Toll-like receptor (TLR) agonist-based lipid compounds, lipid nanoparticles (LNP) comprising same, and methods of use thereof

Abstract

The present disclosure relates to lipid-like compounds comprising toll-like receptor (TLR) agonists, lipid nanoparticles (LNP) comprising the same, and methods of use thereof. In certain embodiments, the LNPs described herein can be used to enhance the therapeutic and / or prophylactic effect of a vaccine composition.

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 509,452, filed June 21, 2023, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference. Statement regarding federally funded research or development This invention was completed with government funding from the TR002776 project granted by the National Institutes of Health in the United States. The government holds certain rights to this invention. Background Technology

[0002] The novel coronavirus disease caused by SARS-CoV-2 has triggered a global health crisis, resulting in millions of deaths and injuries. Vaccines have proven to be a crucial tool in reducing the morbidity and mortality rates of this infectious disease. Currently, various vaccine formulations, including mRNA vaccines, adenoviruses expressing antigens, inactivated viruses, and subunit vaccines, have been approved or are in clinical development. Among these, mRNA vaccines have demonstrated exceptional ability to induce neutralizing antibodies (NAb) and T-cell responses against multiple SARS-CoV-2 variants, and also offer low production costs and short development and manufacturing cycles.

[0003] Although mRNA was discovered as early as 1961, its vaccine development has long been hampered by its susceptibility to enzymatic degradation, low in vivo delivery efficiency, and high innate immunogenicity, with breakthroughs only recently occurring. The first two challenges have been overcome by incorporating mRNA into delivery systems that protect it from degradation and escort it across various biological barriers. Notably, four-component lipid nanoparticles (LNPs) containing ionizable lipids (or lipid-like substances), phospholipids, PEGylated lipids, and cholesterol represent the most advanced mRNA delivery platform in clinical practice, exemplified by Pfizer / BioNTech's BNT162b2 vaccine and Moderna's mRNA-1273 vaccine. The final challenge was addressed by incorporating naturally occurring nucleosides (such as 1-methylpseudouracil nucleoside (m1ψ)) into the in vitro transcribed mRNA sequence to prevent it from being recognized by pattern recognition receptors for innate immunity.

[0004] Although nucleoside-modified mRNAs have improved tolerance and translation, their use has largely impaired the innate immune response and weakened the activation of dendritic cells (DCs), both of which are essential for generating strong adaptive immunity through vaccination.

[0005] Therefore, there is a need in the art for LNP compositions that enhance adjuvant activity and methods of using them. This disclosure fulfills this need. Summary of the Invention

[0006] In one aspect, this disclosure provides a compound of formula (I) or a salt, solvate, stereoisomer, or isotope sequence thereof, wherein R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d The definition can be found in other parts of this article: (I).

[0007] In some embodiments, the compound of formula (I) is the compound of formula (Ia), or a salt, solvate, stereoisomer, or isotopic configuration thereof, wherein R 1 R 2 R 3a R 3b R 4a R 4b R 4c R 4d R 6a R 6b The definitions of L and L can be found in other parts of this article: (Ia).

[0008] In some embodiments, the compound of formula (I) is (C12-TLRa): (C12-TLRa).

[0009] In some embodiments, the compound of formula (I) is (O12-TLRa): (O12-TLRa).

[0010] On the other hand, this disclosure provides a lipid nanoparticle (LNP) comprising: (a) At least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent; (b) at least one ionizable lipid; (c) at least one accessory lipid; (d) cholesterol; and (e) At least one polymer-conjugated lipid.

[0011] On the other hand, this disclosure provides a pharmaceutical composition comprising the lipid nanoparticles (LNPs) of this disclosure and a pharmaceutically acceptable carrier.

[0012] On the other hand, this disclosure provides a method for generating an innate immune response in a subject, the method comprising administering to the subject the lipid nanoparticles (LNPs) of this disclosure or the pharmaceutical composition of this disclosure.

[0013] On the other hand, a method for treating, preventing, and / or improving an infection, disease, or disorder in a subject, the method comprising administering lipid nanoparticles (LNPs) of the present disclosure to the subject, wherein the LNPs comprise at least one cargo molecule. Attached Figure Description

[0014] The accompanying drawings illustrate various embodiments of this application by way of example rather than limitation.

[0015] Figure 1 A schematic diagram depicting an adjuvant lipid-substituted SARS-CoV-2 mRNA-LNP vaccine and its proposed mechanism of action for inducing strong adaptive immunity is provided. In one non-limiting embodiment, an exemplary SARS-CoV-2 mRNA-LNP vaccine is formulated with adjuvant lipids and / or additional lipids, DOPE, DMG-PEG, and cholesterol, as well as m1ψ-modified SARS-CoV-2 mRNA encoding a diproline-modified spike glycoprotein. After injection, the vaccine is internalized by dendritic cells (DCs), where the adjuvant lipids stimulate endosomally localized TLR7 / 8 to activate DCs, and the mRNA is translated into spike antigen, which is processed and presented by the DCs. After migrating to draining lymph nodes, the DCs interact with CD4+. + T cells, CD8 + T cells and B cells work together to coordinate a robust adaptive immune response, including neutralizing antibody (NAb) responses and Th1-biased CD4+ responses. + and CD8 + T cell response, B cell response, and long-lived plasma cell (LLPC) response.

[0016] Figure 2A-2I The synthesis and characterization of exemplary lipids and LNPs are described. Figure 2A The adjuvant lipid C12-TLRa was synthesized via a ring-opening reaction between TLR7 / 8 agonist 1 and C12 epoxide. Figure 2B C12-TLRa LNP-mediated mLuc delivery (n=3). DC2.4 cells were treated for 24 hours with mLuc-loaded C12-TLRa LNP (10-40 ng / well) or free mRNA (40 ng / well). Figure 2C Representative synthesis of polyamines and polyamine-derived lipids C12-113. Figure 2DComparison of C12-TLRa and polyamine-derived lipids in in vitro mLuc delivery (n=3). DC2.4 cells were treated for 24 h with different mLuc-loaded LNPs (10 ng / well) or free mRNA (10 ng / well). Figure 2E Optimization of C12-113 / TLRa LNP formulation (n=3). DC2.4 cells were treated with C12-113 / TLRa LNP loaded with mLuc (10 ng / well) for 24 h, wherein the C12-TLRa substitution rate was 1-17.5 mol%. Figure 2F TLR7 agonistic activity of LNPs was measured in HEK blue reporter cells (n=2). Cells were treated with different concentrations of LNP mRNA for 24 hours. Figure 2G Representative transmission electron microscopy (TEM) image of C12-113 / TLRa LNP. Scale bar, 100 nm. Data are expressed as mean ± SD. Figure 2H-2I Synthesis and characterization of adjuvant lipid O12 TLRa.

[0017] Figures 3A-3H The adjuvant lipid-substituted LNP-mediated in vitro mRNA delivery and DC activation were described. Figures 3A-3B Dose-dependent mLuc delivery; DC2.4 cells ( Figure 3A ) and BMDC ( Figure 3B Treat with a C12-113 LNP or C12-113 / TLRa LNP loaded with mLuc for 24 hours. Figure 3C Representative confocal images of cellular uptake. DC2.4 cells were treated with DiO-labeled LNPs for 2 hours, followed by staining with LysoTracker Deep Red and Hoechst 33342. Scale bar, 20 μm. Figure 3D A schematic diagram is provided to describe the proposed mechanism by which C12-113 / TLRa LNPs enhance endosome escape. The agonist-receptor interaction between C12 TLRa and TLR7 / 8 enhances LNP-mediated endosome disruption. Figure 3E-3F Flow cytometry analysis of mature DC cells. DC2.4 cells ( Figure 3E ) and BMDC ( Figure 3F The cells were treated with LNP (500 ng / mL) loaded with SARS-CoV-2 mRNA for 24 hours, and then stained with the DC maturation markers CD80 and CD86. Figure 3G-3H ELISA analysis of pro-inflammatory cytokines (TNF-α, IL-12p70, and IL-1β). DC2.4 cells ( Figure 3G ) and BMDC ( Figure 3HPatients were treated with LNP (500 ng / mL) loaded with SARS-CoV-2 mRNA for 24 hours, followed by ELISA analysis of cytokine production. Data are presented as mean ± SD (n=3).

[0018] Figures 4A-4H This study depicts adjuvant lipid-substituted LNP-mediated in vivo mRNA delivery and innate immune responses. Figure 4A In vivo bioluminescence imaging at 6 and 24 hours post-treatment with mLuc-loaded LNPs. Mice were subcutaneously injected with mLuc-loaded LNPs (5 μg mRNA / mouse) at the base of the tail. The injection site and the total flux of the two iLNs were quantified. Dashed circles indicate iLNs. Figure 4B In vitro fluorescence and luminescence imaging. Twenty-four hours before euthanasia, mice were subcutaneously injected with DiR-labeled LNPs loaded with mLuc (5 μg mRNA / mouse) at the base of the tail. Major organs and iLNs were collected for in vitro imaging. Figure 4C :( Figure 4B Quantification of fluorescence and luminescence signals in ). Figure 4D-4E Flow cytometry analysis of mature DCs in iLNs. Two iLNs were collected from each mouse 24 hours after injection of LNPs loaded with SARS-CoV-2 mRNA and processed to generate single-cell suspensions stained with CD11c, CD80, and CD86. Figure 4F-4H ELISA analysis of intranodal pro-inflammatory cytokine production. Six or 24 hours after injection of LNPs loaded with SARS-CoV-2 mRNA, two iLNs were collected from each mouse and processed to generate a single-cell suspension. This suspension was cultured for 8 hours, and the supernatant was then collected for TNF-α ( Figure 4F ), IL-12p70 Figure 4G ) and IL-1β( Figure 4H ELISA analysis of the samples. Data are expressed as mean ± SD (n=3).

[0019] Figures 5A-5D Cellular immune responses induced by SARS-CoV-2 mRNA-LNP vaccines with non-limiting exemplary adjuvant lipid substitution are depicted. Figure 5A Schematic diagram of the primary and booster vaccination strategies and analysis of T-cell responses. C57BL / 6J mice were subcutaneously immunized twice on days 0 and 21 with 5 μg of C12-113 mRNA-LNP or C12-113 / TLRa mRNA-LNP vaccine. Figures 5B-5C RBD-specific CD4 + and CD8 +Flow cytometry analysis of T cell responses. On day 35, spleens were harvested and processed to generate single-cell suspensions stimulated with the SARS-CoV-2 RBD peptide pool. T cells were stained for intracellular cytokine expression markers CD107α, Th1 (IFN-γ, IL-2, and TNF-α), Th2 (IL-4, IL-5), and Th17 (IL-17a). Figure 5D Multi-functional CD4 + and CD8 + T cells. Data are presented as mean ± SD (n=4).

[0020] Figures 6A-6H Humoral immune responses induced by SARS-CoV-2 mRNA-LNP vaccines with non-limiting exemplary adjuvant lipids were described. Figure 6A A schematic diagram of the primary and booster vaccination strategies and an analysis of humoral immune responses. C57BL / 6J mice were subcutaneously immunized twice with 5 μg of SARS-CoV-2 mRNA-LNP vaccine on days 0 and 21. Figure 6B RBD-specific IgG titer (n=7). Serum was collected from inoculated mice on day 35, and RBD-specific IgG levels were determined by endpoint dilution ELISA. Figure 6C Neutralizing antibody (NAb) titers (n=5). Serum was collected from inoculated mice on day 35, and NAb levels were measured by a VSV-based pseudovirus neutralization assay. Figure 6D The number of RBD-specific B cells (n=4) was determined. On day 35, spleens were harvested and processed to generate single-cell suspensions stimulated with the SARS-CoV-2 RBD peptide pool. Allotype conversion (IgD) was analyzed by flow cytometry. - IgM - RBD-specific B cells. Figure 6E Number of RBD-specific B cells classified by germinal center (GC) or memory phenotype (n=4). GC B cells are defined as CD38 - GL7 + Memory B cells are defined as CD38. + GL7 - Subgrouping was performed using PD-L2 and CD80 expression. Figure 6F-6H Quantitative analysis of RBD-specific IgG1, IgG2b, and IgG2c antibody-secreting cells (ASCs) in bone marrow (BM) using ELISPOT (n=3). Data are expressed as mean ± SD.

[0021] Figures 7A-7F The interaction between the agonist and TLR7 was described. Figure 7A2D binding posture of TLR7 / 8 agonist 1. Multiple interactions between TLR7 / 8 agonist 1 and TLR7 were observed at the first binding site of TLR7. Figure 7B : 3D binding posture of TLR7 / 8 agonist 1. Figure 7C Front (left) and side (right) views of TLR7 / 8 agonist 1-bound TLR7 dimer. The TLR7 dimer and its dimerizing partner are green and cyan, respectively. The carbon atoms of TLR7 / 8 agonist 1 are black. Figure 7D 2D binding posture of C12-TLRa. Multiple interactions between C12-TLRa and TLR7 were observed at the first binding site of TLR7, which is consistent with... Figure 7A Very similar. Figure 7E : 3D combined pose of C12-TLRa. Figure 7F Front (left) and side (right) views of the C12-TLRa-bound TLR7 dimer. The TLR7 dimer and its dimerizing partner are green and cyan, respectively. The C atoms of C12-TLRa are black.

[0022] Figure 8 TLR7 agonist activity of C12-TLRa was assessed by HEK-mTLR7 cell assay. The image above is a photograph of a 96-well plate. Data are presented as mean ± SD (n=3).

[0023] Figure 9 TNF-α production in LNP-treated DC2.4 cells. 10,000 cells per well were treated with LNP loaded with mRNA for 24 hours. The supernatant was collected for ELISA analysis. Data are presented as mean ± SD (n=3).

[0024] Figure 10 TLR7 agonistic activity of MC3 LNP and MC3 / TLRa LNP was measured in HEK blue reporter cells. Cells were treated with different concentrations of LNP mRNA for 24 hours. Data are presented as mean ± SD (n=3).

[0025] Figure 11 Representative TEM image of C12-113 LNP. Scale bar, 100 nm.

[0026] Figure 12A-12B It describes the cell survival rate. Figure 12A Cell viability of DC2.4 cells after 24 hours of treatment with LNP loaded with mLuc. Figure 12B Cell viability of BMDCs after treatment with mLuc-loaded LNPs for 24 hours. Data are expressed as mean ± SD (n=3).

[0027] Figure 13 Dose-dependent mLuc delivery using non-limiting exemplary LNPs of this disclosure is depicted. DC2.4 cells were treated with MC3 LNPs or MC3 / TLRa LNPs loaded with mLuc for 24 hours. Data are presented as mean ± SD (n=3).

[0028] Figures 14A-14B The gating strategy of a mature DC is described. Figure 14A CD80 + CD86 + Gating strategy for DC2.4 cells. DC2.4 cells were treated with LNP loaded with SARS-CoV-2 mRNA (500 ng / mL) for 24 hours and then stained with DC maturation markers CD80 and CD86. Figure 14B CD80 + CD86 + Gating strategy of BMDCs. BMDCs were treated with LNPs loaded with SARS-CoV-2 mRNA (500 ng / mL) for 24 hours, and then stained with DC surface marker CD11c and DC maturation markers CD80 and CD86.

[0029] Figure 15 In vivo and in vitro chemiluminescence imaging was depicted 24 hours after injection of the non-limiting exemplary LNP of this disclosure. LNP loaded with mLuc (5 μg mRNA / mouse) was subcutaneously injected into the tail root of mice. The interval between different batches exceeded one month. Figures 4A-4B The first batch of LNPs was used. C12-113 / TLRa LNP consistently outperformed C12-113 LNP in terms of injection site and iLN transfection across different LNP batches.

[0030] Figures 16A-16B The in vivo expression kinetics of mLuc-LNP after subcutaneous injection at the tail root were described. Figure 16A Continuous in vivo luminescence imaging of mice treated with C12-113 LNP (left) or C12-113 / TLRa LNP (right) at a dose of 5 μg mRNA per mouse. Figure 16B Quantification of total flux at the tail root. Data are expressed as mean ± SD (n=3).

[0031] Figure 17 The gating strategy of mature dendritic cells (DCs) within lymphatic vessels was depicted. Twenty-four hours after injection of LNPs loaded with SARS-CoV-2 mRNA (5 μg mRNA / mouse), two intracellular LNs (iLNs) were collected from each mouse and processed to generate single-cell suspensions, which were stained with the DC surface marker CD11c and DC maturation markers CD80 and CD86. CD80... + CD11c +and CD86 + CD11c + Cells are gated.

[0032] Figures 18A-18C Provides a description of TNF-α ( Figure 18A ), IL12p70 ( Figure 18B ) and IL-1β( Figure 18C A graph showing serum pro-inflammatory cytokine levels. Serum from immunized mice was collected 6 or 24 hours after injection of LNP loaded with SARS-CoV-2 mRNA (5 μg mRNA / mouse). Data are expressed as mean ± SD (n=3).

[0033] Figure 19 Representative photographs of the immunized mice are provided. C57BL / 6J mice were subcutaneously immunized twice on days 0 and 21 with either 5 μg of C12-113 mRNA-LNP or C12-113 / TLRa mRNA-LNP vaccine. No abnormalities in the appearance of the skin at the base of the tail (such as rashes and lesions) were observed after the primary and booster vaccinations with either LNP.

[0034] Figure 20 The changes in body weight observed in the exemplary mouse study are depicted. C57BL / 6J mice were subcutaneously immunized twice on days 0 and 21 with either 5 μg of C12-113 mRNA-LNP or C12-113 / TLRa mRNA-LNP vaccine. Arrows indicate the day of immunization. No weight loss was observed in any group. Data are presented as mean ± SD (n=4).

