Polyphosphate stabilizer, and composition, application and preparation method thereof

Abstract

The invention provides a stabilizer as well as a composition, a preparation method and application thereof. In one aspect, provided are lipid nanoparticle compositions comprising (a) ionizable lipids, (b) two or more lipids, and (c) a stabilizer of formula (I), as well as methods for preparing the stabilizer and the lipid nanoparticles.

Description

[0001] Cross-reference to related applications This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 537,966, filed September 12, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0002] Lipid nanoparticle (LNP) formulations and nucleic acid-containing lipid nanoparticle (NALNP) formulations are used in a variety of applications, particularly in medical applications, such as oligonucleotide-based therapeutics, including vaccines, immunogenic cell incorporation, and gene therapy. LNP and NALNP formulations are also used in applications such as antibiotics and vitamins.

[0003] However, improved LNP and NALNP formulations are needed. This invention provides improvements over at least some of the shortcomings of the prior art. These and other advantages of the invention will become apparent from the description set forth below. Summary of the Invention

[0004] In one aspect, the present invention provides a stabilizer having the structure of formula (I): (I) Where R is hydrogen, Targeting ligand, hydrophilic group, amphiphilic group or combination thereof; A, B, D and E are each and independently C 1-18 Heteroalkyl; C is C1-C 50 Substituted or unsubstituted heteroalkyl, C1-C 50 Unsaturated heteroalkyl groups, C1-C 50 Charged heteroalkyl groups, C1-C 50 Heterocyclic groups or combinations thereof; and x is an integer from 3 to 1000.

[0005] In some implementations, A, B, D, and E are each and independently C. 1-9 Heteroalkyl; C is C 1-50 Substituted or unsubstituted heteroalkyl, C 1-50 Unsaturated heteroalkyl groups, C 1-50 Charged heteroalkyl groups, C 1-50 Heterocyclic groups or combinations thereof; and x is an integer from 3 to 130.

[0006] In some implementations, C is .

[0007] In some implementations, R is hydrogen, , Peptides, antibodies, sugars, oligosaccharides, aminoglycosides, sterols, phenylboronic acid, or combinations thereof.

[0008] In some implementation schemes, B is m is an integer from 1 to 10, and n is an integer from 1 to 10.

[0009] In some embodiments, the stabilizer is selected from SAF41, SAF44, SAF45, SAF54, SAF84, SAF85, SAF86, SAF87, SAF88, SAF130, SAF132, SAF160, SAF164, SAF165, SAF166, SAF197, SAF198, SAF200, SAF206, SAF207, or combinations thereof.

[0010] In some implementations, the stabilizer of formula (I) is used to stabilize lipid nanoparticles or liposomes.

[0011] In one aspect, the present invention provides a lipid nanoparticle composition comprising: (a) an ionizable lipid; (b) two or more lipids; and (c) a stabilizer of formula (I).

[0012] In some embodiments, the two or more lipids include structural lipids, sterols, or combinations thereof.

[0013] In some embodiments, the lipid nanoparticle composition consists essentially of: (a) an ionizable lipid; (b) two lipids; and (c) the stabilizer.

[0014] In some embodiments, the lipid nanoparticle composition is substantially free of PEG or PEG-R, wherein R is any atom or molecule covalently linked to PEG.

[0015] In some embodiments, the structural lipid is neutrally charged, positively charged, or negatively charged.

[0016] In some embodiments, the ionizable lipid is DODMA, DLin-MC3-DMA, DLin-KC2-DMA, BOCHD-C3-DMA, C12-200, PNI 516, PNI 127, PNI 550, PNI 580, PNI 659, PNI 728, PNI 762, or a combination thereof.

[0017] In some embodiments, the structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cerebroside, cerebroside, or combinations thereof.

[0018] In some embodiments, the structural lipid comprises distearylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine, palmitoyloleoyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearylphosphatidylethanolamine, and 1,2-dipalmitoyl-sn-glycerol. -3-phosphoethanolamine-N-methyl, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N,N-dimethyl, 1,2-ditransoleoyl-sn-glycerol-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, 1,2-ditransoleoyl-sn-glycerol-3-phosphoethanolamine, distearate phosphatidylcholine, dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylglycerol, cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, monosialotetrahexosylganglioside GM1 or combinations thereof.

[0019] In some embodiments, the sterol includes cholesterol, β-sitosterol, 20-α-hydroxysterol, phytosterols, or combinations thereof.

[0020] In some embodiments, the stabilizer has a molecular weight of about 500 Da to about 50,000 Da.

[0021] In some embodiments, the lipid nanoparticle composition includes a second stabilizer, wherein the second stabilizer is polysorbate, N-dodecyl β-D-maltodextrin, D-α-tocopherol polyethylene glycol 1000 succinate, or a combination thereof.

[0022] In some embodiments, the lipid nanoparticle composition comprises about 20 to about 70 mol% ionizable lipids, about 1 to about 25 mol% structured lipids, about 28 to about 50 mol% sterols, and about 0.1 to about 5 mol% stabilizers.

[0023] In one aspect, the present invention provides a lipid nanoparticle comprising the lipid nanoparticle composition and a nucleic acid.

[0024] In some embodiments, the nucleic acid is encapsulated by the lipid nanoparticle composition.

[0025] In some embodiments, the nucleic acid includes antisense oligonucleotides, siRNA, miRNA, self-amplifying RNA (samRNA or saRNA), self-replicating DNA, LNA, DNA, replicon, mRNA, guide RNA, transposon, single gene, vector, plasmid, viral particle, AAV, RNA-RNA binding protein complex, or combinations thereof.

[0026] In some embodiments, the nucleic acid is mRNA encoding an antigen for a preventive or therapeutic vaccine, a nucleic acid for gene therapy, or a nucleic acid for incorporation into immunogenic cells, wherein the immunogenic cells are T cells.

[0027] In some embodiments, the diameter of the lipid nanoparticles is from about 15 nm to about 500 nm.

[0028] In some embodiments, the lipid nanoparticles have a polydispersity index of about 0.01 to about 0.40.

[0029] In some embodiments, the lipid nanoparticles have an encapsulation efficiency of about 50% to about 100%.

[0030] In one aspect, the present invention provides a pharmaceutical composition comprising the lipid nanoparticle composition and a pharmaceutically acceptable carrier.

[0031] In another aspect, the present invention provides a method for preparing lipid nanoparticles, the method comprising: (a) forming a lipid nanoparticle composition by combining ionizable lipids, structural lipids, sterols and stabilizers; (b) preparing lipid nanoparticles by combining the lipid nanoparticle composition and nucleic acids using a microfluidic mixer; and (c) purifying the lipid nanoparticles.

[0032] In some embodiments, the lipid nanoparticle composition and nucleic acid are combined using a flow ratio of about 1:1 to about 10:1 (aqueous phase: organic phase) by volume, an N / P ratio of about 2 to about 20, and a total flow rate of about 2 to about 2000 mL / min.

[0033] In some embodiments, the aqueous phase comprises a low-pH buffer. In some embodiments, the aqueous phase comprises a citrate or acetate buffer.

[0034] In some embodiments, the organic phase comprises 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acid, alcohol, or a combination thereof.

[0035] In some embodiments, the organic phase is an alcohol, and the alcohol includes aqueous or anhydrous alcohols, wherein the alcohol is a primary, secondary or tertiary alcohol having 1-12 branched or unbranched carbons.

[0036] In another aspect, the present invention provides the use of the lipid nanoparticles or the pharmaceutical composition for the prevention, treatment or improvement of a condition or disease, including administering the lipid nanoparticles as a vaccine or as a treatment to prevent or reduce the severity of an infectious disease, administering the lipid nanoparticles as a gene therapy, or administering the lipid nanoparticles to immunogenic cells to treat cancer or infection.

[0037] Further and alternative aspects and features of the disclosed principles will be understood from the following detailed description. As will be understood, the compositions and methods disclosed herein can be performed and used in other and different embodiments and can be modified in various aspects. Therefore, it should be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative and do not limit the scope of the appended claims. Attached Figure Description

[0038] Figure 1A This is a schematic diagram illustrating an study of hEPO expression in mice. In this study, four mice / groups were injected with an LNP expressing EPO, which contained PBS, SAF41, SAF44, SAF85, or SAF87 as stabilizers as described herein. hEPO expression levels were measured 24 h after administration.

[0039] Figure 1B This is a dot plot showing the hEPO expression level in the serum of C57BL / 6 mice (24 h post-administration) following IV administration of an EPO-expressing LNP containing PBS, SAF41, SAF44, SAF85, or SAF87 as a stabilizer as described herein. Mice were administered 0.25 mg / kg and 200 µL / 20 g mouse doses of the ionizable lipid PNI 516 encoding recombinant human EPO in the VO2 composition (N / P-8). The lipid nanoparticle composition used comprised PNI 516-VO2 (lipid concentration: 25 mM). The VO2 composition comprised 47.5 mol% ionizable lipid, 12.5 mol% structural lipid, 38.5 mol% sterol, and 1.5 mol% stabilizer.

[0040] Figure 2A This is a schematic diagram illustrating a SARS-CoV-2 vaccine study in mice. In this study, mice were injected with an LNP expressing SARS-CoV-2, which contained PBS or SAF45 as a stabilizer as described herein, and SARS-CoV-2 antigen-specific IgG levels were measured 21 and 42 days after IV administration.

[0041] Figure 2BThis is a dot plot showing the levels of SARS-CoV-2 spike protein-specific IgG in the serum of mice treated with A5 saRNA lipid nanoparticles (1 µg / 50 µL / 20 g mouse / injection) with different stabilizers (PBS or SAF45) as described herein 14 days after booster injection (42 days after primary immunization). The lipid nanoparticle composition used included PNI516-VO2 (lipid concentration: 25 mM, N / P-8). The VO2 composition used was 47.5 mol% ionizable lipids, 12.5 mol% structural lipids, 38.5 mol% sterols, and 1.5 mol% stabilizer.

[0042] Figure 3 This is a bar chart illustrating the in vitro knockout (KO) efficiency in T cells after treatment with T cell receptor (TCR) single-guide RNA (sgRNA) / Cas9 mRNA LNP with a polyphosphate-based stabilizer for 24 h (10,000 cells / 100 μL, dose: 4 μg / million cells). The composition used was 40 mol% PNI 762, 20 mol% DSPC, 37.5 mol% cholesterol, and 2.5 mol% stabilizer. SAF198 and SAF207 showed higher potency than the PEG-DMG stabilizer in in vitro experiments.

[0043] Figure 4 This is a line graph illustrating the in vitro potency of various polyphosphate-based LNPs (lipid composition: 40 mol% PNI 762, 20 mol% DSPC, 37.5 mol% cholesterol, 2.5 mol% stabilizer) with enhanced green fluorescent protein (eGFP) mRNA payloads. Cell lines tested included BHK (…). Figure 4 SAF198 showed higher potency than PEG-DMG in both in vitro and ex vivo experiments.

[0044] Figures 5A-5B This is a bar chart showing the in vitro activity of various LNPs. 24 h after treatment, various polyphosphate-based LNPs (lipid composition: 40 mol% PNI 762, 20 mol% DSPC, 37.5 mol% cholesterol, 2.5 mol% stabilizer) with enhanced green fluorescent protein (eGFP) mRNA payloads... Figure 5A Transfection efficiency (TE) % and ( Figure 5B Mean fluorescence intensity (MFI) was analyzed by flow cytometry. Cell lines tested included BHK, U937, HeLa, and Jurkat. Primary cells used for in vitro studies were human T cells.

[0045] Figures 6A-6B This is a dot plot showing the expression of SARS-CoV-2 spike protein-specific IgG in C57BL / 6 mice on days 21 and 42 after administration of a LNP containing saRNA encoding the SARS-CoV-2 spike protein, formed from a V62-based composition (40% mol PNI 516, 12.5 mol% DSPC, 46 mol% cholesterol, 1.5 mol% stabilizer) (N / P-8) at a dose of 1 µg / mouse with IM. Stabilizers with comparable / superior performance in vivo compared to PEG-DMG in the V62 composition include SAF130 and SAF132.

[0046] Figures 7A-7B This is a dot plot showing the hEPO expression levels (6 h and 24 h post-administration) in C57BL / 6 mice following IV administration of the V62 composition (40 mol% PNI 516, 12.5 mol% DSPC, 46 mol% cholesterol, 1.5 mol% stabilizer) (N / P-8) at a dose of 0.25 mg / kg. Among the stabilizers in the V62 composition, SAF130 showed superior in vivo performance compared to PEG-DMG.

[0047] Figure 8 This is a dot plot showing the hEPO expression level (6 h post-administration) in C57BL / 6 mice following subcutaneous administration of a composition (N / P-8) containing 40 mol% PNI 516, 12.5 mol% DSPC, 46 mol% cholesterol, and 1.5 mol% stabilizer. SAF85 showed superior performance compared to PEG-DMG in in vivo experiments.

