicle thereto to aid administration to a subject. While the present disclosure relates in part to vitrification of lipid particles, lipid particles containing one or more cargo molecules, mRNA compositions, or mRNA vaccine compositions, another aspect is vitrification of lipid particles, lipid particles containing one or more cargo molecules, mRNA compositions, or mRNA vaccine compositions, and addition of other ingredients necessary to complete the composition after reconstitution, e.g., for administration to a subject. Such subsequent steps may include encapsulating the mRNA in a composition described herein and/or adding additional vehicles, such as adjuvants. For example, the mRNA can be reconstituted and then mixed with a lipid or LNP component to allow encapsulation of the reconstituted mRNA.

The reconstituted lipid particles, lipid particles containing one or more cargo molecules, mRNA composition, or mRNA vaccine composition may be administered systemically or locally to a subject to induce an immune response to an exogenous target. As used herein, a "subject" is an animal, optionally a human, non-human primate, equine, bovine, murine, ovine, porcine, rabbit, or other mammal. Optionally, the subject is a human. The vitrified mRNA can be reconstituted prior to administration, optionally immediately prior to administration, optionally within a syringe or other administration device at or substantially near the point of administration. Administration may be oral, injectable, nasal, vaginal, buccal or other route of administration as desired. Optionally, the reconstituted mRNA may be administered by injection, optionally intramuscular, intradermal, subcutaneous, intraperitoneal or intravenous.

Various aspects of the present disclosure are illustrated by the following non-limiting examples. These examples are for illustrative purposes and are not intended to be limiting of any practice of the invention. It will be understood that variations and modifications may be resorted to without departing from the spirit and scope of the invention.

Examples

For the purpose of examining the amount and activity of mRNA, mRNA encoding Green Fluorescent Protein (GFP) was obtained, which had a 5'cap1 structure and a poly (a) tail at the 3' end (Dasher GFP, from Aldriron, madison Wis.). GFP was chosen because it provides a 26.6kDa expressed protein with bright fluorescence to easily track and analyze mRNA delivery and expression in cells or tissues.

To determine what effect the vitrification process may have on mRNA quantity and mRNA activity, the following variables and controls were established and determined:

fresh mRNA

mRNA-free/vehicle-only

Vitrified mRNA stored at-20 ℃

Non-vitrified mRNA stored at-20 ℃

Vitrified mRNA stored at 28 ℃

Non-vitrified mRNA stored at 28 ℃

Vitrified mRNA stored at 55 ℃

The non-vitrified mRNA was stored at 55 ℃.

In all cases, mRNA was checked after 3, 7 and 14 days of storage under the conditions identified (where appropriate). RNA was quantified by spectrophotometry at 260/280nm and activity was determined visually and quantitatively by measuring relative fluorescent units. For GFP activity, CHO-K1 cells were transfected after harvesting/preparation and allowed to stand for 24 hours for expression. Fig. 9 sets up an overview of the storage and time conditions evaluated, as well as the different controls included for comparison and verification purposes.

The day before each evaluation day, cells were prepared and allowed to set up. 40,000 Chinese Hamster Ovary (CHO) cells per well were seeded in 96-well flat bottom clear tissue culture plates and stored at 2-4 ℃.

For the samples, 3 micrograms (μg) of mRNA was used to allow detection of sufficient amounts of mRNA, while further assessing whether lower final amounts could be adequately recovered.

For the vitrification process, mRNA was mixed in a 1:1 ratio with 2 Xvitrification medium (0.454 g trehalose, 0.023g glycerol and 724. Mu.L PBS) which had been previously sterilized by 0.2 μm PES membrane filter. The vitrification mixture was allowed to vitrify under sterile conditions for 30 minutes in a covered petri dish with a wire mesh therein on a Polyethersulfone (PES) disc membrane.

For storage, once vitrified, the tissue cassette is inserted into an aluminum foil bag with desiccant therein and vacuum sealed.

For the non-vitrified samples, the mRNA stock solution was dispensed into microcentrifuge tubes, placed in aluminum foil bags and vacuum sealed.