[0035] Figures 21A-21C Gating strategies of spleen T cells. Figure 21A Used to identify CD4 + and CD8 + A schematic diagram of T-cell gating. Figure 21B : Identification of CD4 cells expressing antigen-specific cytokines + T cells. Figure 21C : Identification of CD8 cells expressing antigen-specific cytokines + T cells.

[0036] Figure 22 A subset of RBD-specific IgG and IgG2c / IgG1 ratios were characterized. C57BL / 6J mice were subcutaneously immunized twice on days 0 and 21 with 5 μg of SARS-CoV-2 mRNA-LNP vaccine. Serum was collected from immunized mice on day 35, and RBD-specific IgG1 and IgG2 were analyzed by ELISA (n = 2–4).

[0037] Figures 23A-23C: Gating strategies of spleen B cells. (A) Used to identify allotype switching (IgD) - IgM - (A) Schematic diagram of splenic B cell gating. (B) IgD identification using biotinylated protein probes. - IgM - RBD binds to B cells. (C) In total IgD - IgM - and IgD - IgM - RBD + Identification of germinal centers (CD38) in B cells - GL7 + ) and memory phenotype (CD38) + GL7 - Subgroups. Cells with the memory phenotype were further subdivided based on the expression of the memory markers PD-L2 and CD80. Detailed Implementation

[0038] Reference will now be made to certain embodiments of the disclosed subject matter, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the exemplary subject matter is not intended to limit the claims to the disclosed subject matter.

[0039] In this document, values ​​expressed in range format should be interpreted flexibly, including not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges contained within the range, as if each value and subrange were explicitly listed. For example, the range “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted as including not only about 0.1% to about 5%, but also the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. Unless otherwise stated, the expression “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise stated, the expression “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0040] In this document, unless the context clearly specifies otherwise, the terms “a,” “an,” or “the” are used to include one or more. Unless otherwise stated, the term “or” is used to refer to a non-exclusive “or.” The expressions “at least one of A and B” or “at least one of A or B” have the same meaning as “A, B, or A and B.” Furthermore, it should be understood that the wording or terminology used herein (unless otherwise defined) is descriptive and not restrictive. The use of any section headings is for the purpose of aiding reading the document and should not be construed as restrictive; information relating to a section heading may appear within or outside that particular section. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference.

[0041] In the methods described herein, actions may be performed in any order unless the timing or sequence of operations is explicitly stated. Furthermore, specific actions may be performed simultaneously unless the explicit language of the claims states that they may be performed separately. For example, the claimed action of doing X and the claimed action of doing Y may be performed simultaneously in a single operation, and the resulting process will fall within the literal scope of the claimed method.

[0042] describe Lipid nanoparticle (LNP)-formulated mRNA vaccines represent a promising tool for preventing infectious diseases, as evidenced by the recent success of the SARS-CoV-2 mRNA vaccine. To avoid immune stimulation and uncontrolled inflammation, immunosuppressive nucleoside-modified mRNAs are preferred. However, this modification largely negates the innate immune response, which is crucial for coordinating robust adaptive immunity.

[0043] In one aspect, this disclosure describes the development of a novel LNP component (i.e., adjuvant lipid) that can enhance the adjuvant properties of LNP vaccines (such as SARS-CoV-2 mRNA LNP vaccines), and LNPs containing the component.

[0044] This disclosure demonstrates that partially replacing ionizable lipids with adjuvant lipids in LNP formulations not only enhances mRNA delivery but also confers Toll-like receptor 7 / 8 agonist activity to LNPs. This significantly improves innate immunity to mRNA LNP vaccines without causing significant adverse reactions. Furthermore, the vaccine elicited broad-spectrum neutralizing antibodies against multiple SARS-CoV-2 pseudovirus variants, strong Th1-biased cellular immunity, and robust B-cell and long-lived plasma cell responses. In summary, this disclosure describes a novel and safe LNP formulation paradigm for enhancing the immunogenicity of mRNA vaccines.

[0045] definition The term “about” as used herein may allow for the degree of variability of a value or range, such as within 10%, 5%, or 1% of the limit of the value or range, and may include the exact value or range.

[0046] As used herein, the term "alkenyl" refers to straight-chain, branched, and cycloalkyl groups as defined herein, except that there is at least one double bond between two carbon atoms. Thus, alkenyl groups have 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms, or in some embodiments 2 to 8 carbon atoms. Examples include, but are not limited to, vinyl, -CH=C=CCH2, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, etc.

[0047] As used herein, the term "alkoxy" refers to an oxygen atom bonded to an alkyl group, including cycloalkyl groups as defined herein. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and so on. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and so on. Examples of cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, and so on. An alkoxy group may include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to an oxygen atom, and may further include double or triple bonds, and may also include heteroatoms. For example, allyloxy or methoxyethoxy are also alkoxy groups as understood herein, as are methylenedioxy groups where two adjacent atoms of the structure are substituted with them.

[0048] As used herein, the term "alkyl" refers to straight-chain and branched alkyl and cycloalkyl groups having 1 to 40 carbon atoms, 1 to 20 carbon atoms, 1 to 12 carbon atoms, or, in some embodiments, 1 to 8 carbon atoms. Examples of straight-chain alkyl groups include those having 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl. As used herein, the term "alkyl" includes n-alkyl, isoalkyl, and trans-isoalkyl groups, as well as other branched forms of alkyl. Representative substituted alkyl groups may be substituted once or more with any of the groups listed herein, such as amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen.

[0049] As used herein, the term "alkynyl" refers to straight-chain and branched alkyl groups, where at least one triple bond exists between two carbon atoms. Therefore, an alkynyl group has 2 to 40 carbon atoms, 2 to approximately 20 carbon atoms, or 2 to 12 carbon atoms, or in some embodiments, 2 to 8 carbon atoms. Examples include, but are not limited to, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3), etc.

[0050] As used herein, the term "alkylene" or "alkylenyl" refers to a divalent saturated aliphatic group (e.g., -CH2-, -CH2CH2-, and -CH2CH2CH2-, etc.). In some embodiments, the term may be considered as a moiety derived from alkenes by opening a double bond, or from alkanes by removing two hydrogen atoms from the same (e.g., -CH2-) or different (e.g., -CH2CH2-) carbon atoms. Similarly, as used herein, the terms "heteroalkylenyl," "cycloalkylenyl," "heterocycloalkylenyl," etc., refer to a divalent group corresponding to a base group (e.g., heteroalkyl, cycloalkyl, and / or heterocycloalkyl). A divalent group has two open valences at any position of the group, where each group can be located on a carbon atom or a heteroatom. Thus, a divalent group can form a single bond with two different atoms or groups, or a double bond with one atom.

[0051] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an adaptive immune response. This immune response may involve the production of antibodies, or the activation of specific immunogenic cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. Those skilled in the art will understand that any DNA or RNA containing a nucleotide sequence or a portion thereof encoding a protein that elicits an adaptive immune response therefore encodes the term "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is apparent that antigens can be produced, synthesized, or derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0052] As used herein, the term "amine" refers to primary, secondary, and tertiary amines having, for example, the formula N(group)3, wherein each group may be independently H or non-H, such as alkyl, aryl, etc. Amines include, but are not limited to, R-NH2, such as alkylamines, aromatic amines, and alkylaromatic amines; R2NH, wherein each R is independently selected, such as dialkylamines, diarylamines, arylalkylamines, heterocyclic amines, etc.; and R3N, wherein each R is independently selected, such as trialkylamines, dialkylaromatic amines, alkyldiaromatic amines, triaromatic amines, etc. The term "amine" also includes ammonium ions as used herein.

[0053] The term "amino group" used in this article refers to -NH2, -NHR, -NR2, and -NR3. + Substituents of the form, wherein each R is independently chosen, and each has a protonated form (but -NR3). + Except that it cannot be protonated. Therefore, any compound substituted with an amino group can be considered an amine. The term "amino" as used herein can refer to a primary, secondary, tertiary, or quaternary amino group. The "alkylamino" group includes monoalkylamino, dialkylamino, and trialkylamino groups.

[0054] The term "anionic lipid" refers to any lipid that carries a negative charge at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphonic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and palmitoylphosphatidylglycerol (POPG), as well as other anionic modifying groups that bind to neutral lipids.

[0055] As used herein, the term "aryl" refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in its ring. Therefore, aryl includes, but is not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrene, triphenylenyl, pyrene, napthacenyl, chrysenyl, biphenylene, anthracene, and naphthyl. In some embodiments, the aryl group contains about 6 to about 14 carbons in the cyclic portion of the group. The aryl group can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be monosubstituted or substituted more than once, such as, but not limited to, phenyl groups substituted at any one or more positions at the 2-, 3-, 4-, 5-, or 6-positions of the benzene ring, or naphthyl groups substituted at any one or more positions at the 2- to 8-positions.

[0056] As used herein, the terms “aryl-(C1-C6)alkyl” or “aralkyl” refer to a functional group in which one to six carbon-alkylene chains are attached to an aryl group, such as -CH2CH2-phenyl or -CH2-phenyl (or benzyl). Specific examples are aryl-CH2- and aryl-CH(CH3)-. The term “substituted aryl-(C1-C6)alkyl” refers to an aryl-(C1-C6)alkyl functional group in which the aryl group has been substituted. A specific example is substituted aryl (CH2)-. Similarly, the term “heteroaryl-(C1-C6)alkyl” refers to a functional group in which one to three carbon-alkylene chains are attached to a heteroaryl group, such as -CH2CH2-pyridyl. A specific example is heteroaryl-(CH2)-. The terms "substituted heteroaryl-(C1-C6)alkyl" or "heteroaralkyl" refer to heteroaryl-(C1-C6)alkyl functional groups in which the heteroaryl group has been substituted. A specific example is substituted heteroaryl-(CH2)-.

[0057] The term "cationic lipid" refers to any of many lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH around 7.0). Cationic lipids comprising alkyl chains having multiple unsaturated sites (e.g., at least two or three unsaturated sites) have been found to be particularly useful for forming lipid particles with increased membrane fluidity. U.S. Patent Publications 20060083780 and 20060240554; U.S. Patents 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992; and PCT Publications WO1996 / 010390, WO2021077066, WO2021077067, WO2023015200, WO2023056282, and WO2023056418 describe numerous cationic lipids and related analogues that are also useful in this disclosure, the entire contents of which are incorporated herein by reference for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, cationic lipids include a protonable tertiary amine (e.g., titratable pH) head group, C 18 The lipids consist of alkyl chains, head groups, ether bonds between alkyl chains, and 0 to 3 double bonds. Examples of such lipids include, for instance, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0058] As used herein, the term "conjugated lipid" refers to a lipid conjugated to one or more polymer groups that inhibits the aggregation of lipid particles. Such lipid conjugations include, but are not limited to, polyamide oligomers (e.g., ATTA lipid conjugations), PEG lipid conjugations such as PEG coupled with dialkoxypropyl, PEG coupled with diacylglycerol, PEG coupled with cholesterol, PEG coupled with phosphatidylethanolamine, PEG conjugated with ceramide (e.g., U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to lipids or linked to lipids via a linker portion. Any linker portion suitable for coupling PEG to lipids can be used, including, for example, ester-free and ester-containing linker portions. In a preferred embodiment, an ester-free linker portion is used.

[0059] As used herein, the term "cycloalkyl" refers to a cycloalkyl group, such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the cycloalkyl group may have 3 to about 8-12 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups, such as, but not limited to, norbornel, adamantyl, bornel, camphenyl, isocamphenyl, and carenyl, and fused rings, such as, but not limited to, decalinyl, etc. Cycloalkyl groups also include rings substituted with straight-chain or branched alkyl groups as defined herein. Representative substituted cycloalkyl groups may be mono- or poly-substituted, such as, but not limited to, 2,2-, 2,3-, 2,4-, 2,5-, or 2,6-disubstituted cyclohexyl groups or mono-, di-, or tri-substituted norbornyl or cycloheptyl groups, which may be substituted, for example, amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination refers to cyclic alkenyl groups.

[0060] "Disease" is a state of health in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease is not improved.

[0061] In comparison, an animal's "disorder" is a state of health in which the animal maintains homeostasis, but its health is not as good as when it is not in a disorder. Without treatment, a disorder does not necessarily lead to a further decline in the animal's health.

[0062] The disease or disorder is "alleviated" if the severity of the symptoms, the frequency with which the patient experiences such symptoms, or both decrease.

[0063] As used herein, the terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount" refer to a non-toxic but sufficient quantity of an agent to provide the desired biological outcome. This outcome may be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired biological systemic change. The appropriate therapeutic amount in any individual case can be determined by a person skilled in the art using routine laboratory procedures.

[0064] Specifically, in the case of mRNA, the “effective amount” or “therapeutic effective amount” of the therapeutic nucleic acid associated with the mRNA is an amount sufficient to produce the desired effect, for example, an amount of protein in which the mRNA is directed to express a protein that causes the desired biological effect in an organism in which the protein is expressed. For example, in some embodiments, the expressed protein is the active form of a protein normally expressed in cell types in vivo, and the therapeutic effective amount of mRNA is an amount that produces an amount of the protein encoding the protein that is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of protein normally expressed in cell types of healthy individuals. For example, in some embodiments, the expressed protein is a protein normally expressed in cell types in vivo, and the therapeutic effective amount of mRNA is an amount that produces a similar level of expression in individuals with abnormal expression of the protein (i.e., protein-deficient individuals) as observed in healthy individuals. Suitable analyses for measuring the expression of mRNA or protein include, but are not limited to, dot blot hybridization, Northern hybridization, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic analysis known to those skilled in the art.

[0065] As used herein, the term "encode" refers to a product (e.g., protein and RNA) specified by a given nucleotide sequence in a nucleic acid (i.e., DNA and / or RNA) during transcription or translation of DNA or RNA, respectively. In some embodiments, the term "encode" refers to an RNA sequence specified by transcription of a DNA sequence. In some embodiments, the term "encode" refers to an amino acid sequence (e.g., polypeptide or protein) specified by translation of mRNA. In some embodiments, the term "encode" refers to an amino acid sequence specified by transcription of DNA into mRNA and subsequent translation of mRNA encoded by the DNA sequence. In some embodiments, the encoded product may include a direct transcription or translation product. In some embodiments, the encoded product may include post-translational modifications understood or reasonably expected by those skilled in the art.

[0066] The term "fully encapsulated" means that the active agent or therapeutic agent in the lipid particles is not significantly degraded after exposure to serum or by nucleases or proteases that would significantly degrade free DNA, RNA, or proteins. In a fully encapsulated system, in a treatment that would typically degrade 100% of the free active agent or therapeutic agent, preferably less than about 25% of the active agent or therapeutic agent in the particles is degraded, more preferably less than about 10%, and most preferably less than about 5% of the active agent or therapeutic agent in the particles is degraded. In the context of nucleic acid therapeutics, full encapsulation can be determined by OLIGREEN® assay. OLIGREEN® is a highly sensitive fluorescent nucleic acid staining agent for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen, Carlsbad, California). "Fully encapsulated" also means that the lipid particles are serum stable, i.e., they do not rapidly degrade into their component parts upon administration in vivo.

[0067] Unless otherwise stated, the terms “halo,” “halogen,” or “halide” as used herein refer to a fluorine, chlorine, bromine, or iodine atom, either on its own or as part of another substituent.

[0068] As used herein, the term "haloalkyl" includes monohaloalkyl, polyhaloalkyl in which all halogen atoms may be the same or different, and perhaloalkyl in which all hydrogen atoms are replaced by halogen atoms (such as fluorine). Examples of haloalkyl groups include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and so on.

[0069] As used herein, the term "helper lipid" refers to lipids that enhance the effectiveness of delivering lipid-based particles (such as cationic lipid-based particles) to a target site, preferably to cells. Helper lipids can be neutral, positively charged, or negatively charged. In some embodiments, the helper lipid is neutral or negatively charged. Non-limiting examples of helper lipids include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC).

[0070] Unless otherwise stated, the term "heteroalkyl" as used alone or in combination with another term herein refers to a non-cyclic, stable, straight-chain or branched chain, or a combination thereof, comprising at least one carbon atom and at least one heteroatom selected from O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom (e.g., O, N, P, and S) may be placed at any internal position of the heteroalkyl group or at any terminal position where the group is attached to the remainder of the molecule.

[0071] As used herein, the term "heteroaryl" refers to an aromatic ring compound containing five or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S; for example, a heteroaryl ring can have five to about 8-12 ring members. A heteroaryl is a type of heterocyclic group having an aromatic electronic structure. A heteroaryl, called a C2-heteroaryl, can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms, and so on. Similarly, a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so on. The sum of the number of carbon atoms and the number of heteroatoms equals the total number of ring atoms. Heteroaryl groups include, but are not limited to, the following groups: pyrrole, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, thiophene, benzothiophene, benzofuranyl, indolyl, azaindolyl, indolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridyl, isoxazolopyridyl, thianaphthyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl. Heteroaryl groups may be unsubstituted or substituted with groups as discussed herein. Representative substituted heteroaryl groups may be substituted once or multiple times with groups such as those listed herein.