[0048] Figure 9 This is a bar chart showing the expression levels of Fluc protein in the spleen and liver of Hsd:ICR female mice (4 h post-administration) following intravenous administration of a combination (N / P-8) containing 40 mol% PNI 516, 12.5 mol% DSPC, 46 mol% cholesterol, and 1.5 mol% stabilizer. SAF85 and SAF87 showed superior performance compared to PEG-DMG in in vivo experiments. Figure 9 Polyphosphates showed higher extrahepatic selectivity than PEG or PEGylated lipids. Detailed Implementation

[0049] I. Introduction This disclosure provides stabilizers and lipid nanoparticle (LNP) compositions comprising said stabilizers, as well as methods for preparing said stabilizers and lipid nanoparticles. These lipid nanoparticle (LNP) compositions can be configured to encapsulate nucleic acids. The lipid nanoparticle compositions for encapsulating nucleic acids may comprise ionizable lipids, two or more lipids, and polyphosphate stabilizers as discussed herein. Advantageously, in contrast to LNP compositions comprising polyethylene glycol (PEG) or PEGylated lipids (PEG-lipid conjugates) as stabilizers, the LNP and NALNP formulations with polyphosphate stabilizers according to the invention are substantially free of PEG and PEGylated lipids. The inventors have found that the stabilizers of the present invention allow for LNPs with higher compatibility and safety, and lower immunogenicity, compared to LNP products based on PEG or PEGylated lipids. In some cases, said PEGylated lipids are PEG-R, where R is any atom or molecule. In some cases, R is DMG, DSG, DSPE, DOPE, or DPPE. In some cases, the PEGylated lipids include DMG-PEG, DSG-PEG, DSPE-PEG, DOPE-PEG, or DPPE-PEG.

[0050] The resulting encapsulated LNP formulation can be used in a variety of applications, particularly medical applications, such as oligonucleotide-based therapeutics, including vaccines, immunogenic cell incorporation, and gene therapy. The composition surprisingly and unexpectedly offers benefits, including more efficient protein expression compared to conventional PEG-based products.

[0051] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0052] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein pertain. Any reference to standard methods refers to the most recently available version of the method at the time of submission of this disclosure, unless otherwise indicated.

[0053] For any method disclosed herein that includes discontinuous steps, the steps may be performed in any feasible order. And, where appropriate, any combination of two or more steps may be performed simultaneously.

[0054] All headings are for the reader's convenience and should not be used to limit the meaning of the text that follows the heading, unless otherwise indicated.

[0055] The terms "preferred" and "ideally" refer to embodiments of the invention that may provide certain benefits in some circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0056] The terms “comprising,” “having,” “including,” and “containing” shall be interpreted as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise specified. Such terms shall be understood to mean that the specified steps or elements or groups of steps or elements are included, but do not exclude any other steps or elements or groups of steps or elements.

[0057] The singular forms "a", "an", and "the" include plural objects unless the context clearly indicates otherwise. These articles refer to one or more (i.e., at least one). The use of the term "at least one" (e.g., "at least one of A and B") after a list of one or more items should be interpreted as referring to one item (A or B) selected from the listed items or any combination of two or more listed items (A and B), unless otherwise specified herein or the context clearly contradicts it. As used herein, the term "or" is generally used in its common sense, including "and / or", unless the context clearly indicates otherwise. The term "and / or" refers to any one or more items in a list connected by "and / or". As an example, "x and / or y" refers to any element in the triad set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element in the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y, and / or z" means "one or more of x, y, and z".

[0058] When a range is provided, the endpoints include all numbers included within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise specified or otherwise apparent from the context and to those skilled in the art, values ​​represented as ranges in different embodiments of this disclosure may take any specific value or subrange within the specified range, the decimal place of the unit of the lower limit of the precise value range, unless the context clearly indicates otherwise. In the text, "at most" a number (e.g., at most 50) includes that number (e.g., 50). The terms "in the range" or "within the range" (and similar statements) include the endpoints of the specified range.

[0059] Throughout this specification, references to "an aspect (or embodiment)," "aspect (or embodiment)," "certain aspects (or embodiments)," or "some aspects (or embodiments)," etc., indicate that a specific feature, configuration, composition, or characteristic described in connection with that aspect is included in at least one aspect of this disclosure. Therefore, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of this disclosure. Furthermore, the specific features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0060] Unless otherwise specified, all figures used in the specification and claims to represent the quantity of components, molecular weight, etc., shall be understood to be modified by the term "about" in all cases. When used herein in conjunction with the quantity of measurement, the term "about" refers to a variation in the quantity of measurement as would be expected by a person skilled in the art to perform the measurement and to exercise a level of caution commensurate with the purpose of the measurement and the accuracy of the measuring equipment used. When used throughout the specification and claims in conjunction with numerical values, the term "about" indicates an accuracy range familiar and acceptable to those skilled in the art. Generally, such an accuracy range is + / - 10%. Thus, "about" can be understood as being greater than or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the specified value. Therefore, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximate values ​​of the variations in desired properties that can be obtained according to the invention. At least and without attempt to limit the doctrine of equivalence to the scope of the claims, each numerical parameter shall be interpreted at least according to the number of significant figures reported and by applying conventional rounding methods.

[0061] Although the numerical ranges and parameters describing the broad scope of the invention are approximations, the values ​​described in the specific embodiments are reported as accurately as possible. However, all values ​​inherently contain ranges that necessarily arise from the standard deviation present in their respective test measurements.

[0062] The term "exemplary" means provided as a non-limiting embodiment, example, or illustration. As used herein, the terms "e.g." and "for example" refer to a list of one or more non-limiting aspects, embodiments, examples, or illustrations.

[0063] As used herein, the term "substantially" refers to a qualitative state that indicates all or nearly all of the range or extent of the feature or property of interest. Biological and chemical phenomena rarely (if at all) reach completeness and / or proceed to full extent, or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to reflect the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may mean at least about 20%, at least about 10%, or at least about 5% of the feature or property of interest.

[0064] As used herein, the term "administration" means the physical introduction of a pharmaceutical agent, such as the lipid nanoparticles disclosed herein, into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. The phrase "parenteral administration" refers to administration methods other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, spinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, such as oral administration. Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more extended cycles.

[0065] The term "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells within the body. Unregulated cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors.

[0066] The term "in vitro" refers to events that occur in an artificial environment, such as in test tubes, reaction vessels, cell cultures, etc., rather than within a multicellular organism. The term "in vitro cell" refers to any cell cultured in vitro. In particular, in vitro cells may include T cells. The term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal.

[0067] The term "nucleic acid" refers to any polymeric chain of nucleotides. Nucleic acids can be DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid comprises one or more naturally occurring nucleic acid residues. In some embodiments, the nucleic acid comprises one or more nucleic acid analogs. In some embodiments, the nucleic acid is prepared by one or more of the following methods: isolation from a natural source, enzymatic synthesis (in vivo or in vitro) via polymerization based on a complementary template, replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325 The nucleic acid may be 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues long (e.g., 20-100, 20-500, 20-1000, 20-2000, or 20-5000 or more residues). In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element encoding a polypeptide or a sequence complementary to that encoding a polypeptide.

[0068] The term "pharmaceutically acceptable" means a molecule or composition that, when administered to a recipient, is harmless to the recipient or whose beneficial effects outweigh those of the recipient. Regarding carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and harmless to the recipient, or any harmful effects must outweigh the beneficial effects on the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which participates in carrying or transporting a drug agent from one part of the body to another (e.g., from one organ to another). Every carrier present in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the other components of the formulation and harmless to the patient, or that any harmful effects must outweigh the beneficial effects on the recipient. Examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0069] "Treatment" or "treating" for a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, with the aim of reversing, alleviating, improving, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions, or disease-related biochemical indicators. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment may be for subjects who do not show signs of the relevant disease, condition, and / or condition, and / or for subjects who only show early signs of the disease, condition, and / or condition. In some embodiments, such treatment may be for subjects who show one or more identified signs of the relevant disease, condition, and / or condition. In some embodiments, treatment may be for subjects who have been diagnosed with the relevant disease, condition, and / or condition. In some embodiments, treatment may be for subjects who are known to have one or more statistically significant susceptibility factors associated with an increased risk of developing the relevant disease, condition, and / or condition.

[0070] As used herein, "disease" refers to a subject's health condition in which the subject is unable to maintain homeostasis, and in which the subject's health continues to deteriorate if the disease is not improved. In contrast, "symptom" refers to a health condition in which the subject is able to maintain homeostasis, but in which the subject's health condition is more detrimental than in the absence of the symptom. A symptom, without treatment, does not necessarily lead to a further decline in the subject's health condition. A disease or symptom is "reduced" if the severity of the signs or symptoms of the disease or symptom, the frequency with which the subject experiences such signs or symptoms, or both, decreases.

[0071] As used herein, the terms “subject,” “individual,” and “patient” are interchangeable and refer to vertebrates, preferably mammals. For example, in the context of this disclosure, mammals include humans, non-human primates, domesticated animals (e.g., dogs, cats, sheep, cattle, goats, pigs, horses, etc.), laboratory animals (e.g., mice, rats, rabbits, guinea pigs, etc.), and captive animals (e.g., zoo animals). The term “animal” as used herein includes humans. The term “subject” may also include a patient, i.e., an animal with a disease. In exemplary aspects, a subject, individual, or patient refers to a person (e.g., a male, female, or child).

[0072] As used herein, the term "disease prevention" in subjects means, for example, stopping the development of one or more clinical symptoms of a disease or condition in subjects before they become detectable. Preferably, the disease or condition does not develop at all, i.e., no symptoms of the disease or condition are detectable. In some aspects, it may also mean delaying or slowing the development of one or more symptoms of a disease or condition. Alternatively or additionally, it may mean reducing the severity of one or more subsequently developed symptoms.

[0073] The invention is defined in the claims. However, the following is a non-exhaustive list of non-limiting exemplary aspects. Any one or more features of these aspects may be combined with any one or more features of another instance, embodiment, or aspect described herein.

[0074] III. Stabilizers Lipid nanoparticles, including liposomes, cubic lipid nanoparticles, hexagonal lipid nanoparticles, solid lipid nanoparticles, and nanostructured lipid carriers, may require steric stabilizers to maintain colloidal stability in aqueous media and improve pharmacokinetic and biodistribution characteristics. One indicator of colloidal stability is the polydispersity index (PDI), which needs to be low enough to prevent nanoparticle aggregation. A high PDI indicates that the stability and viability of nanoparticles decrease over time.

[0075] Currently, polyethylene glycol (PEG)-lipid conjugates are the most commonly used stabilizers in these systems. However, these PEGylated stabilizers can induce unwanted immunogenic responses and hinder cell-cellular interactions with nanoparticles containing a PEG layer. Surprisingly, compared to PEGylated stabilizers, the inventors have developed a novel class of stabilizers with polyphosphate groups that possess advantageous characteristics as described herein (e.g., size, polydispersity index value, organ selectivity, and encapsulation efficiency) without inducing the same immunogenic responses and without hindering cell-cellular interactions.

[0076] This article describes a method for producing these polyphosphate stabilizers. The polyphosphate stabilizers have a general structure of formula (I): (I) Where R is hydrogen, a targeting ligand, a hydrophilic group, an amphiphilic group, or a combination thereof; A, B, D, and E are each and independently C. 1-18 Heteroalkyl; C is C 1-50 Substituted or unsubstituted heteroalkyl, C 1-50 Unsaturated heteroalkyl groups, C 1-50 Charged heteroalkyl groups, C 1-50 Heterocyclic groups or combinations thereof; and x is an integer from 3 to 1000.

[0077] IV. Lipid Nanoparticle Composition In one aspect of the invention, the lipid nanoparticle composition comprises (a) an ionizable lipid; (b) two or more lipids; and (c) a stabilizer of formula (I): (I) Where R is Hydrogen, targeting ligand, hydrophilic group, amphiphilic group or combination thereof; A, B, D and E are each and independently C. 1-18 Heteroalkyl; C is C 1-50 Substituted or unsubstituted heteroalkyl, C 1-50 Unsaturated heteroalkyl groups, C 1-50 Charged heteroalkyl groups, C 1-50 Heterocyclic groups or combinations thereof; and x is an integer from 3 to 1000.

[0078] In some implementations, A, B, D, and E are each C 1-9 Heteroalkyl; C is C1-C 35 Substituted or unsubstituted heteroalkyl, C1-C 35 Unsaturated heteroalkyl groups, C1-C 35 Charged heteroalkyl groups, C1-C 35 Heterocyclic groups or combinations thereof; and x is an integer from 3 to 130. In some embodiments, C is C 32 Substituted or unsubstituted heteroalkyl, C 32 Unsaturated heteroalkyl groups, C 32 Charged heteroalkyl groups, C 32 Heterocyclic groups or combinations thereof. In some embodiments, C is C 30 Substituted or unsubstituted heteroalkyl, C 30 Unsaturated heteroalkyl groups, C 30 Charged heteroalkyl groups, C 30 Heterocyclic groups or combinations thereof.