The vitrified and non-vitrified groups are divided into groups according to different storage conditions (-20, 28 and 55 ℃), wherein samples are available on days 0, 1, 3, 7 and 14.

To reconstruct the vitrified samples, 50 μ L Fluorobrite DMEM was applied to the mRNA to provide a maximum concentration of 60ng/μl.

mRNA concentrations from both vitrified and non-vitrified samples were then measured for fresh mRNA samples prepared according to manufacturer's instructions. Each mRNA was then normalized to provide transduction of an equal amount of mRNA.

For mRNA transduction, the protocol of the manufacturer (Lipofectamine Messenger MAX-ThermoFisher) was followed. Briefly, 1.25. Mu.g mRNA incubated with 3.75. Mu.L of medium and 1.25. Mu. LLiopfectamine Messenger MAX was incubated with 3.75. Mu.L of medium, and then the two were combined and incubated. mu.L of mRNA-lipofectamine mixture/well was then added to CHO cells. The day after transduction, cells were assessed using a plate reader, stimulated at 495nm and detected at 525 nm. The cells were then imaged using GFP (green) channels.

For each time point, cells were plated the day before the reference time point and fluorometric assays were performed the day after transduction to cells. For example, for day 0, cells were plated on day-1 and assessed for fluorescence on day 1. mRNA was reconstituted immediately after sealing in an aluminum foil bag. For day 1, cells were plated on day 0 and fluorometric on day 2. For day 3, cells were plated on day 2 and fluorometric on day 4. For day 7, cells were plated on day 6 and fluorometric on day 8. For day 14, cells were plated on day 13 and fluorometric on day 15.

Day 0 results:

the vitrification mRNA concentration was measured immediately after reconstitution using a standard 260/280UV protocol. The resulting mRNA concentrations (expected to be 60 ng/. Mu.L, reconstituted in 50. Mu.L based on 3. Mu.g) are set in Table 1.

TABLE 1

The results obtained with fluorescence are not shown.

Day 3 results:

the vitrification mRNA concentration was measured immediately after reconstitution using a standard 260/280UV protocol. FIG. 10 shows the mRNA concentrations obtained (expected to be 60 ng/. Mu.L, reconstituted in 50. Mu.L based on 3. Mu.g). Fig. 11 shows the obtained fluorescence, and fig. 12 provides a captured image of the observed fluorescence. Both showed that after 3 days of storage (even at 55 ℃) vitrified mRNA showed excellent concentration recovery and functional activity after transduction into CHO cells.

Results on day 7.

mRNA concentrations were obtained immediately after reconstitution (fig. 13) and then transduced into CHO cells plated the day before. After 24 hours incubation, fluorescence was measured (fig. 14). Good mRNA can be recovered and exhibit excellent in vitro activity even when stored at 55℃while non-vitrified mRNA exhibits low yield and weak fluorescence even at-20 ℃.

Results on day 14.

On day 1, the mRNA concentrations obtained are shown in fig. 15. Clearly, recovery at all vitrification storage temperatures shows excellent recovery of mRNA, whereas mRNA is rapidly degraded without vitrification according to the methods provided herein. Similar results were observed for functional activity, where vitrification allowed retention of functional translational activity of mRNA even after 14 days of storage at 55 ℃.

mRNA vaccine stability

mRNA encoding the desired antigen may be incubated with ionizable lipids, optionally cholesterol, PEG, and helper lipids to encapsulate the mRNA, or Lipofectamine Messenger MAX (Invitrogen), then with vitrification media and placed on PES membranes. The membrane is placed in a petri dish on a wire mesh to provide heat or rolled into a syringe with a support bracket (as a cylindrical support) to prevent the membrane from resting directly against the syringe wall to allow for thermal gradients (see fig. 7A and 7B). The syringe may then be placed in the heating block or the syringe may have a heating element lower therein.