[0072] Other examples of aryl and heteroaryl groups include, but are not limited to, phenyl, biphenyl, indene, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazole, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracene (1-anthrayl, 2-anthrayl, 3-anthrayl), thiophene (2-thiophene, 3-thiophene), furanyl (2-furanyl, 3-furanyl), indole, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthyl, isonindanyl, diphenylmethyl, acridineyl, thiazolyl, pyrrole (2-pyrrole), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), and triazolyl (1,2,3-triazolyl- 1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolinyl (2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 5-quinolinyl, 6-quinolinyl, 7-quinolinyl, 8-quinolinyl), isoquinolinyl (1-isoquinolinyl) 3-Isoquinolinyl, 4-Isoquinolinyl, 5-Isoquinolinyl, 6-Isoquinolinyl, 7-Isoquinolinyl, 8-Isoquinolinyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl) 2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophene (2-benzo[b]thiophene, 3-benzo[b]thiophene, 4-benzo[b]thiophene, 5-benzo[b]thiophene, 6-benzo[b]thiophene, 7-benzo[b]thiophene), 2,3-dihydro-benzo[b]thiophene, (2-(2,3-dihydro-benzo[b]thiophene), 3-(2,3-dihydro-benzo[b]thiophene), 4-(2,3-dihydro-benzo[b]thiophene), 5-(2,3-dihydro-benzo[b]thiophene), 6-(2,3-dihydro-benzo[b]thiophene), 7 ...3-Dihydro-benzo[b]thiophene), indole (1-indole, 2-indole, 3-indole, 4-indole, 5-indole, 6-indole, 7-indole), indazole (1-indole, 3-indole, 4-indole, 5-indole, 6-indole, 7-indole), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl) azole group, 6-benzimidazolyl group, 7-benzimidazolyl group, 8-benzimidazolyl group), benzoxazolyl group (1-benzoxazolyl group, 2-benzoxazolyl group), benzothiazolyl group (1-benzothiazolyl group, 2-benzothiazolyl group, 4-benzothiazolyl group, 5-benzothiazolyl group, 6-benzothiazolyl group, 7-benzothiazolyl group), carbazole group (1-carbazole group, 2-carbazole group, 3-carbazole group, 4-carbazole group), 5H- Dibenzo[b,f]azapyridine (5H-dibenzo[b,f]azapyr-1-yl, 5H-dibenzo[b,f]azapyr-2-yl, 5H-dibenzo[b,f]azapyr-3-yl, 5H-dibenzo[b,f]azapyr-4-yl, 5H-dibenzo[b,f]azapyr-5-yl), 10,11-dihydro-5H-dibenzo[b,f]azapyridine (10,11) -dihydro-5H-dibenzo[b,f]aza-1-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-2-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-3-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-4-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-5-yl, etc.

[0073] As used herein, the term "heterocycloalkyl" refers to an aliphatic, partially unsaturated, or fully saturated 3- to 14-membered ring system, including monocyclic systems of 3 to 8 atoms, as well as bicyclic and tricyclic systems, wherein at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. A heterocycloalkyl group may include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be substituted. Representative heterocycloalkyl groups include, but are not limited to, the following exemplary groups: pyrrolidinyl, pyrazolinyl, pyrazolyl, imidazolinyl, imidazoalkyl, piperidinyl, piperazinyl, oxazolyl, isoxazolyl, morpholinyl, thiazoalkyl, isothiazolyl, and tetrahydrofuranyl.

[0074] As used herein, the term "heterocyclyl" refers to an aromatic or non-aromatic ring compound containing three or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S. Thus, a heterocyclyl group can be a cycloheteroalkyl or heteroaryl group, or, if polycyclic, any combination thereof. In some embodiments, a heterocyclyl group comprises 3 to about 20 ring members, while other such groups have 3 to about 15 ring members. A heterocyclyl group referred to as a C2-heterocyclyl group can be a 5-ring having two carbon atoms and three heteroatoms, a 6-ring having two carbon atoms and four heteroatoms, and so on. Similarly, a C4-heterocyclyl group can be a 5-ring having one heteroatom, a 6-ring having two heteroatoms, and so on. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. The heterocyclyl ring may also include one or more double bonds. A heteroaryl ring is one embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused-ring species, including those comprising fused-ring aromatic and non-aromatic groups. For example, dioxolane and phenyldioxolane systems (methylenedioxanyl ring systems) are heterocyclic groups within the meaning of this document. This phrase also includes polycyclic systems containing heteroatoms, such as, but not limited to, quinuclidyl groups. Heterocyclic groups may be unsubstituted or may be substituted as discussed herein. Heterocyclic groups include, but are not limited to, pyrrolyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophene, benzothiophene, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, inzolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazolepyridinyl, isoxazolpyridinyl, thianaphthyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl. Representative substituted heterocyclic groups may be monosubstituted or substituted multiple times, for example, but not limited to, piperidinyl or quinolinyl, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted by groups as listed herein.

[0075] As used herein, the terms "hydrocarbon" or "hydrocarbyl" refer to a molecule or functional group that comprises carbon and hydrogen atoms. The term may also refer to a molecule or functional group that typically comprises carbon and hydrogen atoms but in which all hydrogen atoms are replaced by other functional groups.

[0076] As used herein, the term "hydrocarbyl" refers to a functional group derived from a straight-chain, branched, or cyclic hydrocarbon, and may be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. A hydrocarbyl group can be represented as (C... a -C b(C1-C4) hydrocarbon groups, where a and b are integers and mean any of a to b carbon atoms. For example, (C1-C4) hydrocarbon groups mean that the hydrocarbon group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0-C5) hydrocarbon groups can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4). b The presence of a hydrocarbon group means that a hydrocarbon group is not present in some implementations.

[0077] The term "immune cell" as used in this article refers to any cell involved in the immune response. Such cells include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells (such as dendritic cells and macrophages), monocytes, neutrophils, eosinophils, basophils, etc.

[0078] Unless the context clearly indicates otherwise, the term "independently selected from" as used herein means that the mentioned groups are the same, different, or a mixture thereof. Therefore, under this definition, the phrase "X" 1 X 2 and X 3 "Independently selected from inert gases" will include, for example, X 1 X 2 and X 3 They are all the same, among which X 1 X 2 and X 3 They are all different, among which X 1 and X 2 Same but X 3 Different scenarios and other similar arrangements.

[0079] As used herein, the term "ionizable lipid" refers to a lipid (e.g., a cationic lipid) having at least one protonable or deprotonable group, such that the lipid is positively charged at a pH equal to or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably neutral at or above physiological pH. Those skilled in the art will understand that the addition or removal of protons as a function of pH is an equilibrium process, and that references to charged or neutral lipids refer to the properties of the dominant species and do not require all lipids to be present in a charged or neutral form. Typically, ionizable lipids have a pK of a protonable group. a It ranges from about 4 to about 7.

[0080] As used herein, the term "local delivery" refers to the direct delivery of an active agent or therapeutic agent (such as messenger RNA) to a target site within an organism. For example, formulations can be delivered locally by direct injection into disease sites (such as tumors) or other targets (such as sites of inflammation) or target organs (such as the liver, heart, pancreas, kidney, etc.).

[0081] The term “lipid” refers to a group of organic compounds, including but not limited to fatty acid esters, characterized by being insoluble in water but soluble in many organic solvents. They are generally classified into at least three categories: (1) “simple lipids”, which include fats, oils and waxes; (2) “complex lipids”, which include phospholipids and glycolipids; and (3) “derived lipids”, such as steroids.

[0082] In the current context, the term "lipidoid" refers to any compound that exhibits lipid characteristics. In some embodiments, lipidoids may possess a range of secondary and tertiary amines, which increases the net positive charge of the compound.

[0083] As used herein, “lipid encapsulated” can refer to lipid particles that provide an active agent or therapeutic agent (such as a nucleic acid (e.g., a protein cargo)) with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is completely encapsulated within the lipid particle (e.g., to form SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles).

[0084] The term "lipid nanoparticle" refers to a particle having a size of at least one nanometer scale (e.g., 1-1000 nm) that comprises one or more lipids and / or additives.

[0085] As used herein, the term "lipid particle" refers to a lipid formulation that can be used to deliver an active agent or therapeutic agent (such as a nucleic acid (e.g., mRNA)) to a target of interest. In the lipid particles of this disclosure (which are typically formed from cationic lipids, non-cationic lipids, and conjugated lipids to prevent particle aggregation), the active agent or therapeutic agent can be encapsulated within the lipid, thereby protecting the formulation from enzymatic degradation.

[0086] As used in this article, the term "monovalent" refers to a substituent that is attached to the substituted molecule via a single bond. When a substituent is monovalent, such as F or Cl, it is bonded to the atom it substituted via a single bond.

[0087] As used herein, the term "mRNA" or "messenger RNA" refers to a ribonucleic acid sequence encoding a peptide or protein. In some embodiments, the mRNA may comprise a "transcription" that is produced using a DNA template and encodes a peptide or protein. Typically, mRNA includes a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA can be produced from a DNA template by in vitro transcription. Methods of in vitro transcription are known to those skilled in the art. For example, various in vitro transfer kits are commercially available. According to this disclosure, in addition to the modifications according to this disclosure, the mRNA may also be modified by further stabilization modifications and cap formation.

[0088] The term "neutral lipid" refers to any of a number of lipid species that exist as uncharged or neutral zwitterions at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalins, cholesterol, cerebrosides, and diacylglycerols.

[0089] The term "non-cationic lipid" refers to any amphiphilic lipid as well as any other neutral or anionic lipid.

[0090] As used herein, the term "nucleic acid" refers to a polymer containing at least two single-stranded or double-stranded deoxyribonucleotides or ribonucleotides, and includes DNA and RNA. DNA can be, for example, antisense molecules, plasmid DNA, pre-concentrated DNA, PCR products, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or links, which are synthetic, naturally occurring, or non-natural, and have similar binding properties to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphate thioesters, phosphoramidites, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless specifically defined, the term includes nucleic acids containing known natural nucleotide analogs that have similar binding properties to a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly contains variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, homologs, SNPs and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyribonucleotide residues (Batzer et al., Nucleic Acid Res, 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mal. Cell. Probes, 8:91-98 (1994)).

[0091] As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally referred to as oligonucleotides, and longer fragments as polynucleotides. In certain embodiments, the oligonucleotides of this disclosure are about 15 to about 60 nucleotides in length. Nucleic acids may be administered alone in lipid particles of this disclosure, or in combination with lipid particles of this disclosure comprising peptides, polypeptides, or small molecules (such as conventional pharmaceuticals) (e.g., co-administration). In other embodiments, nucleic acids may be administered in a viral vector.

[0092] Nucleotides consist of a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. Bases include purines and pyrimidines, further including natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications involving the placement of new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and halogenated hydrocarbons.

[0093] Unless otherwise indicated, a particular nucleic acid sequence also implicitly contains variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, homologs, SNPs and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyribonucleotide residues (Batzer et al., Nucleic Acid Res, 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0094] The terms “patient,” “subject,” or “individual” are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, applicable to the methods described herein. In a non-limiting embodiment, the patient, subject, or individual is a human being.

[0095] As used herein, the term "pharmaceutically acceptable" means a material (such as a carrier or diluent) that is relatively non-toxic and does not eliminate the biological activity or properties of a compound, so that the material can be administered to an individual without causing undesirable biological effects or interacting with any component of the composition contained therein in a harmful manner.

[0096] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of an applied compound prepared from a pharmaceutically acceptable non-toxic acid or base, including inorganic acids or bases, organic acids or bases, solvates, hydrates or inclusion complexes thereof.

[0097] The term "spacer" or "spacer element" as used in this document when referring to crRNA or sgRNA refers to a polynucleotide sequence that can specifically hybridize with a target nucleic acid sequence. Spacer elements interact with the target nucleic acid sequence via hydrogen bonds between complementary base pairs (i.e., paired bases). Spacer elements bind to selected DNA target sequences. Therefore, spacer elements are DNA target-binding sequences. Spacer elements determine the site-specific binding of Cas proteins and the location of nucleic acid endonucleation. The length of spacer elements ranges from 17 to 84 nucleotides, depending on the Cas protein they are associated with, with an average length of 36 nucleotides. For example, for SpyCas9, the functional length of the spacer guiding specific cleavage is typically about 12–25 nucleotides. The variability in the functional length of spacer elements is known in the art, as illustrated in U.S. Patent Application Publication No. 2014 / 0315985, the entire contents of which are incorporated herein by reference.

[0098] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid (including sulfates and hydrogen sulfates), and phosphoric acid (including hydrogen phosphates and dihydrogen phosphates). Suitable organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic, and sulfonic acid organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, malonic acid, saccharin, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactobionic acid, and galacturonic acid.

[0099] Suitable pharmaceutically acceptable base addition salts of the compounds described herein include, for example, ammonium salts, and metal salts, including alkali metal, alkaline earth metal, and transition metal salts, such as, for example, calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines, such as N,N'-diphenylethyldiamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine), and procaine. All these salts can be prepared from the respective compounds by reacting them with, for example, a suitable acid or base.

[0100] As used herein, the terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refer to pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, stabilizers, dispersants, suspending agents, diluents, excipients, thickeners, solvents, or encapsulating materials, which relate to carrying or transporting the compounds described herein within or to a patient to enable them to perform their intended function. Typically, such compounds are carried or transported from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation, including the compounds described herein, and must not be harmful to the patient. 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; astragalus powder; 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; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and compatible substances used in pharmaceutical formulations. As used herein, "pharmaceuticalally acceptable carrier" also includes any and all coatings, antimicrobial and antifungal agents, and absorption delay agents that are compatible with the activity of the compounds described herein and are physiologically acceptable to patients. Additional active compounds may also be incorporated into the composition. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compounds described herein. Other additional ingredients that may be included in a pharmaceutical composition used with the methods or compounds described herein are known in the art and are described, for example, in Remington's *Pharmaceutical Sciences* (Genaro, editor, Mack Publishing, 1985, Easton, PA), which is incorporated herein by reference.

[0101] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art, for example, peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art, for example, proteins, of which there are many types). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and so on. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0102] The term "placenta" as used in this article refers to the maternal organ that connects the developing fetus to the uterine wall. After birth, the placenta is expelled and is known as the postpartum placenta.

[0103] The term "polymer conjugated lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer conjugated lipid is a PEGylated lipid. PEGylated lipids are known in the art and include 1-(monomethoxy polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), DSPE-PEG-DBCO, DOPE-PEG-azide, DSPE-PEG-azide, DPPE-PEG-azide, DSPE-PEG-carboxyl-NHS, DOPE-PEG-carboxylic acid, DSPE-PEG-carboxylic acid, and so on.

[0104] As used herein, the term "solvent" refers to a liquid capable of dissolving solids, liquids, or gases. Non-limiting examples of solvents include siloxanes, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

[0105] The term "specifically bind," as used herein, refers to an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen of one species may also bind to that antigen of one or more other species. However, this cross-species reaction itself does not change the antibody's specific classification. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reaction itself does not change the antibody's specific classification. In some cases, the terms "specific binding" or "specifically binding" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that this interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than the usual protein. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0106] As used herein, the term "substantially" means most or a large portion, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. As used herein, the term "substantially free of" may mean absent or present in a small amount such that the amount of material present does not affect the material properties of the composition comprising the material, such that the material in the composition is about 0 wt% to about 5 wt%, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.01%, or about 0.001 wt% or less. The term "substantially free" may mean having a small amount such that the material in the composition is about 0 wt% to about 5 wt%, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to or greater than about 4.5 wt%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.01%, or about 0.001 wt% or less, or about 0 wt%.

[0107] The term “substituted” as used herein in conjunction with the definition of a molecule or organic group means a state in which one or more hydrogen atoms are replaced by one or more non-hydrogen atoms. The terms “functional group” or “substituent” as used herein refer to a group that can be substituted into or be substituted into a molecule or organic group. Examples of substituents or functional groups include, but are not limited to, halogens (e.g., F, Cl, Br, and I); oxygen atoms in groups such as hydroxyl, alkoxy, aryloxy, arylalkoxy, oxygen (carbonyl), and carboxyl groups including carboxylic acids, carboxylates, and carboxyl esters; sulfur atoms in groups such as thiols, alkyl and arylsulfides, sulfoxides, sulfones, sulfonyl groups, and sulfonamides; nitrogen atoms in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and heteroatoms in various other groups. Non-limiting examples of substituents that can bind to substituted carbon (or other) atoms include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azide, CF3, OCF3, R, O (oxo), S (thiocarbonyl), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2) 0-2 N(R)C(O)R、(CH2) 0-2 N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, where R can be a hydrogen or carbon-based moiety; for example, R can be hydrogen, (C1-C 100 ) hydrocarbon group, alkyl group, acyl group, cycloalkyl group, aryl group, aralkyl group, heterocyclic group, heteroaryl group or heteroaryl group; or, wherein the two R groups bonded to the nitrogen atom or adjacent nitrogen atom may together with one or more nitrogen atoms form a heterocyclic group.

[0108] "Therapeutic" treatment refers to treating subjects who exhibit pathological signs in order to reduce or eliminate those signs.

[0109] As used herein, the term "therapeutic protein" refers to a protein or peptide that, when administered to a subject in a therapeutically effective amount, has a positive or beneficial effect on the condition or disease state of the subject. In some embodiments, the therapeutic protein or peptide has curative or palliative properties and can be administered to improve, alleviate, reduce, reverse, delay the onset of one or more symptoms of a disease or disorder, or reduce its severity. Therapeutic proteins or peptides may have preventative properties and can be used to delay the onset of a disease or reduce the severity of a disease or pathological condition. The term "therapeutic protein" includes the whole protein or peptide and may also refer to a therapeutically active fragment thereof. It may also include therapeutically active variants of the protein. Exemplary therapeutic proteins include, but are not limited to, analgesic proteins, anti-inflammatory proteins, anti-proliferative proteins, pro-apoptotic proteins, anti-angiogenic proteins, cytotoxic proteins, cytosuppressive proteins, cytokines, chemokines, growth factors, wound-healing proteins, pharmacokines, or prodrug-activating proteins. Therapeutic proteins may include growth factors (EGF, TGF-α, TGF-β, TNF, HGF, IGF, and IL-1-8, etc.), cytokines, antibody-binding sites (paratopes), Fab (fragments, antigen-binding), and antibodies.