[0079] In one aspect of the invention, the lipid nanoparticle composition comprises essentially the following: (a) ionizable lipids; (b) two or more lipids; and (c) a stabilizer of formula (I): (I).

[0080] "Substantially consisting of..." means any element listed after the phrase, and may include other additional elements, limited to those that do not interfere with or promote the activity or behavior of the listed elements as specified in this disclosure. Thus, the phrase "substantially consisting of..." indicates that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or behavior of the listed elements.

[0081] As used herein, the term "targeting ligand" can take the form of a portion of a molecule on the surface of a target cell (e.g., a cell within a target tissue of interest). In some embodiments, the targeting ligand is a peptide, antibody, sugar, oligosaccharide, aminoglycoside, sterol, phenylboronic acid, or a combination thereof.

[0082] In some embodiments of the invention, the hydrophilic group may be in the form of a molecule having a water-soluble portion that may carry a formal charge (ionic) or may be neutral (nonionic).

[0083] In some embodiments of the invention, the ionizable group may be in the form of a molecule, which may be in ionic form by its inherent chemical properties or by the medium in which it is present and / or by changes in the pH of the medium.

[0084] In some embodiments of the present invention, the amphiphilic group may be in the form of a molecule having both hydrophilic and hydrophobic properties.

[0085] As used herein, the term "alkyl group" or "alkyl" can take the form of a branched, straight (linear), or cyclic hydrocarbon group having a specified number of carbon atoms, typically from 1 to 18 or from 1 to 35 carbon atoms. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, etc. In some embodiments, the alkyl group may be substituted. A substituted alkyl group is an alkyl group in which at least one hydrogen atom of the alkyl group has been replaced by at least the following groups: a non-hydrogen group, such as a hydrocarbon group, a heteroatom or a group containing a heteroatom, such as a halogen (e.g., Br, Cl, F or I) or at least one functional group, such as -NR*2, -OR*, -SeR*, -TeR*, -PR*2, -AsR*2, -SbR*2, -SR*, -BR*2, -SiR*, -SiR*3, -GeR*, -GeR*3, -SnR*, -SnR*3, -PbR*3, etc., wherein each R* is independently a hydrocarbon group or a halocarbon group, and two or more R* may be linked together to form a substituted or unsubstituted saturated, partially unsaturated or aromatic cyclic or polycyclic ring structure, or wherein at least one heteroatom has been inserted into the hydrocarbon ring.

[0086] The term "branched alkyl" means that an alkyl group contains a tertiary carbon or a quaternary carbon (a tertiary carbon is a carbon atom bonded to three other carbon atoms; a quaternary carbon is a carbon atom bonded to four other carbon atoms). For example, 3,5,5-trimethylhexylphenyl is an alkyl group (hexyl) with three methyl branches (therefore, one tertiary carbon and one quaternary carbon) and is thus a branched alkyl group bonded to a phenyl group. Unless otherwise specified, branched alkyl groups include all of their isomers.

[0087] As used herein, the term “heteroalkyl group” or “heteroalkyl” may be in the form of an alkyl group as described herein, further wherein one or more carbon atoms (and any associated hydrogen atoms) are each independently replaced by the same or different heteroatom or heteroatom group.

[0088] For example, a heteroalkyl group may include 1, 2, 3, 4, 5, or 6 heteroatom groups, such as 1 heteroatom group. Heteroatoms include, but are not limited to, N, P, O, S, and combinations thereof. Heteroatomic groups include, but are not limited to, -NR-, -O-, -S-, -PH-, -P(O)2-, -S(O)-, -S(O)2-, etc., wherein R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or cycloheteroalkyl. The term "heteroalkyl" includes heterocycloalkyl (cyclic heteroalkyl), alkyl-heterocycloalkyl (linear or branched aliphatic groups attached to cyclic heteroalkyl), etc. Heteroalkyl groups include, but are not limited to, -OCH3, -CH2OCH3, -SCH3, -CH2SCH3, -NRCH3, -CH2NRCH3, etc., wherein R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may optionally be substituted. Heteroalkyl groups contain 1 to 10 carbon atoms and heteroatoms, for example, 1 to 6 carbon atoms and heteroatoms.

[0089] As used herein, the term "cycloalkyl group" or "cycloalkyl" is a subset of "alkyl" and can take the form of a saturated or partially saturated cyclic group having 3 to about 10 carbon atoms and no cyclic heteroatoms, having a single ring or multiple rings, including fused, bridged, and spirocyclic systems, having a specified number of carbon atoms, typically 1 to about 18 or 1 to about 35 carbon atoms. For polycyclic systems having aromatic and non-aromatic rings (without cyclic heteroatoms), the term "cycloalkyl" applies when the linking point is located on a non-aromatic carbon atom (e.g., 5,6,7,8-tetrahydronaphthalene-5-yl). The term "cycloalkyl" includes cycloalkenyl. Examples of cycloalkyl include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and cyclohexenyl.

[0090] As used herein, the term "heterocyclic" can take the form of a cycloalkyl group as described herein, containing 1 to 6 heteroatoms selected from N, O, S, or combinations thereof, with the remaining ring atoms being about 3 to about 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. A heterocyclic ring is a ring structure containing heteroatoms, as opposed to a heteroatom-substituted ring, in which hydrogen atoms on the ring atoms are replaced by heteroatoms. For example, tetrahydrofuran is a heterocyclic ring, while 4-N,N-dimethylamino-phenyl is a heteroatom-substituted ring.

[0091] Substituted heterocycles refer to heterocyclic groups in which at least one hydrogen atom of the heterocyclic group has been replaced by at least the following groups: non-hydrogen groups, such as hydrocarbon groups, heteroatoms or groups containing heteroatoms, such as halogens (e.g., Br, Cl, F or I) or at least one functional group, such as -NR*2, -OR*, -SeR*, -TeR*, -PR*2, -ASR*2, -SbR*2, -SR*, -BR*2, -SiR*, -SiR*3, -GeR*, -GeR*3, -SnR*, -SnR*3, -PbR*3, etc., wherein each R* is independently a hydrocarbon group or a halocarbon group.

[0092] In some implementations, C is .

[0093] In some implementations, R is hydrogen, , Peptides, antibodies, sugars, oligosaccharides, aminoglycosides, sterols, phenylboronic acid, or combinations thereof.

[0094] In some implementation schemes, B is , where m is an integer from 1 to 10, and n is an integer from 1 to 10.

[0095] In some embodiments, the lipid nanoparticle composition comprises two or more lipids, the lipids comprising structural lipids, sterols, or combinations thereof.

[0096] Any suitable ionizable lipid may be present in the lipid nanoparticle composition and the lipid nanoparticles. An ionizable lipid is a lipid that is cationic, or becomes ionizable (protonated) when the pH decreases below the pKa of the lipid's ionizable group, but is more neutral at higher pH values. At pH values ​​below the pKa, the lipid is capable of associating with negatively charged nucleic acids (e.g., oligonucleotides). Ionizable lipids include lipids that exhibit a positive charge when the pH decreases from physiological pH, or lipids that carry a net positive charge at a selected pH.

[0097] In some embodiments, the lipid nanoparticle composition or lipid nanoparticles comprise one or more ionizable lipids, such as two or more ionizable lipids, three or more ionizable lipids, or four or more ionizable lipids. In some embodiments, the ionizable lipid is DODMA (1,2-dioleoyloxy-3-dimethylaminopropane), DLin-MC3-DMA (O-(Z,Z,Z,Z-heptadec-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino)), DLin-KC2-DMA (2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane), BOCHD-C3-DMA (4-(dimethylamino)-,9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester), C12-200 (1,1′-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol), PNI 516 (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecane-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl4-(dimethylamino)butyrate, PNI 127(2R,3S,4R)-2-(((1,4-dimethylpiperidin-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl(9E,9'E,12E,12'E)-bis(octadecane-9,12-dienoate), PNI 550 3-(2-((1,17-bis(2-octylcyclopropyl)heptadecane-9-yl)oxy)-2-oxoethyl)cyclopentyl4-(dimethylamino)butyrate, PNI 580 (2S,3R,4R)-2-(((4-(dimethylamino)butyryl)oxy)methyl)tetrahydrofuran-3,4-diylbis(2-hexyldecanoate), PNI 659 ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl4-(dimethylamino)butanoate, PNI 728 ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl2-(dimethylamino)ethyl)carbamate, PNI 762 ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl(2-(diethylamino)ethyl)carbamate or combinations thereof. In some embodiments, the ionizable lipid is PNI 516, PNI127, PNI 550, PNI 580, PNI 659, PNI 728, PNI 762, or a combination thereof.

[0098] The ionizable lipids may be present in the lipid nanoparticle composition or lipid nanoparticles in any suitable amount or concentration. In some embodiments, the ionizable lipids are present at a concentration of about 20 to about 70 mol%, such as about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, or at a concentration within the range defined by any two of the foregoing values. In some cases, the ionizable lipids are present at a concentration greater than about 10 mol%, greater than about 12 mol%, greater than about 14 mol%, greater than about 16 mol%, greater than about 18 mol%, greater than about 20 mol%, greater than about 22 mol%, greater than about 24 mol%, greater than about 26 mol%, greater than about 28 mol%, or greater than about 30 mol%. In some cases, the ionizable lipids are present at concentrations of less than about 80 mol%, less than about 78 mol%, less than about 76 mol%, less than about 74 mol%, less than about 72 mol%, less than about 70 mol%, less than about 68 mol%, less than about 66 mol%, less than about 64 mol%, less than about 62 mol%, less than about 60 mol%, less than about 58 mol%, less than about 56 mol%, less than about 54 mol%, less than about 52 mol%, or less than about 50 mol%.

[0099] In some embodiments, the lipid nanoparticle composition comprises a structural lipid. Any suitable structural lipid may be present in the lipid nanoparticle composition and the lipid nanoparticles. The structural lipid or phospholipid supports particle formation during manufacturing. In various embodiments, the structural lipid comprises one or more neutral, positive, or negatively charged molecules. In some embodiments, the structural lipid has a net negative charge. In some embodiments, the structural lipid has a net neutral charge. In some embodiments, the structural lipid has a net positive charge.

[0100] In some embodiments, the lipid nanoparticle composition or lipid nanoparticles comprise one or more structural lipids, such as two or more structural lipids, three or more structural lipids, or four or more structural lipids. In some embodiments, the structural lipids comprise diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, or combinations thereof. In some embodiments, the structural lipids are distearylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine, palmitoyloleoyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, and dioleoylphosphatidylcholine. Myristoylphosphoethanolamine, distearyl-phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-methyl, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N,N-dimethyl, 1,2-ditransoleoyl-sn-glycerol-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, 1,2-ditransoleoyl-sn-glycerol-3-phosphoethanolamine, distearylphosphatidylcholine or combinations thereof.

[0101] In some embodiments, the structural lipid comprises any suitable lipid that is negatively charged (anionic) at physiological pH. In some embodiments, the structural lipid comprises dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol, cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, monosialotetrahexosylganglioside GM1, or combinations thereof. In some embodiments, the structural lipid is distearate phosphatidylcholine.

[0102] The structural lipids may be present in the lipid nanoparticle composition in any suitable amount. In some embodiments, the structural lipids are present in the lipid nanoparticle composition at a concentration of about 1 to about 25 mol%, for example, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, or about 25 mol%, or at a concentration within the range defined by any two of the foregoing values. In some cases, the structural lipids are present in the lipid nanoparticle composition at a concentration of less than about 1%. In some cases, the structural lipids are present in the lipid nanoparticle composition at concentrations exceeding about 20 mol%, exceeding about 21 mol%, exceeding about 22 mol%, exceeding about 23 mol%, exceeding about 24 mol%, exceeding about 25 mol%, exceeding about 26 mol%, exceeding about 27 mol%, exceeding about 28 mol%, exceeding about 29 mol%, exceeding about 30 mol%, exceeding about 31 mol%, exceeding about 32 mol%, exceeding about 33 mol%, exceeding about 34 mol%, or exceeding about 35 mol%.

[0103] In some embodiments, the lipid nanoparticle composition comprises a sterol. Any suitable sterol may be present in the lipid nanoparticle composition. In some embodiments, the lipid nanoparticle composition comprises one or more sterols, such as two or more, three or more, or four or more sterols. In some embodiments, the sterol is cholesterol, β-sitosterol, 20-α-hydroxysterol, phytosterol, or a combination thereof. In some embodiments, the sterol is cholesterol.