The pressure of the system was then reduced to about 0.04atm while heat at about 55 ℃ was applied to the vitrification mixture on the PES membrane to prevent freezing of the mRNA-LNP composition. Fig. 5 shows the recovery from the initial drop in expected temperature as the vacuum is applied. Once the temperature of the mRNA-LNP has stabilized, vitrification is complete. The vitrified mRNA-LNP can then be sealed in a sterile container, optionally with a desiccant therein until needed. The sealed vitrified product may optionally be stored at room temperature. Once reconstituted, mRNA-LNP is expected to have little degradation and will exhibit good antigen presentation when administered in vivo.

The mRNA recovery results of Lipofectamine Messenger MAX (Invitrogen) encapsulated mRNA vitrification/reconstitution are shown in figure 17. Excellent recovery of mRNA was achieved even when stored at 28 ℃, whereas all mRNA was degraded without vitrification. As shown in fig. 18, lipofectamine Messenger MAX (Invitrogen) encapsulated mRNA vitrified and stored at all temperatures maintained functional activity in terms of GFP expression capacity after cell transfection.

To further assess the ability to recover mRNA samples from the vitrification process, a small volume of mRNA encoding Green Fluorescent Protein (GFP) was used. For the vitrification process, an 8 μm PES film (capillary substrate) was first cut to a diameter of 1/4 inch and autoclaved. A2X Vitrification Medium (VM) containing 1200mM (or 454 mg/mL) trehalose and 22.7mg/mL glycerol in PBS was prepared and then mixed with an equal volume of mRNA (Dasher GFP mRNA,3870FS Allevon) stock solution. The mixture was incubated for 5 minutes, and then 6 μl was pipetted into each vitrified capillary substrate. After pipetting the solution onto the membrane, the sample is covered with a polymer cover and loaded into the vitrification chamber. For each vitrified sample, a total of 6 μl was loaded onto the membrane prior to vitrification, with 3 μl of bare mRNA stock containing 3 μg mRNA.

After vitrification, the samples were sealed in mylar bags and stored at 55 ℃ prior to testing.

100 days after vitrification storage at 55℃the samples were reconstituted with 50. Mu.L of Fluoobrite medium and vortexed briefly to release mRNA. mRNA was then quantified using a Take3 plate on a BioTek synergy H1 plate reader. Table 2 shows the quantization results obtained.

TABLE 2

Part of the mRNA was then used for transfection or for visualization on agarose gels.

For agarose gel, 3. Mu.L Millennium with 3. Mu.L dye and 5. Mu.L water was used in the first lane of a 1.2% agarose gel ^TM RNA marker (AM 7150) gradient. For positive controls, 3 positive controls were used to dilute the mRNA stock to 125ng/μl: the mRNA stock was diluted to 125 ng/. Mu.L, and then 1. Mu.L of the diluted stock was mixed with 3. Mu.L of dye and 5. Mu.L of water. For vitrified samples, 125ng of reconstituted mRNA was mixed with 3. Mu.L of dye and 5. Mu.L of water. After running the gel at 85V for 1 hour, the gel was stained with SYBR Green II for 30 minutes on a shaker in a BioRad transilluminator. FIG. 19A shows a captured image of a gel, where lanes 2 and 3 are fresh mRNA stored at-80 ℃, lanes 4 and 4 are reconstituted vitrified mRNA, and lanes 5 and 6 are non-vitrified mRNA stored at 55 ℃.

For transfection, 4. Mu.L lipofectamine (lipofectamine)e ^TM MessengerMAX ^TM Transfection reagent, lipofectamine ^TM MessengerMAX ^TM Transfection reagent) was added to 16 μl of medium, allowing incubation for 10 minutes. In another tube, 1 μl of mRNA (fresh sample) was added to 19 μl of medium and incubated for 10 minutes. The two solutions were then mixed and incubated for an additional 5 minutes before being transferred to an inoculation with 0.9x10 ⁶ 10. Mu.L cell plates of individual cells/mL CHO (Chinese hamster ovary) cells. For negative control and vitrified samples, the volume of mRNA was normalized to the volume required to prepare 1 μg mRNA after quantification and added to Lipofectamine after 10 minutes of incubation. FIG. 19B shows the collected GFP expression image, the upper panel being a positive control for fresh mRNA, the middle being a negative control for non-vitrified mRNA stored at 55℃and the lower panel being reconstituted mRNA. Fig. 19C shows the percentage of transfection efficiency obtained relative to that obtained for the positive control.

mRNA vitrified on PES film and stored at 55deg.C for 100 days maintained mRNA integrity, purity and stability, similar to fresh liquid mRNA stored at-80deg.C.