[0110] As used herein, the term "toll-like receptor agonist" refers to a small molecule capable of agonizing, stimulating, and / or activating toll-like receptors (TLRs). In some embodiments, agonism, stimulation, or activation of one or more TLRs (e.g., TLR7 / 8) may generate and / or promote an innate immune response. Small molecule TLR agonists are known in the art, and non-limiting exemplary TLR agonists include TLR7 / 8 agonist 1, imiquimod, resiquimod, and gardiquimod.

[0111] As used herein, the terms “treat,” “treating,” and “treatment” refer to the reduction of the frequency or severity of symptoms of a disease or condition experienced by a subject by administering a formulation or compound to the subject.

[0112] Lipids and lipid compounds Toll-like receptor (TLR) agonist lipids In one aspect, this disclosure provides compounds of formula (I) or their salts, solvates, stereoisomers or isotopic configurations: (I), in: R 1 Selected from H, R5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 2 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 5 R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H and R 5 Halogens, CN, NO2, optionally substituted C1-C6 alkyl groups, optionally substituted C3-C8 cycloalkyl groups, optionally substituted C2-C8 heterocyclic alkyl groups, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 5 yes , Where R 1 R 2 R 3a R 3b R 4a R 4b R4c and R 4d At least one of them is R 5 ,or R 3a and R 3b At least one of them is R 6a ; R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the optionally substituted C6-C 28 Alkyl, optionally substituted C6-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C8 heterocycloalkyl, optionally substituted C6-C 28 alkenyl and optionally substituted C6-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0113] In some embodiments, each occurrence of the optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylalkylene, heteroarylalkylene, optionally substituted arylene, and optionally substituted heteroarylene (if present) is independently and optionally substituted by at least one substituent selected from: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 Halogenated alkyl, C1-C3 haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R')(R''), C(=O)R', C(=O)OR', OC(=O)OR', C(=O)N(R')(R''), S(=O)2N(R')(R''), N(R')C(=O)R'', N(R')S(=O)2R'', C2-C8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R' and R'' is independently selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, benzyl, and phenyl.

[0114] In some implementations, R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d One of them is R. 5 .

[0115] In some implementations, R 1 It is R 5 In some implementations, R 1 It is CH2CH(CH3)2. In some embodiments, R 1 It is CH2C(OH)(CH3)2.

[0116] In some embodiments, the compound of formula (I) is the compound of formula (Ia), or a salt, solvate, stereoisomer, or isotopic configuration thereof: (Ia), where R 2Selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H, halogen, CN, NO2, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocyclic alkyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a; R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0117] In some implementations, R 2 It is n-butyl. In some embodiments, R 2 It is H. In some implementations, R 2 It is CH2OCH2CH3. In some embodiments, R 2 It is CH2NHCH2CH3.

[0118] In some implementations, R 3a It is H. In some implementations, R 3b It is H.

[0119] In some implementations, R 4a R 4b R 4c and R 4d At least one of them is H. In some embodiments, R 4aR 4b R 4c and R 4d At least two of them are H. In some implementations, R 4a R 4b R 4c and R 4d At least three of them are H. In some implementations, R 4a R 4b R 4c and R 4d All are H.

[0120] In some embodiments, L is an optionally substituted C7–C 12 Aranediyl group.

[0121] In some implementations, L is .

[0122] In some implementations, R 6a It is R 7a In some implementations, R 6b It is R 7a In some implementations, R 6a It is R 7b In some implementations, R 6b It is R 7b In some implementations, R 6a It is -CH2CH2C(=O)OR 7a In some implementations, R 6b It is -CH2CH2C(=O)OR 7a .

[0123] In some implementations, R 7a It is an optional substitution of C6-C 28 Alkyl group. In some embodiments, R 7a It is an optional substitution of C6-C 28 Alkenyl. In some embodiments, R 7a It is an optional substitution of C6-C 28 Heteroalkyl groups.

[0124] In some implementations, R 7b It is an optional substitution of C6-C 28 Alkyl group. In some embodiments, R 7b It is an optional substitution of C6-C 28 Alkenyl. In some embodiments, R 7b It is an optional substitution of C6-C 28 Heteroalkyl groups.

[0125] In some implementations, R 6aIt is -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6a It is CH2CH(OH) (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6a It is -CH2CH(OH) (optionally substituted C6-C) 28 (Heteroalkyl). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

[0126] In some implementations, R 6b It is -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6b It is CH2CH(OH) (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6b It is -CH2CH(OH) (optionally substituted C6-C) 28 (Heteroalkyl). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

[0127] In some implementations, R 6a It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 6a It is -CH2CH2C(=O)O(CH2) 11 CH3.

[0128] In some implementations, R 6b It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 6b It is -CH2CH2C(=O)O(CH2)11 CH3.

[0129] In some embodiments, the compound is: (C12-TLRa).

[0130] In some embodiments, the compound is: (O12-TLRa).

[0131] Ionizable lipids and / or cationic lipids The scope of ionizable lipids intended for use in this disclosure is not limited to those ionizable lipids exemplified herein. Many cationic lipids and their related analogues that can also be used in this disclosure have been described in the following documents: U.S. Patent Publications 20060083780 and 20060240554; U.S. Patents 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and 5,785,992; and PCT Publications WO1996 / 010390, WO2021077066, WO2021077067, WO2023015200, WO2023056282 and WO2023056418, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0132] In the lipid nanoparticles disclosed herein, cationic lipids or ionizable lipids may include, for example, one or more of the following: (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLinMC3DMA), [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecane-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecanoate)] [alkyl]amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanool (C12-200), 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3- 4,5-Dimethylaminopropyl)-1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinyl-4-N-methylpiperazine-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-KDMA), 1,2-dilinylcarbamoyloxy-3-dimethylaminopropane (D Lin-C-DAP), 1,2-dilinoleoyl-3-dimethylaminoacetoxypropane (DLin-DAC), 1,2-dilinoleoyl-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyl-3-trimethylaminopropane hydrochloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane hydrochloride (DLin-TAP).Cl), 1,2-dilinoleoxy-3-(N-methylpiperazine)propane (D Lin-MPZ), 3-(N,N-dilinoleamino)-1,2-propanediol (D LinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-D MA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSD) MA), N-(1-(2,3-dioleoxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N'N'-dimethylaminoethane)- Carbamoyl cholesterol (DC-Chol), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleoxy-N-[2-(spermine-formamidoethyl]-N,N-dimethyl-1-1-propanium trifluoroacetate (DOSPA), bis(octadecylaminoglycyl)spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxobutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CL) The cationic lipids are DLinDMA, 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoylbenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In some embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA (“XTC2”), or mixtures thereof. Ionizable lipids are not limited to those described herein, and may further include ionizable lipids known to those skilled in the art or described in PCT applications PCT / US2020 / 056255 and / or PCT / US2020 / 056252, the entire disclosure of which is incorporated herein by reference.

[0133] The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as other cationic lipids, is described in U.S. Application Publication No. 2011 / 0256175, the entire contents of which are incorporated herein by reference for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-CDAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as other cationic lipids, is described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, the entire contents of which are incorporated herein by reference for all purposes. The synthesis of cationic lipids (such as CLinDMA) and other cationic lipids is described in U.S. Patent Publication No. 20060240554, the entire contents of which are incorporated herein by reference for all purposes.

[0134] Non-cationic lipids (auxiliaries and / or cholesterol or its derivatives) In the nucleic acid lipid particles of this disclosure, non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. In some embodiments, non-cationic lipids include one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) phospholipids; or (3) a mixture of phospholipids and cholesterol or a derivative thereof.

[0135] Examples of cholesterol derivatives include, but are not limited to, cholesterol alcohols, cholesterol ketones, cholesterol ketones, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, and mixtures thereof. The synthesis of cholesterol-2'-hydroxyethyl ether is known to those skilled in the art and has been described in U.S. Patents 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 1,114,1378, the entire contents of which are incorporated herein by reference for all purposes.

[0136] Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dihexadecoside, distearate phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), palmitoylphosphatidylglycerol (POPG), and dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal). Dipalmitoyl phosphatidyl ethanolamine (DPPE), bis(tetradecanoyl) phosphatidyl ethanolamine (DMPE), distearate phosphatidyl ethanolamine (DSPE), monomethylphosphatidyl ethanolamine, dimethylphosphatidyl ethanolamine, disqualyl phosphatidyl ethanolamine (DEPE), stearoyl oleyl phosphatidyl ethanolamine (SOPE), lysophosphatidylcholine, dilinoleyl phosphatidylcholine, and mixtures thereof.

[0137] Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids can, for example, be derived from a compound having a C... 10 -C 24 The acyl group of the carbon chain fatty acid, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Other examples of non-cationic lipids include sterols such as cholesterol and their derivatives, such as cholesterolanol, cholesterolanone, cholesterolenone, coprosterol, cholesterolenyl-2'-hydroxyethyl ether, cholesterolenyl-4'-hydroxybutyl ether, and mixtures thereof. In some embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0138] Polymer-conjugated lipids In the nucleic acid-lipid particles of this disclosure, the conjugated lipids that inhibit particle aggregation may include one or more of the following: polyethylene glycol (PEG) lipid conjugates, polyamide (ATTA) lipid conjugates, cationic polymer-lipid conjugates (CPL), or mixtures thereof. In some embodiments, the nucleic acid-lipid particles include PEG-lipid conjugates or ATTA-lipid conjugates.

[0139] PEG is a linear, water-soluble polymer of ethylene-PEG repeating units having two terminal hydroxyl groups. PEG is classified according to its molecular weight; for example, PEG 2000 has an average molecular weight of about 2000 Daltons, and PEG 5000 has an average molecular weight of about 5000 Daltons. PEG is commercially available from Sigma Chemical and other companies, including, for example, monomethoxy polyethylene glycol (MePEGOH), monomethoxy polyethylene glycol succinate (MePEGS), monomethoxy polyethylene glycol imine succinate (MePEG-S-NHS), monomethoxy polyethylene glycol amine (MePEG-NH2), monomethoxy polyethylene glycol toluenesulfonate (MePEG-TRES), and monomethoxy polyethylene glycol imidazole carbonyl (MePEG-IM). Other PEGs, such as those described in U.S. Patents 6774180 and 7053150 (e.g., mPEG(20 kDa)amine), can also be used to prepare the PEG-lipid conjugates of this disclosure. For all purposes, the entire disclosure of these patents is incorporated herein by reference. Furthermore, monomethoxy polyethylene glycol acetic acid (MePEG-CH2COOH) is particularly suitable for the preparation of PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0140] In some embodiments, PEG-lipid conjugates or ATTA-lipid conjugates are used in conjunction with CPL. Conjugated lipids that inhibit particle aggregation may include PEG-lipids, such as PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEGDAA conjugates may be PEG dilauroyloxypropyl (C... 12 ), PEG-dimyristoyloxypropyl (C 14 ), PEG-dipalmitoyloxypropyl (C 16 ), PEG-distearate oxypropyl (C 18 (or mixtures thereof).

[0141] Other PEG-lipid conjugates suitable for use in this disclosure include, but are not limited to, mPEG2000-1,2-diO-alkyl-sn3-carbonylglycerol ester (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT application PCT / US08 / 88676, filed December 31, 2008, the entire disclosure of which is incorporated herein by reference for all purposes. Other PEG-lipid conjugates suitable for use in this disclosure include, but are not limited to, 1-[8'-(1,2-bistetradecanoyl-3-propoxy)-formamide-3',6'-dioxaoctyl]carbamoyl-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent 7,404,969, the entire disclosure of which is incorporated herein by reference for all purposes.

[0142] The PEG moiety of the PEG-lipid conjugates described herein may include an average molecular weight ranging from about 550 Daltons to about 10,000 Daltons. In some examples, the PEG moiety has an average molecular weight ranging from about 750 Daltons to about 5,000 Daltons (e.g., from about 1,000 Daltons to about 5,000 Daltons, from about 1,500 Daltons to about 3,000 Daltons, from about 750 Daltons to about 3,000 Daltons, from about 750 Daltons to about 2,000 Daltons, etc.). In some embodiments, the PEG moiety has an average molecular weight of about 2,000 Daltons or about 750 Daltons.

[0143] In addition to the above, those skilled in the art will readily understand that other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used to replace PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid and derived celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0144] In addition to the foregoing components, the particles of this disclosure (e.g., LNP) may further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (e.g., Chen et al., Bioconj. Chem., 11:433-437 (2000)). Suitable SPLPs and SPLP-CPLs for use in this disclosure, as well as methods for manufacturing and using SPLPs and SPLP-CPLs, are disclosed, for example, in U.S. Patent No. 6,852,334 and PCT Publication WO 00 / 62813, the entire disclosure of which is incorporated herein by reference for all purposes.

[0145] In some examples, the conjugated lipids that inhibit particle aggregation (e.g., PEG-lipid conjugates) may comprise about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 1 mol% to about 1.8 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, about 1.4 mol% to about 1.5 mol%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol% (or any fraction or range thereof) of the total lipids in the particles.

[0146] In the lipid nanoparticles of this disclosure, active agents or therapeutic agents can be completely encapsulated within the lipid portion of the particle, thereby protecting the active agents or therapeutic agents from enzymatic degradation. In some embodiments, nucleic acid-lipid particles comprising nucleic acids such as messenger RNA (i.e., mRNA) are completely encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In some examples, the nucleic acid in the nucleic acid-lipid particles does not substantially degrade after the particles are exposed to nucleases at 37°C for at least about 20, 30, 45, or 60 minutes. In some other examples, the nucleic acid in the nucleic acid-lipid particles does not substantially degrade after the particles are incubated in serum at 37°C for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agent or therapeutic agent (e.g., nucleic acid, such as siRNA) is complexed with the lipid portion of the particle. One of the benefits of the formulation disclosed herein is that the lipid particle composition is substantially non-toxic to mammals, such as humans.

[0147] Lipid nanoparticle (LNP) composition In one aspect, the present invention provides a lipid nanoparticle (LNP). In some embodiments, the LNP comprises at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent. In some embodiments, the LNP comprises at least one ionizable lipid. In some embodiments, the LNP comprises at least one accessory lipid. In some embodiments, the LNP comprises cholesterol. In some embodiments, the LNP comprises at least one polymer-conjugated lipid.

[0148] In some embodiments, the LNP comprises (a) at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent.

[0149] In some embodiments, the LNP comprises (b) at least one ionizable lipid.

[0150] In some embodiments, the LNP comprises (c) at least one accessory lipid.

[0151] In some implementations, LNP includes (d) cholesterol.

[0152] In some embodiments, the LNP comprises (e) at least one polymer-conjugated lipid.

[0153] In some embodiments, the LNP comprises a hydrocarbon-substituted TLR agonist, which is a compound of formula (II): A(B) n (II), in: A is a toll-like receptor (TLR) agonist; Each occurrence of B is independently selected from R. 6a and ; R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a , R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L represents a bond, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and n is an integer selected from 1, 2, 3, and 4.

[0154] In some embodiments, the TLR agonist is 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (TLR7 / 8 agonist 1). In some embodiments, the TLR agonist is 1-isobutyl-1H-imidazo[4,5-c]quinoline-4-amine (imiquimod). In some embodiments, the TLR agonist is 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (resimod). In some embodiments, the TLR agonist is 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (gadmod).

[0155] In some embodiments, at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent is a compound of formula (I), or a salt, solvate, stereoisomer, or isotopic configuration thereof: (I), in: R 1 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 2 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 5 R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H and R 5Halogens, CN, NO2, optionally substituted C1-C6 alkyl groups, optionally substituted C3-C8 cycloalkyl groups, optionally substituted C2-C8 heterocyclic alkyl groups, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 5 yes , Where R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d At least one of them is R 5 ,or R 3a and R 3b At least one of them is R 6a ; R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the optionally substituted C6-C 28 Alkyl, optionally substituted C6-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C8 heterocycloalkyl, optionally substituted C6-C 28 alkenyl and optionally substituted C6-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0156] In some implementations, R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d One of them happens to be R. 5 .

[0157] In some implementations, R 1 It is R 5 In some implementations, R 1 It is CH2CH(CH3)2. In some embodiments, R 1 It is CH2C(OH)(CH3)2.

[0158] In some embodiments, the compound of formula (I) is the compound of formula (Ia), or a salt, solvate, stereoisomer, or isotopic configuration thereof: (Ia), where R 2 Selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H, halogen, CN, NO2, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocyclic alkyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a-(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0159] In some implementations, R 2 It is n-butyl. In some embodiments, R 2 It is H. In some implementations, R 2 It is CH2OCH2CH3. In some embodiments, R 2 It is CH2NHCH2CH3.

[0160] In some implementations, R 3a It is H. In some implementations, R3b It is H.

[0161] In some implementations, R 4a R 4b R 4c and R 4d At least one of them is H. In some embodiments, R 4a R 4b R 4c and R 4d At least two of them are H. In some implementations, R 4a R 4b R 4c and R 4d At least three of them are H. In some implementations, R 4a R 4b R 4c and R 4d All are H.

[0162] In some embodiments, L is an optionally substituted C7-C 12 Aranediyl group.

[0163] In some implementations, L is .

[0164] In some implementations, R 6a It is R 7a In some implementations, R 6b It is R 7a In some implementations, R 6a It is R 7b In some implementations, R 6b It is R 7b In some implementations, R 6a It is -CH2CH2C(=O)OR 7a In some implementations, R 6b It is -CH2CH2C(=O)OR 7a .

[0165] In some implementations, R 7a It is an optional substitution of C6-C 28 Alkyl group. In some embodiments, R 7a It is an optional substitution of C6-C 28 Alkenyl. In some embodiments, R 7a It is an optional substitution of C6-C 28 Heteroalkyl groups.