[0104] The sterol may be present in the lipid nanoparticle composition in any suitable amount. In some embodiments, the sterol is present in the lipid nanoparticle composition at a concentration of about 28 to about 50 mol%, for example, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, or about 50 mol%, or at a concentration within the range defined by any two of the foregoing values. In some cases, the sterol is present at concentrations exceeding about 18 mol%, exceeding about 20 mol%, exceeding about 22 mol%, exceeding about 24 mol%, exceeding about 26 mol%, exceeding about 28 mol%, or exceeding about 30 mol%. In some cases, the sterol is present at concentrations less than about 60 mol%, less than about 58 mol%, less than about 56 mol%, less than about 54 mol%, less than about 52 mol%, less than about 50 mol%, less than about 48 mol%, less than about 46 mol%, less than about 44 mol%, less than about 42 mol%, less than about 40 mol%, less than about 38 mol%, less than about 36 mol%, less than about 34 mol%, less than about 32 mol%, or less than about 30 mol%.

[0105] In some embodiments, the lipid nanoparticle composition comprises one or more stabilizers, such as two or more stabilizers, three or more stabilizers, or four or more stabilizers.

[0106] Any suitable stabilizer can be used in the lipid nanoparticle composition. In some embodiments, the stabilizer is SAF41, SAF44, SAF45, SAF54, SAF84, SAF85, SAF86, SAF87, SAF88, SAF130, SAF132, SAF160, SAF164, SAF165, SAF166, SAF197, SAF198, SAF200, SAF206, SAF207, or a combination thereof. Table 1 provides the structures of the selected stabilizers.

[0107] Table 1 The stabilizer may have any suitable molecular weight. In some embodiments, the stabilizer has a concentration of about 500 to about 50,000 Da, for example, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1000 Da, about 2000 Da, about 4000 Da, about 6000 Da, about 8000 Da, about 10000 Da, about 12000 Da, about 14000 Da, about 16000 Da, about 18000 Da, about 20000 Da, about 22000 Da, about 24000 Da, about 26000 Da, about 28000 Da, about 30000 Da, about 32000 Da, about 34000 Da, about 36000 Da, about 38000 Da, about 40000 Da, about 42000 Da, about 44000 Da, about 46000 Da, about 48000 Da. The molecular weight is approximately 50,000 Da or Da, or a range defined by any two of the foregoing values. In some cases, the stabilizer has a molecular weight of less than approximately 1,000 Da. In some cases, the stabilizer has a molecular weight greater than approximately 1,000 Da, approximately 2,000 Da, approximately 4,000 Da, approximately 6,000 Da, approximately 8,000 Da, or approximately 10,000 Da. In some cases, the stabilizer has a molecular weight of less than approximately 60,000 Da, approximately 58,000 Da, approximately 56,000 Da, approximately 54,000 Da, approximately 52,000 Da, approximately 50,000 Da, approximately 48,000 Da, approximately 46,000 Da, approximately 44,000 Da, approximately 42,000 Da, or approximately 40,000 Da.

[0108] The stabilizer may be present at any suitable concentration. In some cases, the stabilizer has a concentration of about 0.1 mol% to about 10 mol%, for example, about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1 mol%, about 1.2 mol%, about 1.4 mol%, about 1.6 mol%, about 1.8 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, about 7.5 mol%, about 8 mol%, about 8.5 mol%, about 3 mol%, about 9 mol%, about 9.5 mol%, about 10 mol%, or a concentration defined by a range of any two of the foregoing values. In some cases, the stabilizer has a concentration exceeding about 0.02 mol%, exceeding about 0.04 mol%, exceeding about 0.06 mol%, exceeding about 0.08 mol%, exceeding about 0.1 mol%, exceeding about 0.2 mol%, exceeding about 0.3 mol%, exceeding about 0.4 mol%, exceeding about 0.5 mol%, exceeding about 0.6 mol%, exceeding about 0.7 mol%, exceeding about 0.8 mol%, exceeding about 0.9 mol%, or exceeding about 1.0 mol%. In some cases, the stabilizer has a concentration less than about 20 mol%, less than about 18 mol%, less than about 16 mol%, less than about 14 mol%, less than about 12 mol%, less than about 10 mol%, less than about 9 mol%, less than about 8 mol%, less than about 7 mol%, less than about 6 mol%, less than about 5 mol%, less than about 4 mol%, or less than about 3 mol%.

[0109] In some embodiments, the lipid nanoparticle composition comprises a second stabilizer or a stabilizer. In some embodiments, the second stabilizer comprises two or more stabilizers, such as two or more stabilizers, three or more stabilizers, or four or more stabilizers. Any suitable second stabilizer can be used. In some embodiments, the second stabilizer is polysorbate, N-dodecyl β-D-maltodextrin, D-α-tocopherol polyethylene glycol 1000 succinate, or a combination thereof.

[0110] In some embodiments, the lipid nanoparticle composition comprises about 20 to about 70 mol% ionizable lipids, about 1 to about 25 mol% structured lipids, about 28 to about 50 mol% sterols, and about 0.1 to about 5 mol% stabilizers. In some embodiments, the lipid nanoparticle composition comprises about 47.5 mol% ionizable lipids, about 12.5 mol% structured lipids, about 38.5 mol% sterols, and about 1.5 mol% stabilizers. In some embodiments, the lipid nanoparticle composition comprises about 40 mol% ionizable lipids, about 12.5 mol% structured lipids, about 46 mol% sterols, and about 1.5 mol% stabilizers.

[0111] In some embodiments, the lipid nanoparticle composition is used to form lipid nanoparticles in embodiments of the methods described herein. In some embodiments, the diameter of the lipid nanoparticles is from about 15 nm to about 500 nm, for example, about 15 nm, about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, or about 500 nm, or a diameter defined by a range of any two of the foregoing values. Such a diameter can be used to improve the tissue targeting and biodistribution of the lipid nanoparticles. In some cases, the diameter of the lipid nanoparticles is greater than about 10 nm, greater than about 15 nm, greater than about 20 nm, greater than about 25 nm, greater than about 30 nm, greater than about 35 nm, greater than about 40 nm, or greater than about 45 nm. In other cases, the diameter of the lipid nanoparticles is less than about 700 nm, less than about 675 nm, less than about 650 nm, less than about 625 nm, less than about 600 nm, less than about 575 nm, less than about 550 nm, less than about 525 nm, less than about 500 nm, less than about 475 nm, less than about 450 nm, less than about 425 nm, less than about 400 nm, less than about 375 nm, less than about 350 nm, less than about 325 nm, or less than about 300 nm.

[0112] The embodiments of the lipid nanoparticles described herein can have any suitable polydispersity index. In some embodiments, the lipid nanoparticles have a polydispersity index of about 0.01 to about 0.40, for example, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2. A polydispersity index of 0, approximately 0.21, approximately 0.22, approximately 0.23, approximately 0.24, approximately 0.25, approximately 0.26, approximately 0.27, approximately 0.28, approximately 0.29, approximately 0.30, approximately 0.31, approximately 0.32, approximately 0.33, approximately 0.34, approximately 0.35, approximately 0.36, approximately 0.37, approximately 0.38, approximately 0.39, or approximately 0.40, or a polydispersity index defined by a range of any two of the foregoing values. The lipid nanoparticle embodiments described herein can have any suitable encapsulation efficiency. Encapsulation efficiency refers to the percentage of nucleic acids successfully captured in the lipid nanoparticles. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of about 50% to about 100%, such as about 50%, about 52%, about 54%, about 56%, about 58%, about 60%, about 62%, about 64%, about 66%, about 68%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, about 98%, or about 100%, or an encapsulation efficiency defined by a range of any two of the foregoing values.

[0113] The present invention also provides a method for preparing lipid nanoparticles as described herein, comprising (a) forming the lipid nanoparticle composition by combining predetermined amounts of ionizable lipids, structural lipids, sterols and stabilizers; (b) preparing lipid nanoparticles by combining the lipid nanoparticle composition and nucleic acids using a microfluidic mixer; and (c) purifying the lipid nanoparticles.

[0114] Any suitable mixing method can be used to combine specified amounts of ionizable lipids, structural lipids, sterols, stabilizers, and nucleic acids. In some embodiments, the ionizable lipids, structural lipids, sterols, and stabilizers are combined by mixing. In some embodiments, the mixing is performed using a microfluidic mixer. In some embodiments, the specified amounts of ionizable lipids, structural lipids, sterols, and stabilizers are as described herein. In some embodiments, the ionizable lipids, structural lipids, sterols, stabilizers, and nucleic acids are combined using standard T-tube mixing techniques, turbulent mixing, titration mixing, stirring to promote ordered self-assembly, or passive mixing of all components followed by self-assembly of the components into nanoparticles. Various methods have been developed to formulate lipid nanoparticles containing gene therapy drugs.

[0115] In some embodiments, a microfluidic mixing device is used, which may involve uniformly mixing two or more types of fluids together within a microfluidic chip, such as NanoAssemblr® Spark™, ​​NanoAssemblr® Ignite™, and NanoAssemblr® Blaze™, the NanoAssemblr® GMP system, and the NanoAssemblr® commercial formulation system. In some embodiments, lipid nanoparticles formed using a microfluidic mixing device have an encapsulation efficiency of approximately 90 to approximately 100%.

[0116] Any suitable method can be used to combine the lipid nanoparticle composition and the nucleic acid. In some embodiments, the lipid nanoparticle composition and the nucleic acid are combined by mixing. In some embodiments, the mixing is performed using a microfluidic mixer. In some embodiments, the microfluidic mixer includes first and second reagent streams fed into the microfluidic mixer, and the lipid nanoparticles are collected from the outlet.

[0117] In some embodiments, the first stream includes a payload in a first solvent. In some embodiments, the payload may include nucleic acids. In some cases, the payload may include a therapeutic agent. The combination of the payloads in the first solvent may be referred to as an aqueous phase. Any suitable first solvent may be used. A suitable first solvent includes a solvent in which the payload is soluble and which is miscible with a second solvent. In some embodiments, the first solvent comprises an aqueous buffer. In some embodiments, the aqueous buffer is a low-pH buffer. In some embodiments, the low-pH buffer is a citrate or acetate buffer.

[0118] In some embodiments, the second stream comprises an embodiment of a lipid nanoparticle composition as described herein in a second solvent. The combination of the lipid nanoparticle composition and the second solvent may be referred to as an organic phase. Any suitable second solvent may be used. Suitable second solvents include solvents in which the ionizable lipids according to embodiments of the invention are soluble and which are miscible with the first solvent. In some embodiments, the second solvent comprises one or more solvents, two or more solvents, three or more solvents, or four or more solvents. In some embodiments, the second solvent comprises 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, alcohols, or combinations thereof. In some embodiments, the second solvent comprises an aqueous or anhydrous alcohol. In some cases, the alcohol is a primary, secondary, or tertiary alcohol having 1-12 branched or unbranched carbon atoms (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol, 2-methyl-1-propanol, 2-butanol, 2-methylprop-2-ol).

[0119] In some embodiments, suitable devices for mixing include one or more microchannels (i.e., channels with a maximum size of less than 1 mm). In some embodiments, the microchannels have a diameter of about 20 to about 300 μm. In some embodiments, at least one region of the microchannel has a primary flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation forming an angle with the primary direction (e.g., an interlaced herringbone mixer or a bifurcated annular flow mixer). To achieve maximum mixing rate, it is advantageous to avoid excessive fluid resistance before the mixing region. In some embodiments, the device has non-microfluidic channels with a size greater than 1000 μm to deliver fluid to a single mixing channel.