Encapsulated RNA

Next it was assessed how the vector and encapsulated nucleic acid would progress when reconstituted from the vitrification process described herein. Vitrification of lentiviruses is chosen because it provides an encapsulating membrane comprising lipids, carbohydrates and proteins, and provides encapsulated nucleic acids within each viral particle.

Lentiviruses (Lenti-ORF control particles (pLenti-C-mGFP), origin, cat. No. PS100071V 5I) were prepared immediately prior to use. The potential impact of the filter and storage temperature is addressed. The MOI (multiplicity of infection) for lentiviruses was 4, requiring 10. Mu.L of virus stock per well for 25,000 cells/well vaccinated. Lentiviruses were mixed with equal volumes (10. Mu.L and 10. Mu.L) of 1200mM trehalose and 10% w/v glycerol of vitrification medium to prepare each sample. An aliquot (10 μl) of each sample was provided to 24 hours room temperature water washed PES membrane (10 mm) or sterile water and PBST washed bare filter (10 mm). PES membranes were prepared by cutting PES (10 mm diameter), washing in water at room temperature for 24 hours, drying at 37 ℃ for 1 hour, and then autoclaving. The bare filter was prepared by cutting the bare filter (diameter 10 mm), washing in water for 10 minutes at room temperature, then washing in 0.05% pbst for 10 minutes at room temperature, drying at 37 ℃ for 1 hour and then autoclaving.

Vitrification was carried out for 30 minutes with the heated bed temperature set at 37 ℃. After vitrification, storage is carried out at 24℃or 37℃for 1, 2 and 3 weeks. The lentiviral negative controls alone were stored for 1, 2 and 3 weeks at 24℃or 37 ℃. Similarly, lentiviral (non-vitrified) negative controls in vitrification media were also stored for 1, 2 and 3 weeks at 24 ℃ or 37 ℃.

For transduction, 25,000 cells (HEK 293) were seeded on 96-well Costar black clear flat bottom assay plates one day ago. On the day of transduction, cell confluence (70% or more) was confirmed with a microscope. For positive control, 30. Mu.L of lentivirus was mixed with 570. Mu. L C-EMEM and 200. Mu.L was added to each well. For the vitrified samples, both vitrified samples were eluted in 275 μlc-EMEM and cells transduced with the eluted solution in the wells. For negative control only, 30 μl of lentivirus was mixed with 570 μl L C-EMEM, and then 200 μl of negative control solution was added per well to transduce cells. For negative control using vitrification media, 30 μl of lentivirus+30 μl of vitrification media was added to 540 μl of C-EMEM, and then 200 μl of negative control solution was added to each well to transduce cells. Plates were incubated at 37℃for 72 hours. After incubation, post-transduction photographs were taken using a fluorescence microscope and GFP expression was measured using a plate reader.

FIG. 20 shows images of lentivirus alone and percent GFP expression (FIG. 20A) and transduction (FIG. 20B) immediately after vitrification on bare or PES filters. GFP expression after vitrification on bare filters or PES membranes has cell transduction efficiency (based on fluorescence intensity) comparable to fresh liquid lentiviral controls. When cells were transduced immediately after vitrification, the performance of vitrification lentiviruses was as good as that of liquid lentiviruses stored at-80 ℃ regardless of the scaffold (bare filter or PES) used, indicating that the vitrification process did not damage the particles.