[0166] In some implementations, R 7b It is an optional substitution of C6-C 28Alkyl group. In some embodiments, R 7b It is an optional substitution of C6-C 28 Alkenyl. In some embodiments, R 7b It is an optional substitution of C6-C 28 Heteroalkyl groups.

[0167] In some implementations, R 6a It is -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6a It is CH2CH(OH) (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6a It is -CH2CH(OH) (optionally substituted C6-C) 28 (Heteroalkyl). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

[0168] In some implementations, R 6b It is -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6b It is CH2CH(OH) (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6b It is -CH2CH(OH) (optionally substituted C6-C) 28 (Heteroalkyl). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

[0169] In some implementations, R 6a It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 6aIt is -CH2CH2C(=O)O(CH2) 11 CH3.

[0170] In some implementations, R 6b It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 6b It is -CH2CH2C(=O)O(CH2) 11 CH3.

[0171] In some embodiments, the compound is: (C12-TLRa).

[0172] In some embodiments, the compound is: (O12-TLRa).

[0173] In some embodiments, the ionizable lipid is selected from at least one of the following: , , , , and in: R 8a R 8b R 8c R 8d R 8e and R 8f Each of the following is independently selected: -(optionally substituted C1-C6 alkylene)-C(=O)OR 9a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 9a (R) 9b -(optionally substituted C1-C6 alkylene)-C(=O)R 9a -(optionally substituted C1-C6 alkylene)-(R 9a -C(=O)OR 9a -C(=O)N(R) 9a (R) 9b -C(=O)R 9a and R 9a ;and R 9a With R 9b Each occurrence of is independently selected from: optionally substituted C1-C 28 Alkyl, optionally substituted C2-C 28Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 alkenyl groups, and optionally substituted C2-C groups 28 Alkyne group.

[0174] In some implementations, R 8a For R 9a In some implementations, R 8a For R 9b In some implementations, R 8a -CH2CH2C(=O)OR 9a In some implementations, R 8a -CH2CH2C(=O)OR 9b In some implementations, R 8b For R 9a In some implementations, R 8b For R 9b In some implementations, R 8b -CH2CH2C(=O)OR 9a .

[0175] In some implementations, R 8b -CH2CH2C(=O)OR 9b In some implementations, R 8c For R 9a In some implementations, R 8c For R 9b In some implementations, R 8c -CH2CH2C(=O)OR 9a In some implementations, R 8c -CH2CH2C(=O)OR 9b In some implementations, R 8d For R 9a In some implementations, R 8d For R 9b In some implementations, R 8d -CH2CH2C(=O)OR 9a In some implementations, R 8d -CH2CH2C(=O)OR 9b In some implementations, R 8e For R 9a In some implementations, R 8e For R 9b In some implementations, R 8e -CH2CH2C(=O)OR 9aIn some implementations, R 8e -CH2CH2C(=O)OR 9b In some implementations, R 8f For R 9a In some implementations, R 8f For R 9b In some implementations, R 8f -CH2CH2C(=O)OR 9a In some implementations, R 8f -CH2CH2C(=O)OR 9b .

[0176] In some implementations, R 9a C1-C is an optional substitute 28 Alkyl group. In some embodiments, R 9a C1-C is an optional substitute 28 Alkenyl. In some embodiments, R 9a C1-C is an optional substitute 28 Heteroalkyl. In some embodiments, R 9b C1-C is an optional substitute 28 Alkyl group. In some embodiments, R 9b C1-C is an optional substitute 28 Alkenyl. In some embodiments, R 9b C1-C is an optional substitute 28 Heteroalkyl groups.

[0177] In some implementations, R 7a -CH2CH(OH) (optionally substituted C1-C) 28 Alkyl group). In some embodiments, R 7a -CH2CH(OH) (optionally substituted C1-C) 28 Alkenyl). In some embodiments, R 7a -CH2CH(OH) (optionally substituted C2-C) 28 (Heteroalkyl). In some embodiments, R 7a -CH2CH2C(=O)O (optionally substituted C1-C) 28 Alkyl group). In some embodiments, R 7a -CH2CH2C(=O)O (optionally substituted C1-C) 28 Alkenyl). In some embodiments, R 7a -CH2CH2C(=O)O (optionally substituted C2-C) 28 Alkyl group). In some embodiments, R 7b-CH2CH(OH) (optionally substituted C1-C) 28 Alkyl group). In some embodiments, R 7b -CH2CH(OH) (optionally substituted C1-C) 28 Alkenyl). In some embodiments, R 7b -CH2CH(OH) (optionally substituted C2-C) 28 (Heteroalkyl). In some embodiments, R 7b -CH2CH2C(=O)O (optionally substituted C1-C) 28 Alkyl group). In some embodiments, R 7b -CH2CH2C(=O)O (optionally substituted C1-C) 28 Alkenyl). In some embodiments, R 7b -CH2CH2C(=O)O (optionally substituted C2-C) 28 (heteroalkyl groups).

[0178] In some implementations, R 7a It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 7a It is -CH2CH2C(=O)O(CH2) 11 CH3. In some implementations, R 7b It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 7b It is -CH2CH2C(=O)O(CH2) 11 CH3.

[0179] In some embodiments, the at least one ionizable lipid comprises: (C12-113).

[0180] In some embodiments, at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, or about 17.5 mol of the LNP.

[0181] In some embodiments, at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent constitutes less than about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, or about 17.5 mol of the LNP.

[0182] In some embodiments, at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, or about 17.5 mol of the LNP.

[0183] In some embodiments, at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1 mol% of the LNP. In some embodiments, at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1.5 mol% of the LNP. In some embodiments, at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 5 mol% of the LNP. In some embodiments, at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 10 mol% of the LNP. In some embodiments, at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 17.5 mol% of the LNP.

[0184] In some embodiments, the at least one ionizable lipid accounts for less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 of the LNP. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or approximately 99 mol%.

[0185] In some embodiments, the at least one ionizable lipid constitutes a fraction of the LNP greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or approximately 99 mol%.

[0186] In some embodiments, the at least one ionizable lipid comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol of the LNP.

[0187] In some embodiments, the at least one ionizable lipid constitutes less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol% of the LNP.

[0188] In some embodiments, the at least one ionizable lipid constitutes a greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol% of the LNP.

[0189] In some embodiments, the at least one ionizable lipid comprises more than about 34 mol% of the LNP. In some embodiments, the at least one ionizable lipid comprises more than about 32.5 mol% of the LNP. In some embodiments, the at least one ionizable lipid comprises more than about 30 mol% of the LNP. In some embodiments, the at least one ionizable lipid comprises more than about 25 mol% of the LNP. In some embodiments, the at least one ionizable lipid comprises more than about 17.5 mol% of the LNP.

[0190] In some embodiments, the at least one auxiliary lipid comprises dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the at least one auxiliary lipid comprises distearate phosphatidylcholine (DSPC).

[0191] In some embodiments, the at least one auxiliary lipid comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 mol% of the LNP. In some embodiments, the at least one auxiliary lipid constitutes less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 mol% of the LNP. In some embodiments, the at least one auxiliary lipid constitutes a proportion of the LNP greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 mol.

[0192] In some embodiments, the at least one auxiliary lipid comprises about 16 mol% of the LNP.

[0193] In some embodiments, cholesterol accounts for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol% of the LNP.

[0194] In some embodiments, cholesterol constitutes less than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol% of the LNP.

[0195] In some embodiments, cholesterol constitutes a percentage of the LNP greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol%.

[0196] In some embodiments, cholesterol accounts for approximately 46.5 mol% of the LNP.

[0197] In some embodiments, the at least one polymer-conjugated lipid comprises 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000).

[0198] In some embodiments, the at least one polymer-conjugated lipid accounts for approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, and 5% of the LNP. 1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3, 15 0.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9 or approximately 20.0 mol%.

[0199] In some embodiments, the at least one polymer-conjugated lipid constitutes less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5 1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10. 7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3, 1 5.4, ​​15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9 or approximately 20.0 mol%.

[0200] In some embodiments, the at least one polymer-conjugated lipid constitutes a fraction of the LNP greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5 1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10. 7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3, 1 5.4, ​​15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, or approximately 20.0 mol%. In some embodiments, the at least one polymer-conjugated lipid accounts for about 2.5 mol% of the LNP.

[0201] In some embodiments, the molar ratio of (a): (b): (c): (d): (e) in the LNP is approximately 5:30: 16: 46.5: 2.5.

[0202] In some embodiments, the LNP further comprises at least one cargo molecule.

[0203] In some embodiments, the cargo is at least one selected from nucleic acids, small molecules, proteins, therapeutic agents, antibodies, and any combination thereof.

[0204] In some implementations, the cargo is nucleic acid.

[0205] In some embodiments, the nucleic acid is DNA or RNA.

[0206] In some embodiments, the nucleic acid is selected from mRNA, cDNA, pDNA, microRNA, siRNA, modified RNA, antagomir, antisense molecules, and any combination thereof.

[0207] In some embodiments, the cargo is at least partially encapsulated in the LNP.

[0208] In some embodiments, the cargo is mRNA.

[0209] In some embodiments, the total amount of lipids in the LNP (i.e., hydrocarbon-substituted Toll-like receptor agonists and ionizable lipids) to mRNA is about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or about 20:1 (i.e., the weight ratio of (a)+(b):mRNA).

[0210] In some embodiments, the total amount of lipids in the LNP (i.e., hydrocarbon-substituted Toll-like receptor agonists and ionizable lipids) to mRNA is approximately 10:1 (i.e., the weight ratio of (a) + (b): mRNA).

[0211] In some embodiments, the mRNA encodes SARS-CoV-2, its immunogenic fragments (e.g., spike protein), or modified derivatives thereof.

[0212] method In one aspect, this disclosure provides a method for generating an innate immune response in a subject, the method comprising administering to the subject lipid nanoparticles (LNPs) of this disclosure or a pharmaceutical composition of this disclosure.

[0213] In another aspect, this disclosure provides a method for treating, preventing, and / or improving an infection, disease, or disorder in a subject, the method comprising administering lipid nanoparticles (LNPs) to the subject. In some embodiments, the LNP comprises (a) at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent. In some embodiments, the LNP comprises (b) at least one ionizable lipid. In some embodiments, the LNP comprises (c) at least one accessory lipid. In some embodiments, the LNP comprises (d) cholesterol. In some embodiments, the LNP comprises (e) at least one polymer-conjugated lipid. In some embodiments, the LNP comprises (f) at least one cargo molecule.

[0214] In some embodiments, the LNP comprises a hydrocarbon-substituted TLR agonist, which is a compound of formula (II): A(B) n (II), in: A is a toll-like receptor (TLR) agonist; Each occurrence of B is independently selected from R. 6a and ; R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L represents a bond, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and n is an integer selected from 1, 2, 3, and 4.

[0215] In some embodiments, the TLR agonist is 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (TLR7 / 8 agonist 1). In some embodiments, the TLR agonist is 1-isobutyl-1H-imidazo[4,5-c]quinoline-4-amine (imiquimod). In some embodiments, the TLR agonist is 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (resimod). In some embodiments, the TLR agonist is 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (gadmod).

[0216] In some embodiments, at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent is a compound of formula (I), or a salt, solvate, stereoisomer, or isotopic configuration thereof: (I), in: R 1 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 2 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 5 R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H and R 5 Halogens, CN, NO2, optionally substituted C1-C6 alkyl groups, optionally substituted C3-C8 cycloalkyl groups, optionally substituted C2-C8 heterocyclic alkyl groups, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 5 yes , Where R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d At least one of them is R 5 ,or R 3a and R 3b At least one of them is R 6a ; R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a-(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the optionally substituted C6-C 28 Alkyl, optionally substituted C6-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C8 heterocycloalkyl, optionally substituted C6-C 28 alkenyl and optionally substituted C6-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0217] In some implementations, R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d One of them happens to be R. 5 .

[0218] In some implementations, R1 It is R 5 In some implementations, R 1 It is CH2CH(CH3)2. In some embodiments, R 1 It is CH2C(OH)(CH3)2.

[0219] In some embodiments, the compound of formula (I) is the compound of formula (Ia), or a salt, solvate, stereoisomer, or isotopic configuration thereof: (Ia), where R 2 Selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H, halogen, CN, NO2, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocyclic alkyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0220] In some implementations, R 2 It is n-butyl. In some embodiments, R 2 It is H. In some implementations, R 2It is CH2OCH2CH3. In some embodiments, R 2 It is CH2NHCH2CH3.

[0221] In some implementations, R 3a It is H. In some implementations, R 3b It is H.

[0222] In some implementations, R 4a R 4b R 4c and R 4d At least one of them is H. In some embodiments, R 4a R 4b R 4c and R 4d At least two of them are H. In some implementations, R 4a R 4b R 4c and R 4d At least three of them are H. In some implementations, R 4a R 4b R 4c and R 4d All are H.

[0223] In some embodiments, L is an optionally substituted C7-C 12 Aranediyl group.

[0224] In some implementations, L is .

[0225] In some implementations, R 6a It is R 7a In some implementations, R 6b It is R 7a In some implementations, R 6a It is R 7b In some implementations, R 6b It is R 7b In some implementations, R 6a It is -CH2CH2C(=O)OR 7a In some implementations, R 6b It is -CH2CH2C(=O)OR 7a .

[0226] In some implementations, R 7a It is an optional substitution of C6-C 28 Alkyl group. In some embodiments, R 7a It is an optional substitution of C6-C 28 Alkenyl. In some embodiments, R7a It is an optional substitution of C6-C 28 Heteroalkyl groups.

[0227] In some implementations, R 7b It is an optional substitution of C6-C 28 Alkyl group. In some embodiments, R 7b It is an optional substitution of C6-C 28 Alkenyl. In some embodiments, R 7b It is an optional substitution of C6-C 28 Heteroalkyl groups.

[0228] In some implementations, R 6a It is -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6a It is CH2CH(OH) (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6a It is -CH2CH(OH) (optionally substituted C6-C) 28 (Heteroalkyl). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6a It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

[0229] In some implementations, R 6b It is -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6b It is CH2CH(OH) (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6b It is -CH2CH(OH) (optionally substituted C6-C) 28 (Heteroalkyl). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl group). In some embodiments, R 6b It is -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkenyl). In some embodiments, R 6bIt is -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

[0230] In some implementations, R 6a It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 6a It is -CH2CH2C(=O)O(CH2) 11 CH3.

[0231] In some implementations, R 6b It is -CH2CH(OH)(CH2)9CH3. In some embodiments, R 6b It is -CH2CH2C(=O)O(CH2) 11 CH3.

[0232] In some embodiments, the compound is: (C12-TLRa).

[0233] In some embodiments, the compound is: (O12-TLRa).

[0234] In some embodiments, the cargo is at least one selected from nucleic acids, small molecules, proteins, therapeutic agents, antibodies, and any combination thereof.

[0235] In some implementations, the cargo is nucleic acid.

[0236] In some implementations, nucleic acids are DNA or RNA.

[0237] In some implementations, the nucleic acid is selected from mRNA, cDNA, pDNA, microRNA, siRNA, modified RNA, anta-coma, antisense molecules, and any combination thereof.

[0238] In some implementations, the goods are at least partially encapsulated in an LNP.

[0239] In some implementations, the cargo is mRNA.

[0240] In some implementations, the mRNA encodes SARS-CoV-2, its immunogenic fragments (e.g., spike protein), or modified derivatives thereof.

[0241] In some implementations, the infection, disease, or obstacle is SARS-CoV-2 infection.

[0242] In some implementations, an innate immune response is promoted in the subject.

[0243] In some implementations, the subject is a mammal.

[0244] In some implementations, the mammal is a human.

[0245] goods In one aspect, this disclosure relates to an LNP comprising at least one cargo molecule at least partially encapsulated therein. In some embodiments, at least one cargo is completely encapsulated therein.

[0246] Small molecule therapeutic agents In various embodiments, the reagent is a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, it can be obtained using standard methods known to those skilled in the art. These methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis, and in vitro translation systems using methods well known in the art. In some embodiments, small molecule therapeutic agents include organic molecules, inorganic molecules, biomolecules, synthetic molecules, etc.

[0247] Combinatorial libraries of molecularly diverse compounds that may be used to treat a variety of diseases and conditions are well known in the art, as are methods for preparing these libraries. These methods can utilize a variety of techniques well known to those skilled in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, labeling techniques, and generating biased structures for lead compound development while generating unbiased molecular landscapes for lead compound discovery. In some embodiments of this disclosure, combinatorial techniques are used to synthesize and / or identify therapeutic agents.

[0248] In general methods for small library synthesis, activated nuclear molecules condense with multiple building units to form a combined library of covalently linked ensembles of nuclear building units. The shape and stiffness of the nucleus determine the orientation of the building units in shape space. Libraries can be biased to target specific biological structures (“focused libraries”) by altering the nuclear structure, linkage, or building units; or synthesized with less structural bias using flexible nuclei. In some embodiments of this disclosure, therapeutic agents are prepared via small library synthesis.

[0249] Even without a description of salts, the small molecules and small molecule compounds described herein may exist in salt form, and it should be understood that this disclosure includes all salts and solvates of the therapeutic agents described herein, as well as non-salt and non-solvent forms of the therapeutic agents, as is well known to those skilled in the art. In some embodiments, the salts of the therapeutic agents of this disclosure are pharmaceutically acceptable salts.

[0250] Where any therapeutic agent described herein may exist in tautomeric forms, even if only one or more tautomeric forms may be explicitly described, each tautomeric mode is intended to be included in this disclosure. For example, when describing the 2-hydroxypyridinyl moiety, the corresponding 2-pyridone tautomer is also referred to.