[0120] Any suitable flow ratio can be used to combine the lipid nanoparticle composition and nucleic acid. In some embodiments, the lipid nanoparticle composition and nucleic acid are combined using a flow ratio of about 1:1 (or 1) to about 10:1 (or 10) by volume (aqueous phase: organic phase), for example, about 1, about 2, about 3, about 4, about 5, about 6, or about 7, about 8, about 9, about 10, or a flow ratio defined by any two of the foregoing values. In some cases, the flow ratio is greater than about 0.5. In some cases, the flow ratio is less than about 20, less than about 18, less than about 16, less than about 14, less than about 12, less than about 10, or less than about 8. Any suitable N / P ratio can be used to combine the lipid nanoparticle composition and nucleic acid. The N / P ratio is the ratio of positively charged polymeric amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. In some embodiments, the lipid nanoparticle composition and nucleic acid have an N / P ratio of about 2 to about 20, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20, or a combined N / P ratio defined by a range of any two of the foregoing values. In some cases, the N / P ratio is greater than about 1, greater than about 2, greater than about 3, greater than about 4, greater than about 5, greater than about 6, greater than about 7, greater than about 8, or greater than about 9. In some cases, the N / P ratio is less than about 40, less than about 38, less than about 36, less than about 34, less than about 32, less than about 30, less than about 28, less than about 26, less than about 24, less than about 22, less than about 20, less than about 18, less than about 16, less than about 14, less than about 12, or less than about 10. Any suitable total flow rate can be used to combine the lipid nanoparticle composition and nucleic acid.In some embodiments, the lipid nanoparticle composition and nucleic acid are used at a flow rate of about 2 to about 2000 mL / min, for example, about 2 mL / min, about 4 mL / min, about 6 mL / min, about 8 mL / min, about 10 mL / min, about 20 mL / min, about 40 mL / min, about 60 mL / min, about 80 mL / min, or about 100 mL / min, about 120 mL / min, about 140 mL / min, about 160 mL / min, about 180 mL / min, about 200 mL / min, about 220 mL / min, about 240 mL / min, about 260 mL / min, about 280 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, or about 500 mL / min, about 550 mL / min, about 600 mL / min, about 650 mL / min, about 700 mL / min, about 750 mL / min. The total flow rate of the organic and aqueous phases may be approximately 800 mL / min, 850 mL / min, 900 mL / min, 950 mL / min, 1000 mL / min, 1100 mL / min, 1200 mL / min, 1300 mL / min, 1400 mL / min, 1500 mL / min, 1600 mL / min, 1700 mL / min, 1800 mL / min, 1900 mL / min, or 2000 mL / min, or a combination of the total flow rates defined by any two of the foregoing values. In some cases, the total flow rate may be greater than approximately 1 mL / min, 2 mL / min, 4 mL / min, 6 mL / min, 8 mL / min, 10 mL / min, 20 mL / min, or 40 mL / min. In some cases, the total flow rate is less than about 3000 mL / min, less than about 2800 mL / min, less than about 2600 mL / min, less than about 2400 mL / min, less than about 2200 mL / min, less than about 2100 mL / min, less than about 2000 mL / min, less than about 1800 mL / min, less than about 1600 mL / min, less than about 1500 mL / min, less than about 1400 mL / min, less than about 1200 mL / min, less than about 1000 mL / min, or less than about 800 mL / min.In some embodiments, the lipid nanoparticle composition and nucleic acid are combined using a flow ratio of about 1:1 (or 1) to about 10:1 (or 10) (aqueous phase:organic phase) by volume, an N / P ratio of about 2 to about 20, and a total flow rate of about 2 to about 2000 mL / min. In some embodiments, the flow rate is 3 (aqueous phase:organic phase) optimized for a specific payload or molar ratio of lipid components.

[0121] Any suitable method can be used to purify lipid nanoparticles. In some implementations, purification is performed using a filter or centrifuge.

[0122] V. Usage Method In some embodiments, the payload is encapsulated in an exemplary lipid nanoparticle composition. Any suitable payload may be present in the lipid nanoparticle composition. The payload may include nucleic acids. In some cases, the payload may include therapeutic agents. Nucleic acids may be substances intended to have a direct effect in diagnosing, curing, alleviating, treating, or preventing disease, or in restoring, correcting, or altering physiological function, or serving as research reagents. Exemplary nucleic acids include any oligonucleotides or polynucleotides delivered to cells to elicit the desired effect. Nucleic acids may be single-stranded DNA or RNA, or double-stranded DNA or RNA, DNA-RNA hybrids, or combinations thereof. In some embodiments, the lipid nanoparticles contain one or more nucleic acids, two or more nucleic acids, three or more nucleic acids, or four or more nucleic acids. In some embodiments (e.g., gene editing), including more than one nucleic acid may be advantageous. In some embodiments, the nucleic acid is an antisense oligonucleotide, siRNA, miRNA, self-amplifying RNA (samRNA or saRNA), self-replicating DNA, LNA, DNA, replicon, mRNA, guide RNA, transposon, single gene, vector, plasmid, viral particle, AAV, RNA-RNA binding protein complex, or a combination thereof. In some embodiments, the nucleic acid is mRNA encoding an antigen.

[0123] In some embodiments, the lipid nanoparticle composition is a therapeutic composition, such as an mRNA-based therapeutic composition. The therapeutic composition may optionally include one or more therapeutically acceptable carriers, diluents, or excipients, such as salts, buffers, preservatives, anti-adhesives, antioxidants, binders, coating materials, tableting aids, disintegrants, dyes, softeners, emulsifiers, fillers, film-forming agents or coating materials, flavoring agents, fragrances, flow aids, lubricants, adsorbents, suspending agents or dispersants, sweeteners, hydration water, and / or other therapeutic agents. As used herein, the term "excipient" means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other component (and often a component of the delivery medium composition) other than the active pharmaceutical ingredient (API), appropriately selected with respect to the intended form of administration and consistent with conventional pharmaceutical practice. The disclosed compounds may be administered to a subject or patient in a therapeutically effective amount. The complex may be administered alone or as part of a pharmaceutically acceptable composition or formulation. Furthermore, the composition can be administered all at once (e.g., by bolus), administered multiple times, or delivered substantially consistently over a period of time. It should also be noted that the dosage of the compound can vary over time.

[0124] In some embodiments, the lipid nanoparticle composition may encapsulate mRNA encoding an antigen and be used as a vaccine. In some embodiments, the mRNA encoding an antigen is used in prophylactic or therapeutic vaccines. A vaccine may be referred to as a substance used to stimulate antibody production and provide immunity against one or more diseases, prepared from a pathogen of the disease, its products, or synthetic substitutes. A vaccine may further comprise one or more immunological adjuvants. As used herein, the term "immunological adjuvant" refers to a compound or mixture of compounds that, when used in conjunction with an immunogen (e.g., a neoantigen), is used to accelerate, prolong, enhance, or alter an immune response. When administered to a host alone, an adjuvant may be non-immunogenic, but when administered in combination with another antigen, it enhances the host's immune response to said antigen. Specifically, the terms "adjuvant" and "immunological adjuvant" are used interchangeably in the disclosure herein. Adjuvant-mediated enhancement and / or duration of the immune response can be evaluated by any method known in the art, including but not limited to one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with an adjuvant / antigen combination compared to the number of antibodies produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells recognizing the antigen or the adjuvant; and (iii) an increase in the level of one or more cytokines. Adjuvants can be aluminum-based adjuvants, including but not limited to aluminum hydroxide and aluminum phosphate; saponins, such as steroidal saponins and triterpenoid saponins; bacterial flagellins; and some cytokines, such as GM-CSF. Adjuvant selection may depend on the antigen, vaccine, and route of administration.

[0125] In some respects, adjuvants improve adaptive immune responses to vaccine antigens by modulating innate immunity or promoting transport and presentation. Adjuvants act directly or indirectly on antigen-presenting cells (APCs), including dendritic cells (DCs). Adjuvants can be ligands for Toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, preference, breadth, and extent of adaptive immunity. In other instances, adjuvants can signal via pro-inflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation. These adjuvants include inorganic salts, oil emulsions, nanoparticles, and polyelectrolytes, and comprise colloids and molecular assemblies exhibiting complex heterogeneous structures. In one example, the composition further comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant.

[0126] In some cases, the lipid nanoparticle compositions are used in gene therapy. Gene therapy is a medical technique that produces therapeutic effects by manipulating gene expression or by altering the biological properties of cells. In some cases, a gene encoding a therapeutic protein (for incorporation into host DNA) or mRNA encoding a therapeutic protein is administered to treat a disease caused by the loss of a protein and / or by the loss of activity of said protein. In some cases, novel genes or mRNAs are provided that enhance cellular function without altering the disease-causing gene. In other cases, antisense oligonucleotides (ASOs) or small interfering RNA (siRNAs) are used as therapeutic agents to silence the activity of variant proteins that cause disease.

[0127] Gene therapy can be performed at the somatic or germline cell level. It can be administered in vitro or in vivo. Gene therapy can be used through various gene editing technologies (e.g., CRISPR, homologous recombination, zinc finger nucleases, TALEN).

[0128] In some embodiments, nucleic acids are used for incorporation into immunogenic cells. In some embodiments, immunogenic cells include T cells. In some embodiments, immunogenic cells may be modified to express receptors for specific antigens or neoantigens, modified to enhance immunogenic responses or immunogenic cells, and modified to reduce proteins associated with adverse reactions such as neurotoxicity (e.g., reducing cytokines to improve the effects of cytokine release syndrome). In some embodiments, lipid nanoparticles are in an anhydrous form. In some embodiments, lipid nanoparticles are in an anhydrous form consisting of lyophilized cakes. In some embodiments, lipid nanoparticles are in a reconstituted form. In the reconstituted form, the lyophilized lipid nanoparticles may have a pharmaceutically acceptable carrier added to the lyophilized lipid nanoparticles.

[0129] In some implementations, the lipid nanoparticles and pharmaceutically acceptable carriers are pharmaceutical compositions. Examples of pharmaceutically acceptable carriers are provided herein.

[0130] abbreviations hEPO-mRNA: Human erythropoietin protein mRNA eGFP-mRNA: Enhanced green fluorescent protein mRNA saRNA: self-amplified mRNA sgRNA: guide RNA PCSK9 gRNA: PCSK9 gene guide RNA TCR sgRNA: T cell receptor single-guide RNA TTR gRNA: Transthyretin guide RNA SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus Type 2 FLuc-mRNA: Firefly luciferase protein mRNA eGFP: A basic (constitutive fluorescence) green fluorescent protein derived from the Victoria multituberculatus jellyfish. hEPO: Human erythropoietin h: hours HPLC: High Performance Liquid Chromatography MFI: Median fluorescence intensity min: minutes mL: milliliters mmol: millimole µL: microliters RT: Room temperature PBS: Phosphate-buffered saline PDI: Multidispersion Index wt: weight C: Celsius iL: Ionizable lipids IM: Intramuscular administration IV: Intravenous administration KO: knockout MFI: Median fluorescence intensity N / P: Nitrogen-to-Phosphorus ratio GPC conditions: Shodex KD-802 (300 x 8.0) mm, mobile phase: 0.1 mM LiBr in DMF (100% mobile phase), 60°C, diluent: 100% DMF, sample concentration: 10 mg / mL 1 H NMR (400 MHz, MeOD) PNI 516: (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecane-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl4-(dimethylamino)butyrate PNI 550: 3-(2-((1,17-bis(2-octylcyclopropyl)heptadecane-9-yl)oxy)-2-oxoethyl)cyclopentyl4-(dimethylamino)butyrate PNI 580: (2S,3R,4R)-2-(((4-(dimethylamino)butyryl)oxy)methyl)tetrahydrofuran-3,4-dimethylbis(2-hexyldecanoate) PNI 127: (2R,3S,4R)-2-(((1,4-dimethylpiperidin-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl(9E,9'E,12E,12'E)-bis(octadec-9,12-dienoate) PNI 659: ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 4-(dimethylamino)butyrate PNI 728: ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl-2-(dimethylamino)ethyl)carbamate PNI 762: ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl(2-(diethylamino)ethyl)carbamate V101: A cloning vector template for the synthesis of self-amplifying replicon DNA or RNA encoding the target gene (GOI): a parental VEEV TC83 replicon with a subgenomic promoter containing a multiple cloning site for insertion into any GOI. A5: Using the V101 vector, saRNA encoding the SARS-CoV-2 spike protein. PEG-DMG (Avanti Polar Lipids (Alabaster, AL); 2509 Da) The following examples further illustrate the invention, but should not be construed as limiting its scope in any way.

[0131] Example 1 This embodiment illustrates an illustrative synthetic route to obtain the lipid alcohol of compound 9, which is essential for the synthesis of the stabilizer. Starting with compounds 1 and 2, a six-step synthetic procedure is performed to obtain compound 9, which will then be used in the synthesis of the stabilizer.

[0132] Scheme 1. Synthetic procedure for lipid alcohol (compound 9) Synthesis of Compound 3: (2,2-dimethyl-1,3-dioxolane-4-yl)methanol (50.0 g, 0.378 mol) in THF (1000 mL) was added to a 2000-mL double-necked round-bottom flask under an argon atmosphere. 60% NaH (20.0 g, 0.833 mol) was added at 0°C and the mixture was stirred for 30 min. (Bromomethyl)benzene (77.72 g, 0.454 mol) was added dropwise at the same temperature, and the reaction mixture was stirred at RT for 16 h, with the reaction monitored by TLC. The reaction mixture was diluted with aqueous NH4Cl (500 mL) and extracted with EtOAc (2 x 600 mL), washed with brine (80 mL), and the organic layer was dried over Na2SO4 and evaporated on a rotary evaporator to give Compound 3 (90 g) as the crude product. The formation of the desired product (yellow liquid) was achieved by… 1 H NMR confirmed. 1 H NMR (CDCl3) 400 MHz: δ ppm: 1.36 (s, 3H,), 1.42 (s,3H), 3.45-3.57(m, 2H), 3.72-3.76 (m, 1H), 4.04-4.07 (m, 1H), 4.30 (m, 1H, J =6 Hz), 4.57 (d, 2H, J = 6.4 Hz), 7.29-7.35 (m, 5H).