FIG. 21 shows GFP expression at weeks 1, 2 and 3 after storage at 24 ℃. Fig. 22 shows the percentage of fluorescence intensity-based transduction efficiency measured and set after 2 weeks (fig. 22A) and 3 weeks (fig. 22B) of storage at 24 ℃ relative to liquid lentiviral positive control. Vitrification lentiviruses, whatever the scaffold used (bare filter or PES), retained their functional activity by all 3 measures, although stored for 3 weeks at 24 ℃, while negative controls showed a significant decrease in function.

Similarly, figure 23 shows GFP expression at weeks 1, 2 and 3 after storage at 37 ℃, and figure 24 shows the percent fluorescence intensity-based transduction efficiency measured and set after storage at 37 ℃ for weeks 2 (figure 24A) and 3 (figure 24B) relative to a liquid lentivirus positive control. Vitrification lentiviruses, whatever the scaffold used (bare filter or PES), retained their functional activity by all 3 measures, although stored for 3 weeks at 37 ℃, while negative controls showed a significant decrease in function.

Further embodiments

A first aspect of the present disclosure (alone or in combination with any other aspect herein) relates to a method of vitrification of one or more particles at temperatures above cryogenic temperatures, the method comprising: a) Placing a vitrification mixture comprising particles thereof and a vitrification medium in or on a substrate comprising or forming a capillary network, and placing the substrate in a drying chamber; b) Reducing the atmospheric pressure within the drying chamber; c) Providing thermal energy to the lipid particles, wherein the thermal energy is sufficient to prevent the vitrification mixture from undergoing freezing conditions; and d) drying the vitrified mixture by capillary action until the vitrified mixture enters the glassy state.

A second aspect of the disclosure (alone or in combination with any other aspect herein) relates to the method of the first aspect, wherein the particles comprise a polynucleotide.

A third aspect of the disclosure (alone or in combination with any other aspect herein) relates to the method of the second aspect, wherein the polynucleotide comprises mRNA and wherein the mRNA is encapsulated within the particle.

A fourth aspect of the disclosure (alone or in combination with any other aspect herein) relates to the method of any one of the first to third aspects, wherein the particle comprises a viral capsid, a viral envelope, or a portion thereof.

A fifth aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of any one of the first to third aspects, wherein the particle further comprises a cell penetrating peptide or carrier protein.

A sixth aspect of the disclosure (alone or in combination with any other aspect herein) relates to the method of the fifth aspect, wherein the cell penetrating peptide or carrier protein is coupled to the polynucleotide.

A seventh aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of the second or third aspect, wherein the polynucleotide is encapsulated by a lipid membrane comprising a cationic lipid and/or an ionizable lipid.

An eighth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of any one of the first to third aspects, wherein the capillary network is provided by a wavy profile along the surface of the substrate.

A ninth aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of any one of the first to third aspects, wherein the substrate is or is associated with a wall of the drying chamber.

A tenth aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of any one of the first to third aspects, wherein the capillary network within the drying chamber is supported by an underlying solid support substrate.

An eleventh aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of any one of the first to third aspects, wherein vitrification of the vitrified mixture occurs in less than 30 minutes.

A twelfth aspect of the disclosure (alone or in combination with any of the other aspects herein) is directed to the method of the eleventh aspect, wherein vitrification of the vitrified mixture occurs in less than 10 minutes.

A thirteenth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of any one of the first to third aspects, wherein the thermal energy is provided by heating the vitrification mixture.

A fourteenth aspect of the present disclosure relates (alone or in combination with any of the other aspects herein) to the method of any of the first to third aspects, wherein the atmospheric pressure is reduced to a value of about 0.9atm to about 0.005 atm.

A fifteenth aspect of the present disclosure (alone or in combination with any of the other aspects herein) is directed to the method of the fourteenth aspect, wherein the atmospheric pressure is reduced to about 0.004atm.

A sixteenth aspect of the present disclosure relates (alone or in combination with any other aspect herein) to the method of any one of the first to third aspects, wherein the thermal energy provided is sufficient to prevent crystallization within the vitrified mixture during vitrification.

A seventeenth aspect of the present disclosure, alone or in combination with any other aspect herein, relates to the method of any one of the first to third aspects, wherein the thermal energy provided is sufficient to maintain the biological sample at a temperature of about 0 ℃ to about 40 ℃ during the vitrification process.