[0251] This disclosure also includes any or all stereochemical forms, including any enantiomers or diastereomers of the therapeutic agents. References to structures or names herein are intended to cover all possible stereoisomers of the described therapeutic agents. This disclosure also covers all forms of the therapeutic agents, such as crystalline or amorphous forms. It also includes compositions comprising the therapeutic agents of this disclosure, such as compositions of substantially pure therapeutic agents including their specific stereochemical forms, or compositions comprising mixtures of the therapeutic agents of this disclosure in any proportion, including mixtures of two or more stereochemical forms, such as racemic or non-racemic mixtures.

[0252] This disclosure also includes any or all active analogs or derivatives, such as prodrugs, of any therapeutic agent described herein. In some embodiments, the therapeutic agent is a prodrug. In some embodiments, the small molecules described herein are candidates for derivatization. Therefore, in some cases, small molecule analogs described herein that have modulating potency, selectivity, and solubility are also included, providing useful lead compounds for drug discovery and drug development. Therefore, in some cases, new analogs are designed considering issues such as drug delivery, metabolism, novelty, and safety during the optimization process.

[0253] In some cases, the small molecule therapeutic agents described herein are derivatives or analogs of known therapeutic agents, as is well known in the fields of combinatorial chemistry and medicinal chemistry. Analogs or derivatives can be prepared by adding and / or substituting functional groups at different positions. Therefore, the small molecules described herein can be converted into derivatives / analogs using well-known chemical synthetic methods. For example, all hydrogen atoms or substituents can be selectively modified to generate new analogs. Furthermore, linking atoms or groups can be modified to have longer or shorter linkers with a carbon skeleton or heteroatoms. Additionally, cyclic groups can be modified to have different numbers of atoms in the ring and / or include heteroatoms. Furthermore, aromatic hydrocarbons can be converted into cyclic rings and vice versa. For example, the ring can be 5-7 atoms and can be a carbon ring or a heterocycle.

[0254] As used herein, the terms "analog," "analogue," or "derivative" refer to a compound or molecule produced from a parent compound or molecule through one or more chemical reactions. Therefore, an analog may be a structure having a structure similar to that of the small molecule therapeutic agents described herein, or may be based on a scaffold of the small molecule therapeutic agents described herein but differ from it in certain components or structural configurations that may have similar or opposite metabolic effects. Analogs or derivatives of any small molecule inhibitor according to this disclosure may be used to treat diseases or disorders.

[0255] In some embodiments, the small molecule therapeutic agents described herein can be independently derivatized or analogs prepared therefrom by modifying hydrogen groups with other substituents independently of each other. That is, each atom on each molecule can be modified independently relative to other atoms on the same molecule. Any conventional modifications used to generate derivatives / analytes can be used. For example, atoms and substituents can be independently composed of hydrogen, alkyl, aliphatic, straight-chain aliphatic, aliphatic with chain heteroatoms, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic with one or more heteroatoms, aromatic, heteroaromatic, polyaromatic, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halogenated aliphatic, etc. Furthermore, any cyclic group on the compound can be derivatized to increase and / or decrease the ring size and change the skeletal atom to a carbon atom or a heteroatom.

[0256] Nucleic acid therapy In some embodiments, the compositions of this disclosure comprise in vitro transcribed (IVT) RNA molecules. For example, in some embodiments, the compositions of this disclosure comprise IVT RNA molecules encoding reagents. In some embodiments, the IVT RNA molecules of the compositions are nucleoside-modified mRNA molecules. In some embodiments, the reagents are used to target immune cells to pathogens or tumor cells of interest. In some embodiments, the IVT RNA molecules encode chimeric antigen receptors (CARs).

[0257] In some embodiments, the CAR is specific for binding to one or more antigens. In some embodiments, the antigen includes at least one viral antigen, bacterial antigen, fungal antigen, parasitic antigen, influenza antigen, tumor-associated antigen, tumor-specific antigen, or any combination thereof.

[0258] However, this disclosure is not limited to any particular reagent or combination of reagents. In some embodiments, the composition comprises an adjuvant. In some embodiments, the composition comprises a nucleic acid molecule encoding an adjuvant. In some embodiments, the composition comprises nucleoside-modified RNA encoding an adjuvant.

[0259] In some embodiments, the composition comprises at least one RNA molecule encoding a combination of at least two reagents. In some embodiments, the composition comprises a combination of two or more RNA molecules encoding a combination of two or more reagents.

[0260] In some embodiments, this disclosure provides a method for inducing an immune response in a subject. For example, the method can be used to provide immunity against viruses, bacteria, fungi, parasites, cancer, etc., in a subject. In some embodiments, the method includes administering to the subject a composition comprising one or more LNP molecules formulated for targeting immune cells in vivo, the LNP molecules comprising one or more RNAs encoding at least one antigen, adjuvant, or a combination thereof.

[0261] In some embodiments, this disclosure provides a method for gene editing of a subject's immune cells. For example, the method may be used to deliver one or more components of a gene editing system (e.g., components of a CRISPR system) to the subject's immune cells. In some embodiments, the method includes administering to the subject a composition comprising one or more ionizable LNP molecules formulated for targeted T cell delivery, and comprising one or more nucleoside-modified RNA molecules for gene editing.

[0262] In some embodiments, the method includes administering the composition to a subject. In some embodiments, the method includes administering multiple doses to a subject. In some embodiments, the method includes administering a single dose of the composition, wherein the single dose is effective in delivering the targeted therapeutic agent.

[0263] In other relevant aspects, the therapeutic agent is an isolated nucleic acid. In some embodiments, the isolated nucleic acid molecule is either a DNA molecule or an RNA molecule. In some embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA, or miRNA molecule. In some embodiments, the isolated nucleic acid molecule encodes a therapeutic peptide, such as a thrombotic regulatory protein, an endothelial protein C receptor (EPCR), an antithrombotic protein including plasminogen activator and its mutants, or an antioxidant protein including catalase, superoxide dismutase (SOD), and iron chelating proteins. In some embodiments, the therapeutic agent is an siRNA, miRNA, shRNA, or antisense molecule that inhibits target nucleic acids, including nucleic acids encoding proteins involved in the exacerbation of the pathological process.

[0264] In some embodiments, the nucleic acid contains a promoter / regulatory sequence that enables the nucleic acid to direct its expression. Therefore, this disclosure includes expression vectors and methods for introducing exogenous nucleic acids into cells and simultaneously expressing them within the cells, such as those described by Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), as well as those described elsewhere herein.

[0265] In some implementations, siRNA is used to reduce the level of target proteins. RNA interference (RNAi) is a phenomenon that introduces double-stranded RNA (dsRNA) into various organisms and cell types, leading to the degradation of complementary mRNA. In cells, long dsRNA is cleaved into short interfering RNAs, or siRNAs, by a ribonuclease called Dicer. The siRNA then assembles with protein components to form an RNA-induced silencing complex (RISC), which unfolds in the process. The activated RISC then binds to the complementary transcript via base-pairing interactions between the antisense strand of the siRNA and the mRNA. The bound mRNA is cleaved, and the sequence-specific degradation of the mRNA results in gene silencing. For example, see U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG14(7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) described a chemical modification of siRNA that facilitates intravenous systemic delivery. Optimizing siRNA requires consideration of overall G / C content, terminal C / T content, Tm, and nucleotide content of the 3' dangling nucleotides. For example, see Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, this disclosure also includes methods for reducing PTPN22 levels using RNAi technology.

[0266] In one aspect, this disclosure includes a vector comprising siRNA or an antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide. The incorporation of the desired polynucleotide into the vector and the choice of the vector are well known in the art, for example, by Sambrook et al. (2012) and Ausubel et al. (1997) and elsewhere herein.

[0267] In some embodiments, the expression vector described herein encodes a short hairpin RNA (shRNA) therapeutic agent. shRNA molecules are well known in the art and target the mRNA of a target, thereby reducing the expression of the target. In some embodiments, the encoded shRNA is expressed by a cell and then processed into siRNA. For example, in some cases, the cell possesses a natural enzyme (such as dicer) that cleaves the shRNA to form siRNA.

[0268] To assess the expression of siRNA, shRNA, or antisense polynucleotides, the expression vector introduced into cells may also contain a selective marker gene or a reporter gene, or both, to facilitate the identification of expressing cells from a cell population seeking transfection or infection using the delivery vector of this disclosure. In other embodiments, the selective marker may be carried on a separate DNA fragment or contained within the delivery vector. Both the selective marker and the reporter gene may be flanked by suitable regulatory sequences to enable their expression in host cells. Useful selective markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance.

[0269] Therefore, in one aspect, the delivery vector may comprise a vector containing a nucleotide sequence or construct to be delivered. The choice of vector will depend on the host cell into which it will be subsequently introduced. In a particular embodiment, the vector of this disclosure is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In a particular embodiment, the expression vector is selected from viral vectors, bacterial vectors, and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors can be used in this disclosure to produce polynucleotides or their homologous polypeptides. Many such systems are commercially available.

[0270] For example, the vector that introduces the nucleic acid sequence can be a plasmid, which, when introduced into a cell, may or may not integrate into the host cell's genome. Illustrative, non-limiting examples of vectors into which nucleotide sequences or gene constructs of this disclosure may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0271] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In one particular embodiment, the vector is a vector that can be used to transform animal cells.

[0272] In some implementations, the recombinant expression vector may also contain nucleic acid molecules encoding peptides or peptide-like molecules.

[0273] Promoters can be promoters naturally associated with a gene or polynucleotide sequence, which can be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such promoters can be referred to as "endogenous." Similarly, enhancers can be enhancers naturally associated with a polynucleotide sequence, located downstream or upstream of that sequence. Optionally, certain advantages can be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a polynucleotide sequence in their natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters and enhancers isolated from any other prokaryotic, viral, or eukaryotic cells, and promoters or enhancers that are not "naturally present," i.e., those containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to synthesizing nucleic acid sequences for the production of promoters and enhancers, recombinant cloning and / or nucleic acid amplification technologies (including PCR™) can be used in conjunction with the compositions disclosed herein to produce sequences (US Patent 4,683,202, US Patent 5,928,906). Furthermore, control sequences that guide the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts can also be considered.

[0274] Of course, it is important to use promoters and / or enhancers that effectively guide the expression of DNA fragments in the selected cell type, organelle, and organism for expression. Those skilled in the art of molecular biology generally know how to use combinations of promoters, enhancers, and cell types for protein expression; see, for example, Sambrook et al. (2012). The promoters employed can be constitutive, tissue-specific, inducible, and / or, under appropriate conditions, advantageous for guiding high-level expression of the introduced DNA fragment, such as in the large-scale production of recombinant proteins and / or peptides. Promoters can be heterologous or endogenous.

[0275] Recombinant expression vectors can also contain selective marker genes, which aids in host cell selection. Suitable selective marker genes are protein-encoding genes, such as G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof, such as the Fc portion of immunoglobulins, preferably IgG. Selective markers can be introduced from the nucleic acid of interest into separate vectors.

[0276] After the siRNA polynucleotide is generated, those skilled in the art will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, siRNA polynucleotides can be further designed to resist degradation by modifying them to include thiophosphates or other linkages, methylphosphonates, sulfones, sulfates, ketones, dithiophosphates, phosphoramidates, phosphates, etc. (see, for example, Agrawal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett.26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

[0277] Any polynucleotide can be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, adding flanking sequences at the 5' and / or 3' ends; using thiophosphates or 2'O-methyl groups in the backbone instead of phosphodiester linkages; and / or including non-traditional bases such as inosine, queuosine, and wybutosine, as well as acetylation, methylation, thiolation, and other modifications of adenine, cytidine, guanine, thymine, and uridine.

[0278] In some embodiments of this disclosure, the antisense nucleic acid sequence expressed by a plasmid vector is used as a therapeutic agent to inhibit the expression of a target protein. The antisense expression vector is used to transfect mammalian cells or mammals themselves, thereby resulting in a reduction in the endogenous expression of the target protein.

[0279] Antisense molecules and their use in suppressing gene expression are well known in the art (see, for example, Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). An antisense nucleic acid is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule, as the term is defined elsewhere herein (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule, thereby suppressing gene translation.

[0280] The use of antisense methods to suppress gene translation is known in the art, as described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). As taught in Inoue, 1993, U.S. Publication No. 5,190,931, such antisense molecules can be delivered to cells via gene expression using DNA encoding antisense molecules.

[0281] Optionally, the antisense molecules of this disclosure can be synthesized and then provided to cells. Antisense oligomers of about 10 to about 30 nucleotides are preferred, more preferably about 15 nucleotides, because they are readily synthesized and introduced into target cells. The synthetic antisense molecules contemplated in this disclosure include oligonucleotide derivatives known in the art that exhibit improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0282] In some embodiments of this disclosure, ribozymes are used as therapeutic agents to inhibit the expression of target proteins. Ribozymes that can be used to inhibit the expression of target molecules can be designed by incorporating the target sequence into a basic ribozyme structure, such as one that is complementary to the mRNA sequence encoding the target molecule. Ribozymes targeting target molecules can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA), or they can be expressed by DNA genes encoding them.

[0283] In some embodiments, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, wherein a guide RNA (gRNA) targeting a gene encoding a target molecule and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the target gene. In some embodiments, the therapeutic agent comprises gRNA or a nucleic acid molecule encoding gRNA. In some embodiments, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0284] In some embodiments, the formulation comprises miRNA or a miRNA mimic. In some embodiments, the formulation comprises a nucleic acid molecule encoding a miRNA or a miRNA mimic.

[0285] miRNAs are small non-coding RNA molecules that can induce post-transcriptional silencing of specific genes in a cell by inhibiting translation or degrading target mRNAs. miRNAs can be perfectly complementary to their target nucleic acids or have non-complementary regions, resulting in a "bulge" in the non-complementary region. miRNAs can suppress gene expression by inhibiting translation, for example, when the miRNA is not perfectly complementary to the target nucleic acid, or by inducing degradation of the target RNA; degradation is believed to occur only when the miRNA binds to its target in a perfectly complementary manner. This disclosure may also include double-stranded precursors of miRNAs. The length of miRNAs or pri-miRNAs can be 18-100 nucleotides or 18-80 nucleotides. The length of mature miRNAs can be 19-30 nucleotides or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. Pre-miRNAs typically have a length of about 70-100 nucleotides and exhibit a hairpin conformation. miRNAs are generated in vivo from pre-miRNAs by the enzymes Dicer and Drosha, which specifically process long pre-miRNAs into functional miRNAs. The hairpin or mature microRNA, or pri-miRNA reagents of this disclosure can be synthesized in vivo via cell-based systems or in vitro via chemical synthesis.

[0286] In various embodiments, the reagent comprises an oligonucleotide containing a nucleotide sequence of a disease-associated miRNA. In some embodiments, the oligonucleotide comprises a nucleotide sequence of a disease-associated miRNA in mature or hairpin-form premicroRNA. In other embodiments, combinations of oligonucleotides comprising sequences of one or more disease-associated miRNAs, any premiRNA, any fragment, or any combination thereof are contemplated.

[0287] miRNAs can be synthesized to include modifications that impart desired characteristics. For example, such modifications can improve stability, hybridization thermodynamics with target nucleic acids, targeting specific tissues or cell types, or cell permeability, for example, through mechanisms that are dependent on or independent of endocytosis.

[0288] Modifications can also improve sequence specificity, thereby reducing off-target effects. Methods for synthesis and chemical modification are described in more detail below. If desired, miRNA molecules can be modified to stabilize the miRNA against degradation, extend its half-life, or otherwise enhance its efficacy. Desired modifications are described, for example, in U.S. Patent Publications 20070213292, 20060287260, 20060035254, 20060008822, and 2005028824, the entire contents of each of which are incorporated herein by reference. To enhance nuclease resistance and / or binding affinity to targets, the single-stranded oligonucleotide reagents of this disclosure may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphate thioester linkages. Including locked nucleic acids (LNAs), vinyl nucleic acids (ENAs), such as 2'-4'-vinyl-bridged nucleic acids, and certain nucleotide modifications can also increase binding affinity to targets. Adding pyranose to the oligonucleotide backbone can also reduce endonuclease cleavage. Oligonucleotides can be further modified by including a 3' cationic group or by reversing the 3'-terminal nucleoside linker to the 3'-3' end. Alternatively, the 3' end can be blocked with an aminoalkyl group. Other 3' conjugates can inhibit 3'-5' exonuclease cleavage. While not theoretically constrained, the 3' may inhibit exonuclease cleavage by sterically blocking the binding of exonucleases to the 3' end of the oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5' exonucleases.

[0289] In some embodiments, the miRNA comprises a 2'-modified oligonucleotide containing oligodeoxynucleotide internucleotides, some or all of which are modified with phosphate thioesters for nuclease resistance. The presence of methylphosphonate modification increases the affinity of the oligonucleotide for its target RNA, thereby reducing IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It should be understood that the methods and reagents of this disclosure can be used in conjunction with any techniques that may be developed to enhance the stability or efficacy of repressive nucleic acid molecules.

[0290] miRNA molecules include nucleotide oligomers containing a modified backbone or non-natural internucleotide links. Oligomers with modified backbones include oligomers that retain a phosphorus atom in the backbone and oligomers that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides without a phosphorus atom in their internucleotide backbone are also considered nucleotide oligomers. Nucleotide oligomers with modified oligonucleotide backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), phosphonites, phosphoramidates, thiophosphatates, thioalkylphosphonates, thioalkylphosphate triester polymers, and boron phosphates. Various salts, mixed salts, and free acid forms are also included.

[0291] The miRNAs described herein, whether in mature or hairpin form, can be provided as naked oligonucleotides. In some cases, formulations that facilitate the delivery of miRNAs or other nucleotide oligomers to cells may be required (see, for example, U.S. Patent Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, the entire contents of each of which are incorporated herein by reference).