[0133] Synthesis of Compound 4: 4-((benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (45.0 g, 202.44 mmol), water (62 mL), and acetic acid (250 mL) were added to a 250-mL double-necked round-bottom flask and stirred at RT for 16 h. The reaction progress was monitored by TLC. The reaction mixture was then concentrated on a rotary evaporator to give the crude compound. The crude compound was purified by CombiFlash using 2.0% MeOH in DCM as the eluent to give Compound 4 (30.0 g) (yield, 81.32%) as a brown liquid. The desired product was obtained by... 1 H NMR analysis and confirmation. 1 H NMR (CDCl3) 400 MHz: δppm: 3.56-3.65 (m, 3H,) 3.69-3.72 (dd, 1H, J1 = 11.4 Hz, J2 = 3.6Hz), 3.88-3.91 (m, 1H), 4.55 (br s, 2H), 7.30-7.36 (m, 5H).

[0134] Synthesis of Compound 5: 3-(benzyloxy)propane-1,2-diol (30.0 g, 164.83 mmol) dissolved in 450 mL toluene was added to a 1000-mL double-necked round-bottom flask under argon atmosphere at RT. Potassium tert-butoxide (74.0 g, 659 mmol) and bromotetradecane (137.0 g, 494.0 mmol) were added, and the reaction mixture was maintained at 110°C and stirred for 12 h. The reaction progress was monitored by TLC. After completion, the reaction mixture was cooled to RT, concentrated, and toluene was removed under reduced vacuum. 100 mL of water and 100 mL of ethyl acetate were added, and the organic layer was separated. The aqueous layer was washed again with ethyl acetate (2 x 250 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude compound was purified by rapid column chromatography by elution with 14% EtOAc in hexane to give compound 5 (60.0 g) (yield, 63.8%) as a grayish-white solid. 1 H NMR (CDCl3) 400 MHz: δ ppm: 0.88 (t, 6H, J = 6.8 Hz) 1.18-1.31 (m,45H), 1.51-1.58 (m, 4H), 3.42 (t, 2H, J = 6.4 Hz), 3.50-3.59 (m, 6H), 4.55(br s, 2H), 7.28-7.34(m, 5H).

[0135] Synthesis of Compound 6: 2,3-bis(tetradecyloxy)prop-1-ol (20 g, 34.782 g) in 250 mL ethanol and 4.5 g of 10% Pd / C were added to a 500 mL Parsley reactor. The reaction mixture was hydrogenated at 50 psi for 5 h. The reaction progress was monitored by TLC. The reaction mixture was filtered through a diatomaceous earth bed and washed with 200 mL of ethanol. The filtrate was concentrated under reduced pressure to give Compound 6 (15.0 g) (91% yield) as a brown liquid. The desired product was obtained by… 1 H NMR confirmed. 1 H NMR (CDCl3) 400 MHz: δ ppm: 0.88 (t, 6H, J = 6.8 Hz) 1.18-1.31 (m,46H), 1.51-1.60 (m, 4H), 3.42 (m, 1H), 3.43-3.59 (m, 6H), 3.61-3.63 (m, 3H).

[0136] Synthesis of Compound 8: 2,3-bis(tetradecyloxy)prop-1-ol (2.0 g, 4.12 mmol) and 3-(benzyloxy)propionic acid (0.817 g, 4.53 mmol) in DCM (40.0 mL) were added to a 100-mL double-necked round-bottom flask. DCC (0.936 g, 4.53 mmol) was added to this solution at 0°C, followed by DMAP (0.050 g, 0.41 mmol). The reaction mixture was then stirred at RT for 16 h. The reaction progress was monitored by TLC. The reaction mixture was diluted with DCM (40 mL) and filtered through diatomaceous earth. The filtrate was then washed with aqueous NaHCO3 (2 x 20 mL) and brine (20 mL), dried over Na2SO4, and then evaporated under reduced pressure on a rotary evaporator to give the crude compound. The crude compound was purified by CombiFlash using 5–10% EtOAc in hexane as the eluent to give 1.2 g (yield, 45%) as a brown solid. The desired product was then obtained and purified by… 1 H NMR confirmed. 1 H NMR (CDCl3) 400 MHz: δ ppm: 0.88 (t, 6H, J = 6.8Hz) 1.18-1.31 (m, 46H), 1.49-1.58(m, 4H), 2.64 (t, 2H, J = 6.4 Hz), 3.39-3.47(m, 4H), 3.53 (t, 2H, J=6.8 Hz), 3.59-3.64 (m, 1H), 3.75 (t, 2H, J = 6.4 Hz), 4.10-4.15 (m, 1H), 4.23-4.26 (dd, J1=4.4, J2=11.6), 4.53 (br s, 2H), 7.28-7.34 (m, 5H).

[0137] Synthesis of Compound 9: 2,3-bis(tetradecyloxy)propyl 3-(benzyloxy)propionate (1.0 g, 1.545 mmol) in 20 mL methanol and 10% Pd / C (0.325 g) were added to a 50-mL double-necked round-bottom flask, followed by hydrogenation at RT under 40 psi for 16 h using a Paasche shaker. The reaction progress was monitored by TLC. The reaction mixture was filtered through a diatomaceous earth bed, washed with methanol, and the filtrate was concentrated under reduced pressure. The crude compound was purified by CombiFlash column chromatography and eluted with 38% ethyl acetate in n-hexane to give 0.5 g (yield, 58.13 g) of Compound 9 as a brown solid. The desired product was obtained by… 1H NMR confirmed. 1 H NMR (CDCl3) 400 MHz: δ ppm: 0.88 (t, 6H, J = 6.8 Hz)1.18-1.40 (m, 46H), 1.49-1.58(m, 4H), 2.56-2.64 (m, 2H), 3.42-3.56 (m, 4H), 3.85-3.88 (m, 2H), 4.12-4.16 (m, 1H), 4.31-4.34 (m, 1H).

[0138] Example 2 This embodiment illustrates an illustrative synthetic route to obtain compound 12 (2-((2-methoxyethyl)amino)-1,3,2-dioxaphosphatane 2-oxide), which is the subunit required for the synthesis of the stabilizer.

[0139] Scheme 2. Synthetic procedure for cyclic monomers 2-Methoxyethylamine (3.35 mL, 38.596 mmol) and triethylamine (5.35 mL, 38.595 mmol) in 100 mL tetrahydrofuran were added to a 500 mL double-necked round-bottom flask. 2-Chloro-1,3,2-dioxaphosphatane 2-oxide (5.0 g, 35.087 mmol) dissolved in 25 mL THF was added dropwise, and the reaction mixture was stirred at RT for 16 h. The reaction progress was monitored by TLC. The reaction mixture was filtered to remove HCl salts. The filtrate was concentrated under reduced pressure. The crude compound was reprecipitated with DCM in diethyl ether to give 3.4 g (yield, 53.0%) of the desired viscous, clear liquid, which was obtained by... 1 H NMR confirmed. 1 H NMR (CDCl3) 400 MHz: δ ppm: 3.08-3.19 (m, 2H), 3.24 (br s,1H), 3.37 (br s, 3H), 3.45 (t, 2H, J = 5.2 Hz), 4.28-4.33(m, 2H), 4.41-4.45(m, 2H). 31 P NMR (CDCl3) 161.8 MHz: shows a single peak at 26.22 ppm.

[0140] Example 3 This example illustrates an illustrative synthetic route to obtain stabilizers SAF45, SAF85, SAF86, and SAF87. This synthesis requires the subunits prepared in Examples 1 and 2.

[0141] Scheme 3. General synthetic procedure for compounds SAF45, 85, 86 and 87 Compound 12,2-((2-methoxyethyl)amino)-1,3,2-dioxaphosphatane 2-oxide (0.500 g, 2.762 mmol) was placed in a Schlenk tube under a nitrogen atmosphere. Compound 9,2,3-bis(tetradecyloxy)propyl 3-hydroxypropionate (0.153 g, 276 mmol) in 3 mL of DCM and triazabicyclodecene (TBD) in 1.0 mL of DCM were added to the tube at °C. The reaction mixture was then stirred at 0 °C for 5 h. The reaction mixture was quenched with 2 mL of DCM and concentrated on a rotary evaporator. The crude compound was washed with diethyl ether, followed by dialyzing for 6 h and lyophilization. The desired product SAF 45 (0.300 g) was formed and obtained by... 1 HNMR and 31 PNMR confirms this. 1 H NMR (400 MHz, MeOD) δ0.80 (t, 6H), 1.3 (br, 48H), 1.55 (br, 4H), 1.95 (m, 6H), 2.5 (br, 18H), 3.1(br, 32H), 3.45 (br, 105H), 3.8 (br, 3H), 4.2 (br, 88H). 31 P NMR (CDCl3, 161.8MHz): δ 10.93 ppm. End-group analysis was used to obtain... 1 The molecular weight determined by ¹H NMR was 4358 Da. GPC analysis was performed (Shodex KD-802, 300 x 8.0 mm, mobile phase: 0.1 mM LiBr in DMF (100% mobile phase), 60°C, sample concentration: 10 mg / mL). The retention time was measured to be 8.7 min.

[0142] Compound 12,2-((2-methoxyethyl)amino)-1,3,2-dioxaphosphatane 2-oxide (1.383 g, 7.643 mmol) was placed in a Schlenk tube under a nitrogen atmosphere. Compound 9,2,3-bis(tetradecyloxy)propyl 3-hydroxypropionate (0.250 g, 0.449 mmol) in 6 mL DCM at 0°C and TBD (0.124 g, 0.899 mmol) in 2.0 mL DCM were added. The reaction mixture was then stirred at 0°C for 5 h. The reaction mixture was quenched with 5 mL DCM and concentrated on a rotary evaporator to obtain 0.880 g of crude product. The crude compound was washed with diethyl ether, followed by dialyzing 0.200 g of the crude compound from 0.880 g for 2 h and then lyophilized for 48 h. After lyophilization, the compound was a gel-like semi-solid of 0.120 g. The remaining compound was reprecipitated three times in diethyl ether (15 mL) with DCM (2 mL) to give 0.350 g of a colloidal semi-solid. The desired product SAF 85 (0.350 g) was formed and then... 1 H NMR (12 repeat units) and 31 PNMR confirms this. 1 H NMR (400MHz, MeOD) δ 0.80 (t, 6H), 1.3 (br, 45H), 1.6 (br, 4H), 1.95 (m, 4H), 2.7(br, 3H), 3.1 (br, 25H), 3.5 (br, 82H), 4.2 (br, 57H). 31 P NMR (CDCl3, 161.8MHz): δ 10.94 ppm. End-group analysis was used to obtain... 1 The molecular weight determined by 1H NMR was 2728 Da. GPC analysis was performed (Shodex KD-802, 300 x 8.0 mm, mobile phase: 0.1 mM LiBr in DMF (100% mobile phase), 60°C, sample concentration: 10 mg / mL). The retention time was measured to be 9.4 min.

[0143] By optimizing purification conditions (through longer dialysis), compound SAF 86 (0.120 g) was obtained from the synthetic route described herein and purified by [method name missing]. 1 H NMR (11 repeat units) and 31 PNMR confirms this. 1H NMR (400 MHz, MeOD) δ 0.80(t, 6H, br), 1.3 (br, 44H), 1.6 (br, 4H), 1.95 (m, 3H), 2.8 (br, 2H), 3.1(br, 22H), 3.5 (br, 32H), 3.8 (m, 6H), 4 (br, 6H), 4.2 (br, 46H). 31 P NMR (CDCl3, 161.8 MHz): δ 9.71 ppm. End-group analysis was used to obtain... 1 The molecular weight determined by ¹H NMR was 2547 Da. GPC analysis was performed (Shodex KD-802, 300 x 8.0 mm, mobile phase: 0.1 mM LiBr in DMF (100% mobile phase), 60°C, sample concentration: 10 mg / mL). The retention time was measured to be 9.4 min.

[0144] Compound 12,2-((2-methoxyethyl)amino)-1,3,2-dioxaphosphatane 2-oxide (0.500 g, 2.762 mmol) was placed in a Schlenk tube under a nitrogen atmosphere. Compound 9,2,3-bis(tetradecyloxy)propyl 3-hydroxypropionate (0.153 g, 276 mmol) in 3 mL of DCM was added at 0°C, followed by TBD (0.076 g, 0.550 mmol) in 1.0 mL of DCM. The reaction mixture was then stirred at 0°C for 5 h. The reaction mixture was quenched with 2 mL of DCM and concentrated on a rotary evaporator. The compound was dissolved in 3 mL of water and placed in a dialysis bag, then stirred in 200 mL of water for 2 h. The compound was then lyophilized. The desired product SAF 87 (0.400 g) (16 repeating units) was formed and obtained by... 1 H NMR and 31 P NMR confirmed. 1 H NMR (400 MHz, MeOD) δ 0.80 (t, 6H), 1.3 (br, 47H), 1.6 (br, 4H), 1.95 (m, 3H), 2.8 (br, 4H), 2.9 (m, 1H), 3.1 (br, 33H), 3.4 (m, 93H), 3.5 (br, 4H), 3.6 (m, 4H), 3.8 (m, 8H), 3.9 (m, 10H), 4.2 (br, 63H). 31PNMR (CDCl3, 161.8 MHz): δ 10.90 ppm. End-group analysis was used to obtain... 1 The molecular weight determined by 1H NMR was 3452 Da. GPC analysis was performed (Shodex KD-802, 300 x 8.0 mm, mobile phase: 0.1 mM LiBr in DMF (100% mobile phase), 60°C, sample concentration: 10 mg / mL). The retention time was measured to be 9.257 min.