An eighteenth aspect of the present disclosure relates (alone or in combination with any other aspect herein) to the method of any one of the first to third aspects, wherein the vitrification medium comprises disaccharides, optionally trehalose, glycerol and betaine and/or choline.

A nineteenth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of any one of the first to third aspects, wherein the capillary network is hydrophilic.

A twentieth aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of any one of the first to third aspects, wherein the capillary network comprises a continuous capillary channel.

A twenty-first aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of any one of the first to third aspects, wherein the particulate composition is stored at a temperature of 60 ℃ or less for a period of at least three weeks after vitrification.

A twenty-second aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of the twenty-first aspect, wherein the particles are reconstituted in an aqueous medium and retain an activity equivalent or near equivalent to the particles or their contents prior to step a).

A twenty-third aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of any one of the first to third aspects, wherein the vitrification medium comprises trehalose and glycerol suspended in a cellular medium.

The twenty-fourth aspect of the present disclosure, alone or in combination with any other aspect herein, relates to the method of the twenty-third aspect, wherein the vitrification medium comprises 500 to 1500mM trehalose and 5 to 20% weight/volume (percent weight by volume) glycerol in the cell culture medium.

A twenty-fifth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of any one of the first to third aspects, further comprising, after step d), placing the capillary network in a dark environment.

A twenty-sixth aspect of the present disclosure, alone or in combination with any other aspect herein, is directed to the method of the twenty-fifth aspect, wherein the dark environment is maintained at an atmosphere of less than 5% Relative Humidity (RH).

A twenty-seventh aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of the twenty-sixth aspect, wherein the dark environment is maintained at 2% rh or less.

A twenty-eighth aspect of the present disclosure (alone or in combination with any of the other aspects herein) relates to a method of inducing an immune response in a subject, comprising: a) Reconstructing the vitrified mixture obtained from any one of the first to twenty-seventh aspects by providing a volume of solution to the vitrified mixture on a capillary network to obtain an eluted vitrified mixture; b) Obtaining an eluted vitrification mixture from a capillary network; and c) administering the eluted vitrification mixture to a subject.

A twenty-ninth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of the twenty-eighth aspect, wherein the particles comprise an attenuated virus.

A thirty-first aspect of the present disclosure (alone or in combination with any other aspects herein) relates to the method of the twenty-first aspect, wherein the particle comprises a polynucleotide encoding at least a portion of a viral protein, optionally an mRNA.

A thirty-first aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the method of the thirty-first aspect, wherein the polynucleotide is coupled to a cell-penetrating peptide.

A thirty-second aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of the thirty-first aspect, wherein the polynucleotide is encapsulated by a lipid membrane.

A thirty-third aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the method of the thirty-first aspect, wherein the lipid membrane comprises a cationic lipid.

A thirty-fourth aspect of the present disclosure (alone or in combination with any of the other aspects herein) is directed to the method of the thirty-first aspect, wherein the lipid membrane comprises an ionizable lipid.

A thirty-fifth aspect of the present disclosure (alone or in combination with any other aspects herein) relates to a vitrified polynucleotide composition comprising a polynucleotide molecule encapsulated in a particle and a dehydrated vitrification medium.

A thirty-sixth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the vitrified vaccine composition of the thirty-fifth aspect, wherein the composition is vitrified without freezing the polynucleotide molecule.

A thirty-seventh aspect of the present disclosure (alone or in combination with any other aspect herein) relates to the vitrified vaccine composition of the thirty-fifth aspect, wherein the particles comprise an attenuated virus.

A thirty-eighth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the vitrified vaccine composition of any one of the thirty-fifth to thirty-seventh aspects, wherein the polynucleotide molecule comprises mRNA encoding at least a portion of a viral protein.

A thirty-ninth aspect of the present disclosure (alone or in combination with any other aspect herein) is directed to the vitrified vaccine composition of any one of the thirty-fifth to thirty-seventh aspects, wherein the polynucleotide molecule is coupled to a cell penetrating peptide.