[0292] In some instances, the miRNA composition is at least partially crystalline, homogeneously crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another instance, the miRNA composition is in an aqueous phase, such as in a solution comprising water. The aqueous phase or crystalline composition may be incorporated into a delivery carrier, such as liposomes (particularly for the aqueous phase) or particles (e.g., microparticles suitable for the crystalline composition). Typically, the miRNA composition is formulated in a manner compatible with the intended method of administration. The miRNA composition may be formulated in combination with another reagent, such as another therapeutic agent or a reagent for stabilizing oligonucleotides, such as a protein complexed with an oligonucleotide reagent. Other reagents include chelating agents, such as EDTA (e.g., for removing divalent cations, such as Mg), salts, and RNase inhibitors (e.g., broad-spectrum specific RNase inhibitors). In some embodiments, the miRNA composition includes another miRNA, such as a second miRNA composition (e.g., a microRNA different from the first). Other formulations may include at least three, five, ten, twenty, fifty, or one hundred or more different oligonucleotide species.

[0293] In some embodiments, the composition comprises an oligonucleotide composition that mimics miRNA activity. In some embodiments, the composition comprises an oligonucleotide having nucleobase identity with the nucleobase sequence of the miRNA and is therefore designed to mimic miRNA activity. In some embodiments, the oligonucleotide composition mimicking miRNA activity comprises a double-stranded RNA molecule that mimics a mature miRNA hairpin or a processed miRNA double-strand.

[0294] In some embodiments, the oligonucleotide shares identity with the nucleobase sequence of an endogenous miRNA or miRNA precursor. The selected oligonucleotide used in the compositions disclosed herein can be of one of several lengths. Such an oligonucleotide can be 7 to 100 linked nucleosides. For example, an oligonucleotide sharing nucleobase identity with the miRNA can be 7 to 30 linked nucleosides. An oligonucleotide sharing identity with the miRNA precursor is at most 100 linked nucleosides. In some embodiments, the oligonucleotide comprises 7 to 30 linked nucleosides. In some embodiments, the oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In some embodiments, the oligonucleotide comprises 19 to 23 linked nucleosides. In some embodiments, the oligonucleotide is 40 to 50, 60, 70, 80, 90, or 100 linked nucleosides.

[0295] In some embodiments, the oligonucleotide has a sequence that is identical to the miRNA or its precursor. The nucleotide sequence of the mature miRNA described herein and its corresponding stem-loop sequence are sequences found in miRBase, an online searchable database of miRNA sequences and annotations. Entry in the miRBase sequence database represents a predicted hairpin portion of the miRNA transcript (stem-loop), containing information about the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (premiRNAs) and may in some cases include flanking sequences of the presumed primary transcript and the presumed primary transcript. The miRNA nucleotide sequence described herein includes any version of the miRNA, including sequences described in miRBase sequence database version 10.0 and sequences described in any earlier releases of the miRBase sequence database. The release of the sequence database may lead to the renaming of certain miRNAs. The release of the sequence database may lead to variations in the mature miRNA sequence. The compositions disclosed herein comprise oligomeric compounds containing oligonucleotides that are identical to any version of the nucleotide sequence of the miRNA described herein.

[0296] In some embodiments, the oligonucleotide has a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to that of the miRNA in a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Therefore, in some embodiments, the oligonucleotide's nucleotide sequence may have one or more nucleotides that are different from those of the miRNA.

[0297] In some embodiments, the composition comprises a nucleic acid molecule encoding a miRNA, precursor, mimic, or fragment thereof. For example, the composition may comprise a viral vector, plasmid, granule, or other expression vector suitable for expressing the miRNA, precursor, mimic, or fragment thereof in desired mammalian cells or tissues.

[0298] combination In some embodiments, the combinations of the reagents disclosed herein include the reagent combinations described herein. In some embodiments, compositions comprising the reagent combinations described herein have an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual reagent. In other embodiments, compositions comprising the reagent combinations described herein have a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual reagent.

[0299] A composition comprising a combination of reagents contains individual reagents in any suitable proportion. For example, in some embodiments, the composition contains two individual reagents in a 1:1 ratio. However, the composition is not limited to any particular proportion. Rather, any proportion that has proven effective is included.

[0300] Pharmaceutical Composition Formulations of the pharmaceutical compositions described herein may be prepared by any method known or subsequently developed in the field of pharmacology. Generally, such preparation methods involve the step of binding the active ingredient with a carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into desired single-dose or multi-dose units.

[0301] Although the description of the pharmaceutical compositions provided herein is primarily directed toward pharmaceutical compositions suitable for ethical human administration, those skilled in the art will understand that such compositions are generally suitable for administration to all animal species. Modifications to pharmaceutical compositions suitable for human administration are well known to be made to make them suitable for administration to a variety of animals, and such modifications can be designed and performed by a reasonably skilled veterinary pharmacologist through routine (if any) experiments. Subjects intended to administer the pharmaceutical compositions of this disclosure include, but are not limited to, humans and other primates, mammals, including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0302] Pharmaceutical compositions that can be used in the methods disclosed herein can be prepared, packaged, or marketed as formulations suitable for ocular, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, oral, intravenous, intradermal, intramuscular, or other routes of administration. Other formulations considered include projected nanoparticles, liposome formulations, resealed red blood cells containing the active ingredient, and immunogenicity-based formulations.

[0303] The pharmaceutical compositions disclosed herein can be prepared, packaged, or sold in bulk, in single unit doses, or in multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to a subject or a convenient portion of such a dose, such as half or one-third of such a dose.

[0304] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any adjuncts in the pharmaceutical compositions disclosed herein will vary depending on the identity, size, and condition of the treated subject, and further depending on the route of administration of the composition. For example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0305] In addition to the active ingredient, the pharmaceutical compositions disclosed herein may further comprise one or more other pharmaceutically active agents.

[0306] The controlled-release or sustained-release formulations of the pharmaceutical compositions disclosed herein can be prepared using conventional techniques.

[0307] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical destruction of the subject’s tissues and administration of the pharmaceutical composition through ruptures in the tissues. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection, administration through a surgical incision, administration through a non-surgical wound penetrating tissue. In particular, parenteral administration is intended to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal, intratumoral, intravenous, intraventricular, and renal dialysis infusion techniques.

[0308] Formulations of pharmaceutical compositions intended for parenteral administration comprise an active ingredient bound to a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or marketed in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or marketed in unit dosage forms, such as in ampoules or multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may also contain one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In some embodiments of formulations for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0309] Pharmaceutical compositions can be prepared, packaged, or marketed as sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated using known techniques and may contain additional components, such as dispersants, wetting agents, or suspending agents as described herein, in addition to the active ingredient. Such sterile injectable formulations can be prepared using non-toxic, parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic monoglycerides or diglycerides. Other available parenterally applicable formulations include those containing the active ingredient in microcrystalline form, liposome formulations, or formulations in which the active ingredient is a component of a biodegradable polymer system. Compositions intended for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0310] The pharmaceutical compositions disclosed herein can be prepared, packaged, or sold as formulations suitable for oral administration to the lungs. Such formulations may comprise dry particles containing the active ingredient, with a diameter ranging from about 0.5 to about 7 micrometers, preferably from about 1 to about 6 micrometers. These compositions are conveniently presented as dry powders for administration using devices including dry powder reservoirs into which a propellant stream can be directed to disperse the powder, or using self-propelled solvent / powder dispensing containers, such as devices containing the active ingredient dissolved or suspended in a low-boiling-point propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 micrometers, and at least 95% by number of the particles have a diameter less than 7 micrometers. More preferably, at least 95% by weight of the particles have a diameter greater than 1 micrometer, and at least 90% by number of the particles have a diameter less than 6 micrometers. The dry powder compositions preferably comprise a solid fine powder diluent, such as sugar, and are conveniently provided in unit doses.

[0311] Low-boiling-point propellants typically include liquid propellants with a boiling point below 65°F at atmospheric pressure. Typically, the propellant comprises 50 to 99.9% (w / w) of the composition, and the active ingredient comprises 0.1 to 20% (w / w) of the composition. The propellant may also contain additional components, such as liquid nonionic or solid anionic surfactants or solid diluents (preferably having a particle size on the same order of magnitude as the particles containing the active ingredient).

[0312] Formulations of pharmaceutical compositions intended for parenteral administration comprise an active ingredient bound to a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or marketed in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or marketed in unit dosage forms, such as in ampoules or multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may also contain one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In some embodiments of formulations for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0313] Pharmaceutical compositions can be prepared, packaged, or marketed as sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated using known techniques and may contain additional components, such as dispersants, wetting agents, or suspending agents as described herein, in addition to the active ingredient. Such sterile injectable formulations can be prepared using non-toxic, parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic monoglycerides or diglycerides. Other available parenterally applicable formulations include those containing the active ingredient in microcrystalline form, liposome formulations, or formulations in which the active ingredient is a component of a biodegradable polymer system. Compositions intended for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0314] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous carriers and solvents; oily carriers and solvents; suspending agents; dispersants or wetting agents; emulsifiers, demulsifiers; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions disclosed herein are known in the art, for example, as described in Remington’s Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0315] Application / Dosage The method of administration may affect the composition of the effective dose. Therapeutic agents can be administered to patients before or after the onset of disease or disorder. Furthermore, several fractionated doses may be administered daily or sequentially, as well as alternating doses, or the dose may be administered continuously by infusion or by bolus. Additionally, the dose of the therapeutic agent may be increased or decreased proportionally, as indicated by the urgency of the treatment or prevention situation.

[0316] The administration of the compositions disclosed herein to patients (such as mammals, such as humans) may be performed using known procedures at doses and durations for effective treatment of the diseases or disorders described herein. The effective amount of the therapeutic agent (i.e., the composition) required to achieve a therapeutic effect may vary depending on factors such as the activity of the particular therapeutic agent used; the time of administration; the excretion rate of the composition; the duration of treatment; other drugs, compounds, or materials used in combination with the composition; the state of the disease or disorder; age; sex; weight; condition; general health status; and the patient's medical history, as well as similar factors known in the medical community. Dosing methods may be adjusted to provide an optimal therapeutic response. For example, several fractionated doses may be administered daily, or the dose may be reduced proportionally as indicated by the urgency of the treatment situation. A non-limiting example of an effective dose range for the therapeutic compositions of this disclosure is from about 0.01 mg / kg to 100 mg / kg body weight / day of the active agent (i.e., nucleic acid). Those skilled in the art will be able to investigate the relevant factors and determine the effective amount of the therapeutic composition without inappropriate experimentation.

[0317] This composition can be administered to animals several times a day, or at a less frequent frequency, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months, or even once a year or less. It should be understood that, in non-limiting examples, the daily dose of the composition can be administered daily, every other day, every two days, every three days, every four days, or every five days. For example, for every other day administration, a dose of 5 mg daily could be administered starting on Monday, followed by the first dose of 5 mg daily on Wednesday, the second dose of 5 mg daily on Friday, and so on. The frequency of dosage will be apparent to a skilled technician and depends on many factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the animal.

[0318] The actual dose level of the active ingredient in the pharmaceutical composition disclosed herein can be varied to obtain an effective amount of the active ingredient to achieve the desired therapeutic response for a particular patient, composition, and administration mode without toxicity to the patient.

[0319] A physician (e.g., a doctor or veterinarian) with ordinary skills in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may start with a dose of the disclosed compound in the pharmaceutical composition below the required level to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0320] In certain embodiments, preparing the compound in dosage units is particularly advantageous for ease of administration and dosage uniformity. As used herein, dosage unit form refers to physically discrete units suitable as a single dose to a patient to be treated; each unit contains a predetermined amount of the therapeutic composition to be combined with a desired drug carrier to produce the desired therapeutic effect. The dosage unit form of this disclosure depends on and is directly dependent on (a) the unique properties of the therapeutic composition and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the field of compounding / formulating such therapeutic compositions for treating a patient's disease or disorder.

[0321] In some embodiments, the dosage range for administering the compositions of this disclosure to a patient is from 1 to 5 times daily or more. In other embodiments, the dosage range for administering the compositions of this disclosure to a patient includes, but is not limited to, once daily, once every two days, once every three days to once weekly, and once every two weeks. It will be apparent to those skilled in the art that the frequency of administration of the various combinations of this disclosure will vary from subject to subject to many factors, including but not limited to age, disease or disorder to be treated, sex, general health, and other factors. Therefore, this disclosure should not be construed as limiting to any particular dosing method, and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking into account all other factors concerning the patient.

[0322] The amount of active agent used in the compositions of this disclosure for application may be in the range of about 1 µg to about 7500 mg, about 20 µg to about 7000 mg, about 40 µg to about 6500 mg, about 80 µg to about 6000 mg, about 100 µg to about 5500 mg, about 200 µg to about 5000 mg, about 400 µg to about 4000 mg, about 800 µg to about 3000 mg, about 1 mg to about 2500 mg, about 2 mg to about 2000 mg, about 5 mg to about 1000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all of the increments therein.

[0323] In some embodiments, the amount of the active agent (i.e., nucleic acid) present in the compositions of this disclosure is from about 0.5 µg to about 5000 mg. In some embodiments, the amount of the active agent present in the compositions of this disclosure used in the compositions described herein is less than about 5000 mg, or less than about 4000 mg, or less than about 3000 mg, or less than about 2000 mg, or less than about 1000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound as described herein is less than about 1000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and all or part of the increments thereof.

[0324] In some embodiments, this disclosure relates to packaged pharmaceutical compositions comprising a container holding a therapeutically effective amount of the disclosed composition alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or alleviate one or more symptoms of a patient’s disease or disorder.

[0325] The term "container" includes any container used to contain a pharmaceutical composition or to manage stability or absorbency. For example, in some embodiments, a container is a package that includes a pharmaceutical composition, such as a liquid (solution and suspension), semi-solid, lyophilized solid, solution, powder, or lyophilized formulation present in a two-chamber configuration. In other embodiments, the container is not a package containing a pharmaceutical composition; that is, the container is a receiver, such as a box or bottle containing a packaged or unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. Furthermore, packaging techniques are known in the art. It should be understood that instructions for use of the pharmaceutical composition may be included on the package containing the pharmaceutical composition, and thus the instructions for use form an additional functional relationship with the packaged product. However, it should be understood that the instructions for use may contain information relating to the compound's ability to perform its intended function, such as its ability to treat, prevent, or reduce disease or disorder in a patient.

[0326] application Routes of administration for any of the compositions disclosed herein include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, tongue, oral cavity, urethra, vagina (e.g., vagina and perivasal), nasal (internal) and (trans)rectal), intravesical, intrapulmonary, intraduodenal, gastrointestinal, intrathecal, epidural, intrathoracic, intraperitoneal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical application.

[0327] Suitable compositions and dosage forms include, for example, tablets, capsules, pouches, pills, gel caps, sleeves, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma, lozenges, creams, pastes, ointments, lotions, tablets, suppositories, liquid sprays for nasal or oral administration, dry powder or nebulized formulations for inhalation, and compositions and formulations for intravesical administration. It should be understood that the formulations and compositions useful in this disclosure are not limited to the specific formulations and compositions described herein.

[0328] External application As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical destruction of the subject’s tissues and administration of the pharmaceutical composition through destruction of the tissues. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection, administration through a surgical incision, administration through tissue penetration of a non-surgical wound, etc. In particular, parenteral administration is considered to include, but is not limited to, intraventricular, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal, and renal dialysis infusion techniques. In some embodiments, the compositions of this disclosure are administered intraventricularly.

[0329] Formulations of pharmaceutical compositions suitable for parenteral administration include an active ingredient combined with a pharmaceutically acceptable carrier (such as sterile water or sterile isotonic saline). Such formulations may be prepared, packaged, or marketed in forms suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or marketed in unit dosage forms, such as in ampoules or in multi-dose containers containing preservatives. Injectable formulations may also be prepared, packaged, or marketed in devices such as patient-controlled analgesia (PCA). Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further include one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Pharmaceutical compositions may be prepared, packaged, or marketed in the form of sterile injectable aqueous or oily suspensions or solutions. The suspension or solution may be formulated according to known techniques and may include, in addition to the active ingredient, additional components such as dispersants, wetting agents, or suspending agents as described herein. Such sterile injectable formulations can be prepared using non-toxic, parenterally acceptable diluents or solvents (such as water or 1,3-butanediol). Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixative oils (such as synthetic mono- or diglycerides). Other useful parenterally applicable formulations include those containing the active ingredient in microcrystalline form in recombinant human albumin, fluid gelatin, liposome formulations, or those containing the active ingredient as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymers or hydrophobic materials such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0330] Example Various embodiments of this application can be better understood by referring to the following illustrative examples. The scope of this application is not limited to the embodiments given herein.

[0331] Materials and Methods Material TLR7 / 8 agonist 1,2-dihydrochloride was purchased from Cayman Chemical (Ann Arbor, MI). 1,2-Epoxydodecane (C12) and cholesterol were purchased from Sigma-Aldrich (St. Louis, MO). Core 200 was custom-made from Enamine (Monmouth Junction, NJ), and other polyamine cores were purchased from Sigma-Aldrich and TCI (Tokyo, Japan). Anti-mouse CD16 / 32 antibody, APC anti-mouse CD11c antibody, FITC anti-mouse CD80 antibody, and PE anti-mouse CD86 antibody were purchased from Biolegend (San Diego, CA). Uncoated ELISA assays for mouse IL-1β, mouse IL-12 p70, and mouse TNF-α, LysoTracker® Deep Red, 3,3'-bis(octadecyloxacarbonyl)cyanine perchlorate (DiO), and 1,1'-dioctyl-3,3,3',3'-tetramethylindolecarbonylcyanine perchlorate (DiR) were purchased from Invitrogen (Carlsbad, CA). 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000), and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL). DLin-MC3-DMA was purchased from MedChem Express (Monmouth Junction, NJ). m1ψ-modified luciferase mRNA and SARS-CoV-2 mRNA were produced according to methods known to those skilled in the art as described in the literature.