[0145] Scheme 5. General synthetic procedure for compound SAF41 Synthesis of 2-ethoxy-1,3,2-dioxaphosphatane 2-oxide (cyclic monomer) Triethylamine (7.2 mL, 52.40 mmol) and ethanol (3.8 mL, 65.2 mmol) in THF (200 mL) were added to a 500 mL double-necked round-bottom flask under a nitrogen atmosphere at -78°C. 2-Chloro-1,3,2-dioxaphosphatane 2-oxide (4.0 mL, 43.5 mmol) in 70 mL THF was added dropwise and the mixture was stirred for 5 h. The reaction mixture was brought to -68°C and stirred at the same temperature for 2 h, then stirred at RT for 14 h. The reaction mixture was filtered through anhydrous THF to remove salts, and the filtrate was concentrated to give a pale yellow liquid. The crude compound was distilled under reduced pressure at 140°C to give 4.6 g (71.87%) of a colorless liquid. The desired product was formed and... 1 H NMR confirmed. 1 H NMR (CDCl3) 400 MHz: δppm: 1.38 (t, 3H, J = 7.2 Hz) 4.20-4.24 (m, 2H), 4.34-4.39 (m, 2H), 4.43-4.46(m, 2H). 31 P NMR (CDCl3 161.8 MHz) showed a single peak at 18.153 ppm.

[0146] Compound 2-ethoxy-1,3,2-dioxaphosphatrazine 2-oxide (0.410 g, 2.697 mmol) was placed in a Schlenk tube under an argon atmosphere. 2,3-bis(tetradecyloxy)propyl 3-hydroxypropionate (0.100 g, 0.179 mmol) in anhydrous DCM (4.5 mL) was added at RT. The reaction mixture was then stirred at 0°C. DBU (0.027 g, 0.179 mmol) in 0.5 mL of DCM was added, and the reaction mixture was stirred at 30°C for 16 h. The reaction mixture was quenched with 2 mL of DCM and concentrated on a rotary evaporator. The crude compound was washed with diethyl ether, dialyzed for 5 h, and then lyophilized. The desired product was obtained and obtained by... 1 H NMR and 31 P NMR and GPC confirmed the yield. Yield: 0.055 g. 1 H NMR(400 MHz, MeOD) δ 0.80 (t, 6H), 1.2 (br, 25H), 1.4 (br, 357H), 1.55 (br, 7H), 1.75 (br, 13H), 2.5 (br, 1H), 2.9 (br, 3H), 3.4 (br, 7H), 3.8 (br, 11H), 3.95 (br, 9H), 4.25 (br, 762H). 31 P NMR (CDCl3, 161.8 MHz): δ -0.67 ppm. End-group analysis was used to obtain... 1 The molecular weight determined by 1H NMR was 19,556 Da. GPC analysis was performed (Shodex KD-802, 300 x 8.0 mm, mobile phase: 0.1 mM LiBr in DMF (100% mobile phase), 60°C, sample concentration: 10 mg / mL). The retention time was measured to be 8.4 min.

[0147] The remaining SAF (x = 3 to 1000) can be synthesized in a similar manner by changing the ratio of compound 12 and compound 9.

[0148] Example 4 This example illustrates an illustrative method for preparing lipid nanoparticles.

[0149] The components of a lipid nanoparticle composition comprising ionizable lipids, structural lipids, sterols, and the stabilizer or control stabilizer (PEG-DMG) of this invention are mixed together at different molar ratios. The lipid nanoparticle composition is prepared in ethanol by incorporating prescribed amounts of lipids from individual lipid ethanol reservoirs. The lipid nanoparticle composition and nucleic acid are then used to prepare LNPs via a NanoAssemblr® Ignite™ microfluidic mixer. Using a microfluidic mixer from the NanoAssemblr® platform, a lipid nanoparticle (LNP) formulation is produced by mixing the lipid nanoparticle composition (PNI 516V02, with DSPC or DOPE as structural lipids; (lipid concentration: 25 mM)) with a nucleic acid (e.g., mRNA or saRNA) solution (100 mM acetate buffer; pH 4) at a 3:1 volume aqueous:organic solution ratio. The formulation is diluted with 25x PBS and then stored at 4°C for 30 minutes. To purify the LNPs, the formulation is centrifuged at 2200 x g for 45 minutes using UF Amicon tubes. Store EPOmRNA LNPs at 4°C until further use. Mix A5 saRNA LNPs with appropriate cryopreservation buffer and hold at -80°C until further use.

[0150] The mixing of nucleic acid and lipid nanoparticle compositions was performed as follows. Ionizable lipids, structural lipids, sterols, and stabilizers were mixed at a molar ratio of V02 or V62 in 100% ethanol (Table 3). An aqueous phase was prepared by diluting nucleic acids, such as mRNA / saRNA / pDNA solutions, in 100 mM sodium acetate buffer (pH 4). Solutions were combined using NanoAssemblr® Ignite™ with Ignite™ NxGen™ (DVBM) column cores at a flow ratio of 3:1 (aqueous phase:organic phase), an N / P ratio of 8 or 10, and a total flow rate of 12 mL / min unless otherwise noted. The resulting LNPs were diluted 25–40 times in 1xPBS (pH 7.4), and the mixture underwent downstream processing. Downstream processing included ethanol removal by dialysis in PBS (pH 7), or by using an Amicon™ centrifuge filter (Millipore, USA) at 2500 RPM, or by using a tangential flow filtration system. Concentrate the particles to the required target dose.

[0151] Example 5 This embodiment illustrates an illustrative method for measuring the size, polydispersity index (PDI), and encapsulation efficiency (EE) of lipid nanoparticles (LNPs).

[0152] The size and PDI of the LNPs were measured using a ZetaSizer™ Nano ZS™ (Malvern Instruments) via dynamic light scattering (DLS). A 633 nm He / Ne laser was used as the light source. Data were measured from scattering intensity data obtained in backscattering detection mode (measurement angle = 173°). Samples of 0.5–2 µL were placed in cuvettes and diluted with PBS (0.3 mL). Measurements were the average of 10 runs over two cycles for each sample. The Z-mean size is reported as the particle size and is defined as the harmonic mean particle size. The EE of the LNPs was measured using the Quant-iT™ RiboGreen® RNA reagent. These LNP characteristics, as well as the nucleic acid EE results of the LNPs in various lyophilization buffers (LB), are described in the following examples.

[0153] The size, PDI, and EE of lipid nanoparticles with polyphosphate and polyphosphoramide (amidide) stabilizers and different nucleic acid payloads were measured and are provided in Table 2. Lipid nanoparticle compositions used for EPO mRNA and A5 saRNA studies included PNI 516-VO2 (lipid concentration: 25 mM) (Table 3). Lipid nanoparticle compositions used for TTR sgRNA / Cas9 mRNA studies included PNI 516 V62 (lipid concentration: 25 mM). The N / P ratio for the EPO mRNA and A5 saRNA formulations was 8. The N / P ratio for the TTR sgRNA / Cas9 mRNA formulations was 6. The sgRNA:Cas9 mRNA ratio was 1:1 by weight. LNP size was measured using a ZetaSizer™ Nano ZS™ (Malvern Instruments, UK) via dynamic light scattering (DLS). EE measurements were performed using a modified Ribogreen® assay (Quanti-iT RiboGreen® RNA Assay Kit, Thermo Fisher).

[0154] Table 2. Physicochemical properties of LNPs formulated with different stabilizers in VO2 or V62 compositions using PNI 516 or PNI 580 for various nucleic acid payloads. Table 2 Table 3. Exemplary lipid nanoparticle compositions Example 6 This example illustrates an illustrative procedure for evaluating EPO expression in vivo using LNPs that express erythropoietin (EPO).

[0155] LNP was administered intravenously to mice (6-week-old female C57 BL6 mice) at a single dose of 0.25 mg / kg. Serum samples were collected 6 h post-injection via tail cutting. For serum preparation, after whole blood collection, the blood was allowed to clot by incubating the collection tube at room temperature for 15–30 minutes. The clot was removed by centrifuging the tube at 1000–2000 xg for 10 minutes at 4°C. The clear, golden-yellow supernatant was carefully removed and transferred on ice to sterile screw-cap clear polypropylene tubes. The serum was then stored at -80°C until further use. A final blood collection was performed 24 h post-injection. Erythropoietin (EPO) protein levels in the serum samples were determined using the Ella kit (ProteinSimple, Catalog # SPCKB-PS-000487). LNP containing SAF 41, 44, 85, and 87 showed effective EPO expression in mice 24 h post-administration. Figure 1A-1B The EPO activity in LNPs containing these SAFs is higher than that in LNPs containing PEG-DMG.

[0156] Example 7 This example shows an illustrative procedure for evaluating the expression of SARS-CoV-2 in vivo using A5 PNI saRNA-LNP for expressing SARS-CoV-2.

[0157] LNP was administered intramuscularly to four groups of mice (N = 4, 6-week-old male BALB / c mice) on day 0 at an initial dose of 0.05 mg / kg (1 µg / 50 µL / 20-g mice) and on day 28 at a booster dose of 0.05 mg / kg (1 µg / 50 µL / 20-g mice). Serum samples were collected on days 21 and 42. For serum preparation, after whole blood collection, the blood was allowed to clot by incubating the collection tubes at room temperature for 15–30 minutes. The clot was removed by centrifuging the tubes at 1000–2000 xg for 10 minutes at 4°C. The clear, golden-yellow supernatant was carefully removed and transferred on ice to sterile screw-cap clear polypropylene tubes. The serum was then stored at -80°C until further use. The SARS-CoV-2 antigen-specific IgG levels in the serum were determined using an enzyme-linked immunosorbent assay (ELISA). LNPs containing SAF 45 induce efficient expression of SARS-CoV-2 spike protein-specific IgG titers in mice. Figure 2A-2B(and enhance the response. LNPs containing SAF-45 induce SARS-CoV-2 spike protein-specific IgG expression comparable to LNPs containing PEG-DMG.)

[0158] Example 8 This example illustrates the physicochemical properties of the selected composition. The size and PDI of the LNP were measured using a ZetaSizer™ Nano ZS™ (Malvern Instruments) via dynamic light scattering (DLS). A 633 nm He / Ne laser was used as the light source. Data were obtained from scattering intensity measurements taken in backscattering detection mode (measurement angle = 173°). Measurements were averaged over 10 runs of two cycles for each sample. The Z-mean size is reported as the particle size and is defined as the harmonic mean particle size. The encapsulation efficiency (EE) of the LNP was measured using the Quant-iT™ RiboGreen® RNA reagent.

[0159] Table 4. Properties of various LNPs formed based on polyphosphate stabilizers Example 9 This example illustrates an in vitro T-cell knockout study. Cryopreserved primary human pan-T cells were thawed, activated with a CD3 / CD28 / CD2 T-cell activator, and cultured for 3 days in a 96-well plate (37°C, 95% humidity, and 5% CO2, 0.2 mL / well, 0.125 million cells / well). Cells were then treated with LNP loaded with TCR sgRNA / Cas9 mRNA (4 μg / well) for 24 h, followed by washing to remove the activator. The washed cells were then incubated for 3 days in complete medium (T-cell expansion medium containing IL-2) before staining and flow cytometry analysis. For cell staining, cells were stained with anti-CD3 (PE conjugate), anti-TCR α / β (FITC conjugate), and FVS660 dye (APC conjugate) according to the manufacturer's protocol. The number of live cells and TCRα / β+ cells was calculated using CytExpert software, and the T cell receptor (TCR) knockout efficiency (%) of the LNP samples was calculated and displayed. Figure 3 middle.

[0160] Example 10 This example illustrates an in vitro study of GFP expression. BHK cells were cultured and seeded (96-well plates, 0.1 mL / well, 12,000 cells / well). Cells were then treated with LNP loaded with GFP mRNA for 24 h, followed by analysis using fluorescence microscopy. Results showed… Figure 4 middle.