A fortieth aspect of the present disclosure, alone or in combination with any of the other aspects herein, is directed to the vitrified vaccine composition of any of the thirty-fifth to thirty-seventh aspects, wherein the particles comprise a cationic lipid.

A fortieth aspect of the present disclosure, alone or in combination with any other aspects herein, is directed to a kit for providing an immune response in a subject comprising a vitrified mixture prepared by any one of the first to twenty-seventh aspects.

A forty-second aspect of the present disclosure (alone or in combination with any of the other aspects herein) is directed to the kit of the forty-first aspect, wherein the vitrified mixture is stored in a dark, dry container.

A fortieth aspect of the present disclosure, alone or in combination with any other aspects herein, is directed to the kit of the fortieth aspect, further comprising a sterile solvent suitable for reconstitution of the vitrified mixture, said solvent being suitable for administration to a subject.

The forty-fourth aspect of the present disclosure (alone or in combination with any of the other aspects herein) is directed to the kit of any one of the forty-first to forty-third aspects, further comprising a vial.

Various modifications of the disclosure in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

It is to be understood that all reagents may be obtained from sources known in the art unless otherwise indicated.

It is also to be understood that this disclosure is not limited to the particular aspects and methods described herein, as the particular components and/or conditions may, of course, vary. Furthermore, the terminology used herein is for the purpose of describing particular aspects of the present disclosure only and is not intended to be limiting in any way. It will be further understood that, although the terms "first," "second," "third," etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a "first element," "component," "region," "layer" or "section" discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein. Similarly, as used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, including "at least one" unless the content clearly indicates otherwise. "or" means "and/or". As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof. The term "or a combination thereof" is intended to encompass a combination of at least one of the foregoing elements.

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Reference is made in detail to the exemplary compositions, aspects and methods of the present disclosure, which constitute the best modes of practicing the disclosure presently known to the inventors. The figures are not necessarily drawn to scale. However, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the disclosure and/or as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

Patents, publications and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains. These patents, publications, and applications are herein incorporated by reference to the same extent as if each individual patent, publication, or application was specifically and individually indicated to be incorporated by reference.

The foregoing description is illustrative of specific embodiments of the present disclosure, but is not meant to be limiting thereof. The following claims, including all equivalents thereof, are intended to define the scope of this disclosure.

Claims

1. A method of vitrification of one or more particles at a temperature above cryogenic temperatures, the method comprising:

a) Placing a vitrification mixture comprising particles thereof and a vitrification medium in or on a substrate comprising or forming a capillary network, and placing the substrate in a drying chamber;

b) Reducing the atmospheric pressure within the drying chamber;

c) Providing thermal energy to the lipid particles, wherein the thermal energy is sufficient to prevent the vitrification mixture from undergoing freezing conditions; and

d) The vitrification mixture is dried by capillary action until the vitrification mixture enters the glassy state.

2. The method of claim 1, wherein the particles comprise polynucleotides.

3. The method of claim 2, wherein the polynucleotide comprises mRNA and wherein the mRNA is encapsulated within the particle.

4. A method according to any one of claims 1 to 3, wherein the particle comprises a viral capsid, a viral envelope or a part thereof.

5. A method according to any one of claims 1 to 3, wherein the particles further comprise a cell penetrating peptide or carrier protein.

6. The method of claim 5, wherein the cell penetrating peptide or carrier protein is coupled to the polynucleotide.

7. The method of claim 2 or 3, wherein the polynucleotide is encapsulated by a lipid membrane comprising a cationic lipid and/or an ionizable lipid.

8. A method according to any one of claims 1 to 3, wherein the capillary network is provided by a wave-like profile along the surface of the substrate.

9. A method according to any one of claims 1 to 3, wherein the substrate is or is associated with a wall of the drying chamber.

10. A method according to any one of claims 1 to 3, wherein the capillary network within the drying chamber is supported by an underlying solid support substrate.

11. The method of any one of claims 1-3, wherein vitrification of the vitrification mixture occurs in less than 30 minutes.

12. The method of claim 11, wherein vitrification of the vitrification mixture occurs in less than 10 minutes.

13. A method according to any one of claims 1 to 3, wherein the thermal energy is provided by heating the vitrification mixture.

14. The method of any one of claims 1-3, wherein the atmospheric pressure is reduced to a value of about 0.9atm to about 0.005 atm.

15. The method of claim 14, wherein the atmospheric pressure is reduced to about 0.004atm.

16. A method according to any one of claims 1 to 3, wherein the thermal energy provided is sufficient to prevent crystallisation within the vitrification mixture during vitrification.

17. The method of any one of claims 1-3, wherein the thermal energy provided is sufficient to maintain the biological sample at a temperature of about 0 ℃ to about 40 ℃ during the vitrification process.

18. A method according to any one of claims 1 to 3, wherein the vitrification medium comprises disaccharides, optionally trehalose, glycerol and betaine and/or choline.

19. A method according to any one of claims 1 to 3, wherein the capillary network is hydrophilic.

20. A method according to any one of claims 1 to 3, wherein the capillary network comprises a continuous capillary channel.

21. A method according to any one of claims 1 to 3, wherein the lipid particle composition is stored at a temperature of 60 ℃ or less for a period of at least three weeks after vitrification.

22. The method of claim 21, wherein the lipid particles are reconstituted in an aqueous medium and retain an activity equivalent or near equivalent to the particles or their contents prior to step a).

23. A method according to any one of claims 1 to 3, wherein the vitrification medium comprises trehalose and glycerol suspended in the cell culture medium.

24. The method of claim 23, wherein the vitrification medium comprises 500 to 1500mM trehalose and 5 to 20% weight/volume glycerol in the cell culture medium.

25. The method of any one of claims 1-3, further comprising placing the capillary network in a dark environment after step d).

26. The method of claim 25, wherein the dark environment is maintained at an atmosphere of less than 5% Relative Humidity (RH).

27. The method of claim 26, wherein the dark environment is maintained at 2% rh or less.

28. A method of inducing an immune response in a subject, comprising:

a) Reconstituting the vitrified mixture obtained from any one of claims 1-27 by providing a volume of solution to the vitrified mixture on a capillary network to obtain an eluted vitrified mixture;

b) Obtaining an eluted vitrification mixture from a capillary network; and

c) The eluted vitrification mixture is administered to a subject.

29. The method of claim 28, wherein the particle comprises an attenuated virus.

30. The method of claim 28, wherein the particle comprises a polynucleotide encoding at least a portion of a viral protein, optionally mRNA.

31. The method of claim 30, wherein the polynucleotide is coupled to a cell penetrating peptide.

32. The method of claim 31, wherein the polynucleotide is encapsulated by a lipid membrane.

33. The method of claim 31, wherein the lipid membrane comprises a cationic lipid.

34. The method of claim 31, wherein the lipid membrane comprises an ionizable lipid.

35. A vitrified polynucleotide composition comprising a polynucleotide molecule encapsulated in a particle and a dehydrated vitrification medium.

36. The vitrified vaccine composition of claim 35, wherein the composition is vitrified without freezing the polynucleotide molecule.

37. The vitrified vaccine composition of claim 35, wherein the particles comprise an attenuated virus.

38. The vitrified vaccine composition of any one of claims 35-37, wherein the polynucleotide molecule comprises mRNA encoding at least a portion of a viral protein.

39. The vitrified vaccine composition of any one of claims 35-37, wherein the polynucleotide molecule is coupled to a cell penetrating peptide.

40. The vitrified vaccine composition of any one of claims 35-37, wherein the particles comprise cationic lipids.

41. A kit for providing an immune response in a subject comprising the vitrified mixture prepared according to any one of claims 1-27.

42. The kit of claim 41, wherein the vitrification mixture is stored in a dark, dry container.

43. The kit of claim 41, further comprising a sterile solvent suitable for reconstitution of the vitrified mixture, said solvent being suitable for administration to a subject.

44. The kit of any one of claims 41-43, further comprising a vial.