[0161] Example 11 This example illustrates an in vitro GFP expression study on T cells. Cryopreserved primary human pan-T cells were thawed, activated with CD3 / CD28 / CD2 T cell activators, and cultured in a 96-well plate (37°C, 95% humidity, and 5% CO2) for 3 days (0.2 mL / well, 0.125 million cells / well). Cells were then treated with GFP mRNA-loaded LNP (4 μg / well) for 24 h, followed by staining and flow cytometry analysis. For cell staining, cells were stained with FVS660 dye (BDBiosciences) according to the manufacturer's protocol. Data were analyzed using CytExpert software, and the transfection efficiency (TE) % and mean fluorescence intensity (MFI) of the LNP samples were calculated and displayed. Figures 5A-5B middle.

[0162] Example 12 This example illustrates an in vivo vaccine study. This study describes the procedure for evaluating SARS-CoV-2 expression in vivo using A5 pNIsaRNA-LNP for expressing SARS-CoV-2. LNP was administered intramuscularly to mice (6-week-old male BALB / c mice) at a primary immunization dose of 0.05 mg / kg (1 µg / 20-g mouse) on day 0 and at a booster dose of 0.05 mg / kg (1 µg / 20-g mouse) on day 28 (7 days after the first serum collection). Serum samples were collected on days 21 and 42 post-primary immunization. For serum preparation, after collecting whole blood, the blood was allowed to clot by incubating the collection tube at room temperature for 15–30 minutes. The clot was removed by centrifuging the tube at 1000–2000 xg for 10 minutes at 4°C. The clear, golden-yellow supernatant was carefully removed and transferred on ice to sterile screw-cap clear polypropylene tubes. The serum was then stored at -80°C until further use. The level of SARS-CoV-2 antigen-specific IgG in serum was measured and displayed using an enzyme-linked immunosorbent assay (ELISA). Figures 6A-6B middle.

[0163] Example 13 This example illustrates an in vivo protein substitution study. This study describes the procedure for evaluating EPO expression in vivo using LNP, which expresses erythropoietin (EPO). LNP was administered intravenously at a single dose of 0.25 mg / kg to mice (6-week-old female C57 BL6 mice). Serum samples were collected 6 h post-injection using the tail-cutting method. For serum preparation, after collecting whole blood, the blood was allowed to clot by incubating the collection tube at room temperature for 15–30 minutes. The clot was removed by centrifuging the tube at 1000–2000 xg for 10 minutes at 4°C. The clear, golden-yellow supernatant was carefully collected and transferred on ice to sterile screw-cap clear polypropylene tubes. The serum was then stored at -80°C until further use. A final blood collection was performed 24 h post-injection. Erythropoietin (EPO) protein levels in the serum samples were determined using the Ella kit (ProteinSimple, Catalog # SPCKB-PS-000487) and displayed. Figures 7A-7B middle.

[0164] Example 14 This example illustrates an in vivo subcutaneous study. This study describes the procedure for evaluating EPO expression in vivo using LNP, which expresses erythropoietin (EPO). LNP was subcutaneously injected into mice (6-week-old female C57 BL6 mice) at a single dose of 0.25 mg / kg. Serum samples were collected 6 hours post-injection using the tail-cutting method. For serum preparation, after collecting whole blood, the blood was allowed to clot by placing the collection tube at room temperature for 15–30 minutes. The clot was removed by centrifuging the tube at 1000–2000 xg for 10 minutes at 4°C. The clear, golden-yellow supernatant was carefully removed and transferred on ice to sterile screw-cap clear polypropylene tubes. The serum was then stored at -80°C until further use. A final blood collection was performed 24 hours post-injection. Erythropoietin (EPO) protein levels in the serum samples were determined and displayed using the Ella kit (ProteinSimple, Catalog #SPCKB-PS-000487). Figure 8 middle.

[0165] Example 15 This example illustrates an in vivo biodistribution (BD) study. This study describes the procedure for evaluating the in vivo expression of Fluc by LNP, which expresses firefly luciferase (Fluc). LNP was administered intravenously at a single dose of 0.1 mg / kg to mice (6-week-old female Hsd:IRC mice). Four hours after administration, mice were given an intraperitoneal injection of D-luciferin substrate solution (150 mg / kg, 200 µL / 20 g), followed by isoflurane anesthesia and imaging 12 minutes after administration for a 30 s exposure. After imaging, blood and organs (liver, lung, spleen, kidney, heart) were collected, rinsed with PBS, and imaged ex vivo for 1 s, 5 s, 10 s, and 30 s exposures. Liver and blood (500 µL) were imaged separately. Bioluminescence values ​​were quantified by measuring photon flux (photons / second) for both in vivo and ex vivo imaging using the Living Image® software program. Figure 9 Polyphosphates showed higher extrahepatic selectivity than PEG-DMG.

[0166] All references cited in this article, including publications, patent applications and patents, are incorporated into this article by reference to the same extent that each reference is individually and specifically indicated to be incorporated by reference and to be presented in its entirety in this article.

[0167] This document describes preferred embodiments of the invention, including the best modes known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors intend that those skilled in the art will use such variations where appropriate, and the inventors intend that the invention be practiced in ways other than those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the appended claims, as permitted by applicable law. Furthermore, the invention includes any combination of all possible variations of the foregoing elements, unless otherwise specified herein or otherwise obviously contradicted by the context.

Claims

1. A stabilizer comprising a structure of Formula (I): Formula (I). (I) wherein R is hydrogen, a targeting ligand, a hydrophilic group, an amphiphilic group, or a combination thereof; A, B, D, and E are each separately and independently C 1-18 substituted or unsubstituted heteroalkyl, C 1-50 substituted or unsubstituted heteroalkyl, C 1-50 substituted or unsubstituted heteroalkyl, C 1-50 substituted or unsubstituted heteroalkyl, C 1-50 heterocyclyl, or a combination thereof; and x is an integer from 3 to 1000.

2. The stabilizing agent of claim 1, wherein A, B, D, and E are each and independently of the other C 1-9 heteroalkyl; C is C1-C 35 substituted or unsubstituted heteroalkyl, C1-C 35 unsaturated heteroalkyl, C1-C 35 charged heteroalkyl, C1-C 35 heterocyclyl, or combinations thereof; and x is an integer from 3 to 130.

3. The stabilizer of claim 1 or 2, wherein C is 。 4. The stabilizer of any one of claims 1-3, wherein R is hydrogen, , a peptide, an antibody, a sugar, an oligosaccharide, an aminoglycoside, a sterol, a phenylboronic acid, or a combination thereof.

5. The stabilizer of any one of claims 1-4, wherein B is m is an integer from 1 to 10, and n is an integer from 1 to 10.

6. The stabilizer of any one of claims 1-5, wherein the stabilizer is selected from SAF41, SAF44, SAF45, SAF54, SAF84, SAF 85, SAF 86, SAF 87, SAF 88, SAF130, SAF132, SAF160, SAF164, SAF165, SAF166, SAF197, SAF198, SAF200, SAF206, SAF207, or a combination thereof.

7. The stabilizer of any one of claims 1-6, wherein the stabilizer is used to stabilize a lipid nanoparticle or a liposome.

8. A lipid nanoparticle composition comprising: (a) an ionizable lipid; (b) two or more lipids; and (c) the stabilizer of any one of claims 1-7.

9. The lipid nanoparticle composition of claim 8, wherein the two or more lipids comprise a structural lipid, a sterol, or a combination thereof.

10. The lipid nanoparticle composition of claim 8 or 9, consisting essentially of: (a) an ionizable lipid; (b) two lipids; and (c) the stabilizer of any one of claims 1-7.

11. The lipid nanoparticle composition of claim 10, wherein the lipid nanoparticle composition is substantially free of PEG or PEG-R, wherein R is any atom or molecule covalently linked to PEG.

12. The lipid nanoparticle composition of any one of claims 9-11, wherein the structural lipid is neutral charged, positively charged, or negatively charged.

13. The lipid nanoparticle composition of any one of claims 8-12, wherein the ionizable lipid is DODMA, DLin-MC3-DMA, DLin-KC2-DMA, BOCHD-C3-DMA, C12-200, PNI 516, PNI 127, PNI 550, PNI 580, PNI 659, PNI 728, PNI 762, or a combination thereof.

14. The lipid nanoparticle composition of any one of claims 8-13, wherein the structural lipid comprises a diacyl phosphatidylcholine, a diacyl phosphatidylethanolamine, a diacyl phosphatidylglycerol, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a plasmalogen, a cerebroside, or a combination thereof.

15. The lipid nanoparticle composition of any one of claims 8-14, wherein the structural lipid comprises distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine, palmitoyloleo ylphosphatidylcholine, l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, palmitoyloleoyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)- cyclohexane-l-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, distearoyl-phosphatidylethanolamine, l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-methyl, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N,N- dimethyl, l,2-ditallolyl-sn-glycero-3-phosphoethanolamine, l-stearoyl-2-oleoyl- phosphatidylethanolamine, l,2-ditallolyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol, cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, monosialoganglioside GM1, or a combination thereof.

16. The lipid nanoparticle composition of any one of claims 8-15, wherein the solid sterol comprises cholesterol, beta-sitosterol, 20-alpha-hydroxysterol, phytosterol, or a combination thereof.

17. The lipid nanoparticle composition of any one of claims 8-16, wherein the stabilizer has a molecular weight of about 500 Da to about 50,000 Da.

18. The lipid nanoparticle composition of any one of claims 8-17, further comprising a second stabilizer, wherein the second stabilizer is a polysorbate, N-dodecyl beta-D-maltoside, D-alpha- tocopheryl polyethylene glycol 1000 succinate, or a combination thereof.

19. The lipid nanoparticle composition of any one of claims 8-18, wherein the lipid nanoparticle composition comprises about 20 to about 70 mol% ionizable lipid, about 1 to about 25 mol% structural lipid, about 28 to about 50 mol% solid sterol, and about 0.1 to about 5 mol% stabilizer.

20. A lipid nanoparticle comprising the lipid nanoparticle composition of any one of claims 8-19 and a nucleic acid.

21. The lipid nanoparticle of claim 20, wherein the nucleic acid is encapsulated by the lipid nanoparticle composition.

22. The lipid nanoparticle of claim 20 or 21, wherein the nucleic acid is an antisense oligonucleotide, siRNA, miRNA, self-amplifying RNA (SAM or saRNA), self-replicating DNA, LNA, DNA, replicon, mRNA, guide RNA, transposon, single gene, vector, plasmid, viral particle, AAV, complex of RNA with RNA binding protein, or a combination thereof.

23. The lipid nanoparticle of any one of claims 20-22, wherein the nucleic acid is an antigen-encoding mRNA for a prophylactic or therapeutic vaccine, a nucleic acid for gene therapy, or a nucleic acid for immunogenic cell incorporation, wherein the immunogenic cell is a T cell.

24. The lipid nanoparticle of any one of claims 20-23, wherein the lipid nanoparticle diameter is about 15 nm to about 500 nm.

25. The lipid nanoparticle of any one of claims 20-24, wherein the lipid nanoparticle has a polydispersity index of about 0.01 to about 0.

40.

26. The lipid nanoparticle of any one of claims 20-25, wherein the lipid nanoparticle has an encapsulation efficiency of about 50% to about 100%.

27. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of claims 20-26 and a pharmaceutically acceptable carrier.

28. A method for making the lipid nanoparticle of any one of claims 20-26 or the pharmaceutical composition of claim 27, the method comprising: (i) forming a lipid nanoparticle composition by combining an ionizable lipid, a structural lipid, a sterol, and a stabilizer; (ii) preparing a lipid nanoparticle by combining the lipid nanoparticle composition and a nucleic acid using a microfluidic mixer; and (iii) purifying the lipid nanoparticle.

29. The method of claim 28, wherein the lipid nanoparticle composition and the nucleic acid are combined using a flow ratio of about 1 : 1 to about 10: 1 (aqueous phase: organic phase) by volume, an N / P ratio of about 2 to about 20, and a total flow rate of about 2 to about 2000 mL / min.

30. The method of claim 29, wherein the aqueous phase comprises a low pH buffer.

31. The method of claim 29 or 30, wherein the aqueous phase comprises a citrate or acetate buffer.

32. The method of any one of claims 29-31, wherein the organic phase comprises 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, an acid, an alcohol, or a combination thereof.

33. The method of any one of claims 29-32, wherein the organic phase is an alcohol, and the alcohol comprises an aqueous or anhydrous alcohol, wherein the alcohol is a primary, secondary, or tertiary alcohol having 1-12 branched or unbranched carbons.

34. Use of the lipid nanoparticle of any one of claims 20-26 or the pharmaceutical composition of claim 27 for preventing, treating, or ameliorating a condition or disease, comprising administering the lipid nanoparticle as a vaccine or as a treatment to prevent or lessen the severity of an infectious disease, administering the lipid nanoparticle as a gene therapy, or administering the lipid nanoparticle to an immunogenic cell to treat cancer or an infection. ​

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