[0332] Synthesis of adjuvant lipids By using ring-opening reactions (i.e., S-reaction of epoxides and amines) N2) The adjuvant lipid C12 TLRa was synthesized by reacting dodecane oxide (C12) with TLR7 / 8 agonist 1 dihydrochloride. Briefly, 10 mg of TLR7 / 8 agonist 1 dihydrochloride was dissolved in 0.8 mL of ethanol in a glass vial equipped with a magnetic stir bar. 8 μL of triethylamine was added to neutralize the hydrochloride, followed by the addition of 20 mg of C12. The vial was sealed, and the mixture was stirred at 80 °C for 48 hours. The crude product was purified by a CombiFlash NextGen300+ chromatographic system (Teledyne ISCO, Lincoln, NE) with gradient elution from CH2Cl2 to 75:22:3CH2Cl2 / MeOH / NH4OH (aqueous solution). The desired fraction (yield 44%) was collected. C12 TLRa was analyzed by mass spectrometry (calculated MS: 728.12, finding [M+2H)). 2+ Characterized by (=365.25) and nuclear magnetic resonance spectroscopy. 1 H-NMR (400 MHz, DMSO-d6) δ:7.78 (d, J = 8.3 Hz, 1H), 7.57 (dd, J = 8.4, 1.3 Hz, 1H), 7.35 – 7.29 (m, 1H), 7.27 (d, J = 7.9 Hz, 2H), 7.05 – 7.00 (m, 1H), 6.98 (d, J = 8.0 Hz, 2H), 5.84 (s, 2H), 3.61 – 3.47 (m, 2H), 3.44 (s, 2H), 2.90 (t, J = 7.7 Hz, 2H), 2.68 (q, J= 1.9 Hz, 2H), 2.34 (t, J = 2.8 Hz, 2H), 1.69 (p, J = 7.6 Hz, 2H), 1.37 (dt, J =14.9, 7.5 Hz, 2H), 1.22 (s, 36H), 0.90 – 0.81 (m, 9H).

[0333] General methods for the synthesis of polyamine-derived lipids As previously mentioned, the polyamine nucleus reacts with the saturated amine requiring an excess molar amount of C12. Taking C12-113 as an example, the 113 nucleus (1 equivalent) and C12 (4.8 equivalents) were mixed at 80°C under pristine conditions for 48 hours. The crude product was used for initial library screening. To purify the best-performing C12-113 lipids, the crude product was separated using a CombiFlash NextGen 300+ chromatography system as described above. The fully saturated product was collected and identified by mass spectrometry (calculated MS: 854.49, [M+2H] was found). 2+ =429.13), and used for subsequent experiments.

[0334] Structural simulation of agonist-TLR7 interaction For the first time, the structures of TLR7 / 8 agonist 1 and C12 TLRa were optimized using molecular dynamics simulations under the CHARMm force field. The precise TLR7 protein crystal structure was derived from the structure of the TLR7 / R848 complex (PDB ID: 5GMH), with any ligands or solvent molecules removed. Structural simulations of the TLR7 dimer and agonist were performed using CDocker docking simulations and in-situ structural superposition. An overview of potential nonvalent interactions, binding capsules, and binding sites between the TLR7 dimer and the corresponding agonist was generated using BIOVIA Discovery Studio 2018.

[0335] LNP formulations As previously described, LNPs were prepared via microfluidic mixing. In short, within a microfluidic chip device, an ethanol phase containing lipids (with or without C12-TLRa substitution), DOPE, cholesterol, and DMG-PEG in a molar ratio of 35:16:46.5:2.5 was mixed with an aqueous phase containing mRNA (10 mM citrate buffer, pH 3) at a flow rate of 1:3 and a lipid / RNA weight ratio of 10:1. LNPs were dialyzed against 1x PBS for 2 hours in a 20 kDa MWCO cartridge, sterilized through a 0.22 μM filter, and stored at 4°C. DiO or DIR-labeled LNPs were obtained by mixing DiO or DIR (1 mol% of total lipids) with the LNPs prior to dialyze.

[0336] LNP features Hydrodynamic dimensions, polydispersity index (PDI), and zeta potential of LNPs were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The morphology of LNPs was characterized by transmission electron microscopy (JEOL 1010, Tokyo, Japan). The mRNA encapsulation efficiency and p-value of LNPs were determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen) and a 6-(p-toluidine)naphthalene-2-sulfonic acid (TNS) assay, respectively. Ka .

[0337] Cell culture and animal research HEK-blue mTLR7 cell lines were purchased from InvivoGen (#hkb-mTLR7). These cells were maintained according to the supplier's instructions. Mouse macrophage DC2.4 cell lines were purchased from American Type Culture Collection (ATCC) and stored in Dulbecco-modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. All cells were cultured at 37°C in a humidified incubator with 5% CO2 and routinely tested for mycoplasma contamination.

[0338] BMDCs were generated from C57BL / 6 mice. Briefly, bone marrow cells were flushed from the femur and tibia of mice, lysed with ACK buffer to remove erythrocytes, and then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin, 1% HEPES, 0.1% mM β-mercaptoethanol, 20 ng / mL interleukin-4 (IL-4, #214-14, PeproTech), and 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF, #315-03, PeproTech). On day 6, non-adherent and loosely adherent cells were collected for studies.

[0339] C57BL / 6 female mice (6-8 weeks old, 18-20g body weight) were purchased from Jackson Laboratory.

[0340] In vitro mLuc delivery DC cells or BMDCs were seeded overnight in 96-well plates at a density of 10,000 cells per well, followed by treatment with LNP loaded with mLuc at a specified dose for 24 hours. Luciferase expression was assessed using the Luciferase Reporter 1000 assay system (E4550, Promega) according to the manufacturer's protocol, and cell viability was measured using the CellTiter Glo luminescent cell viability assay (G7572, Promega). Relative luciferase expression was reported as relative light units (RLU) normalized relative to cell viability. Free mRNA was used as a control.

[0341] TLR7 reporter gene testing According to the manufacturer's instructions, the TLR7 agonist activity of C12-TLRa was tested on HEK-Blue mTLR7 reporter cells using the HEK-Blue Detection Kit (#hb-det2, InvivoGen). TLR7 / 8 agonist 1 was used as a positive control. Similarly, the TLR7 agonist activity of C12-113 LNP or C12-113 / TLRa LNP was tested.

[0342] Cellular uptake DC2.4 cells were seeded in 35 mm glass dishes for 24 hours, then treated with DiO-labeled C12-113 LNPs or DiO-labeled C12-113 / TLRa LNPs at a concentration of 500 ng / mL mRNA for 2 hours. Cells were then stained sequentially with LysoTracker Deep Red (100 nM) for 30 minutes and Hoechst 33342 (10 μg / mL) for 5 minutes. Images were immediately captured using a confocal laser scanning microscope (LSM 710, Zeiss).

[0343] Analysis of DC in vitro maturation and cytokine production DC2.4 cells at 1x10 6 Cells were seeded at a density of 10 cells / well in 12-well plates overnight, then treated with LNP (500 ng / mL) loaded with SARS-CoV-2 mRNA for 24 hours. Cell cultures were collected for ELISA analysis of TNF-α, IL-12p70, and IL-1β. Cells were collected, blocked with anti-mouse CD16 / 32 antibody, stained with FITC anti-mouse-CD80 antibody and PE anti-mouse-CD86 antibody at 4°C for 30 min, and then analyzed by flow cytometry (BD, LSR II). BMDCs were seeded at 1x10⁻⁶ cells / well. 6 Cells were seeded at a density of 100 cells / well in 12-well plates and incubated for 24 hours with LNP loaded with SARS-CoV-2 mRNA (500 ng / mL). Cell cultures were collected for ELISA analysis. Cells were collected, blocked with anti-mouse CD16 / 32 antibody, and then stained with APC anti-mouse CD11c antibody, FITC anti-mouse-CD8- antibody, and PE anti-mouse-CD86 antibody at 4°C for 30 minutes, followed by flow cytometry analysis.

[0344] Analysis of in vivo DC maturation and cytokine production Twenty-four hours after injecting LNPs loaded with SARS-CoV-2 mRNA (5 μg mRNA / mouse) into the tail root, two iLNs were collected from each mouse and gently mechanically disrupted with a sterile pestle in 0.1 mL RPMI complete medium in a 1.5 mL tube. The resulting cell suspension was collected, blocked with anti-mouse CD16 / 32 antibody, and then stained with APC anti-mouse CD11c antibody, FITC anti-mouse-CD80 antibody, and PE anti-mouse-CD86 antibody at 4°C for 30 minutes, followed by flow cytometry analysis.

[0345] Blood was collected via retroocular approach into serum separation tubes (BD#365967) at 6 and 24 hours post-immunization. After incubation at room temperature for 30 minutes, serum was separated from blood, and the samples were centrifuged at 10,000 g for 5 minutes. Serum was stored at -20°C until use. To analyze intravascular cytokine production, cell suspensions generated by iLN were placed in 96-well plates at a density of 10,000 cells / 100 μL / well and cultured for 8 hours. The supernatant was collected and analyzed together with serum samples for ELISA of TNF-α, IL-12p70, and IL-1β.

[0346] Distribution and transfection of LNPs in vivo Mice were subcutaneously injected with mLuc-loaded LNPs at the base of the tail at a dose of 5 μg mRNA per mouse. Six hours or 24 hours post-injection, mice were intraperitoneally (ip) injected with potassium D-luciferin (150 mg / kg), and bioluminescence imaging was performed on an IVIS imaging system (PerkinElmer). To achieve simultaneous bioluminescence and fluorescence imaging, DiR-labeled mLuc-loaded LNPs were injected into mice. Twenty-four hours post-injection, mice were intraperitoneally (ip) injected with potassium D-luciferin, and major organs and iLNs were collected for bioluminescence and fluorescence imaging.

[0347] In vivo immunity Mice were subcutaneously immunized with LNPs loaded with SARS-CoV-2 mRNA at a dose of 5 μg mRNA per mouse, using a primary-boost strategy with three-week intervals. Body weight was recorded twice weekly during the experiment. Serum was collected using serum separation tubes as described above and stored at -20°C for ELISA and virus neutralization assays. Two weeks after the second vaccination, mice were anesthetized with isoflurane, and spleens were collected for flow cytometry analysis.

[0348] Anti-RBD antibody titer was determined using ELISA. High Bind Stripwell™ Corning 96-well clear polystyrene microplates were coated overnight with 1 μg / mL purified SARS-CoV-2 His-labeled RBD. The plates were washed once with wash buffer (0.05% Tween-20 in PBS) and blocked for two hours at room temperature with heat-inactivated IgG-depleted protease-free bovine serum albumin (2% w / v BSA in PBS). After blocking, the plates were washed three times, serially diluted with mouse serum in blocking solution, and incubated for 2 hours at room temperature. The plates were washed three times before adding HRP-conjugated anti-mouse secondary antibodies against total IgG (1:10000) or subclasses (IgG1:10000, IgG2c:10000) in blocking buffer. The plates were incubated for 1.5 hours, washed three times, and then 100 µL of KPL TMB substrate was added to each well for 8 minutes. The reaction was stopped by adding 50 µL of 2N sulfuric acid, and the absorbance was measured at 450 nm using a SpectraMax™ 190 microplate reader. The RBD-specific IgG endpoint dilution titer was defined as the highest serum dilution that yielded an OD greater than the cutoff OD value determined using the Frey method.

[0349] fake virus neutralization test As described, VSV pseudotypes were produced using SARS-CoV-2S. Antibody neutralization assays were performed using VSVΔG-RFP SARS-CoV-2: Vero E6 cells stably expressing TMPRSS2 were loaded at 2.5 x 10⁻⁶ cells. 4 Cells / well were seeded at 100 μL in 96-well collagen-coated plates. The next day, serially diluted serum samples (2-fold) were mixed with VSVΔG-RFPSARS-CoV-2 pseudovirus encoding the D614G spike (β or δ variant) (50–200 focal-forming units / well) and incubated at 37°C for 1 hour. This mixture also included 8G5F11, a mouse anti-VSV Indiana G at 100 ng / ml (absolute antibody #Ab01401-2.0), to neutralize any potential VSV-G-carrying virus. The antibody-virus mixture was then used instead of the culture medium on VeroE6 TMPRSS2 cells. Twenty hours post-infection, cells were washed and fixed with 4% PFA before visualization on an S6 FluoroSpot analyzer (CTL, Shaker Heights OH). Individual infection foci were counted and their values ​​compared to control wells without antibodies. Focal reduction neutralizing titers of 50% (FRNT) were considered neutralizing. 50 The focal count was measured as a reduction of at least 50% in the maximum serum dilution relative to control cells infected with pseudovirus in the absence of mouse serum. The technique was repeated twice on different days, measuring FRNT for each sample.50 Titer.

[0350] Flow cytometry analysis of T cells and B cells T cells. Spleens were collected and treated as single cells, filtered through a 70µm cell filter in an intact RPMI-1640, centrifuged, and lysed in ACK lysis buffer to obtain a clear single-cell suspension. To measure antigen-specific T cells, 2 million spleen cells were stimulated for 6 hours at 37°C, 5% CO2, and with 2 mg / mL anti-CD28 (Tonbo#40-0281-M001) in FACS tubes to provide co-stimulation. Stimulation was performed for 1 hour, followed by the addition of 5 mg / mL brefeldin A (Biolegend#420601), 2 mM monensin (Biolegend#420701), and 5 mg / mL anti-CD107a-Alexa Fluor 647 (Biolegend#121610) for 5 hours. DMSO was used as a negative control, and a combination of 50 mg / mL phorbol 12-myristate 13-acetate and 1 mg / mL ionomycin was used as a positive control. After a total of 6 hours, samples were washed with PBS, stained with Live / Dead Aqua for 5 minutes, blocked with anti-mouse CD16 / 32 antibody for 20 minutes, and filtered extracellularly with antibody for 30 minutes (Fig. 21D). Cells were washed in FACS buffer, fixed and infiltrated using the Cytofix / Cytoperm kit (BD Biosciences #554714), and stained intracellularly with antibody for 30 minutes (Fig. 21D). After intracellular staining, cells were washed twice, fixed with 300 µL (1% paraformaldehyde), and samples were collected on a BD LSR II equipped with 4 laser lines and 18 PMTs. Gating strategy ( Figures 21A-21C The antibody list and fluorophore information are shown in Table 1.

[0351] Table 1. T cell antibody groups Memory B cells. Spleens were collected and treated as single cells, filtered through a 40µm cell filter in a intact RPMI-1640, centrifuged at 300g for 5 min, lysed with ACK for 1 min, washed twice, and counted. 2 million cells per sample were incubated with anti-mouse CD16 / 32 antibody at 4°C for 20 min. Cells were then washed with FACS buffer (1% BSA in PBS) and stained with the group shown in Table 2 for 1 h. After staining, cells were washed twice, fixed with 300µL (1% paraformaldehyde), and samples were collected on a BD LSR II equipped with 4 laser lines and 18 PMTs. This article provides the gating strategy and a list of antibodies, internal fluorescent RBD probes (…). Figures 23A-23C (and Table 2).

[0352] Table 2. T cell antibody groups ELISpot assay Bone marrow was flushed from the femur and tibia into FACS buffer and filtered through a 63µm Nitex mesh. Red blood cells were lysed in ACK buffer on ice for 5 min and washed twice with FACS buffer. The resulting cells were counted using a Beckman Coulter ViCell. MultiScreenHTS IP plates, 0.45µm (Millipore Sigma, catalog: MSIPS4W10), were coated for 1 h at 37°C with 10 μg / mL RBD protein antigen in sodium carbonate / sodium bicarbonate buffer (35 mM NaHCO3 and 15 mM Na2CO3) at pH 9.6. The plates were then washed three times with 200µL PBS per well and blocked in complete RPMI for 30 min at 37°C. Bone marrow cells were seeded at 6 halved dilutions, starting with 1 million total BM cells per well and incubated overnight in complete RPMI. The plate was then washed five times with wash buffer (1x PBS + 0.1% Tween 20), and biotinylated anti-IgG detection antibody (goat anti-mouse IgG human ads-BIOT: Southern Biotech cat# 1030-08) was added to PBS + 2% BSA at a final dilution of 3 μg / mL, and incubated at room temperature for 1 hour. The plate was washed five more times, and streptavidin alkaline phosphatase (diluted 1:20000 in PBS + 2% BSA) was added before incubation at room temperature for 30 minutes. The plate was then washed five times with wash buffer, and 50 µL / well BCIP / NBT single solution (Sigma B1911-100 mL) was added for approximately 10 minutes, or until spots appeared, at which point the reaction was quenched with 100 µL 1M sodium dihydrogen phosphate solution. After rinsing the plate with deionized water and drying overnight, it was scanned and counted using CTL Immunospot hardware and software.

[0353] Statistical analysis Data are presented as mean ± standard deviation. Student's t-test or one-way ANOVA followed by Tukey's test was used to compare two or more groups. p < 0.05 was considered statistically significant.

[0354] Example 1: Synthesis of adjuvant lipids and polyamine-derived lipids The adjuvant lipid C12-TLRa was synthesized via a ring-opening reaction between an amine-containing TLR7 / 8 agonist 1 and a C12 epoxide. Figure 2A The purity and structure of C12-TLRa were verified by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. Structural simulations based on computational analysis showed that C12-TLRa can form multiple interactions with TLR7 at the first binding site (e.g., with Asp). 555and Thr 586 hydrogen bonds and with Phe 408 π-π stacking), similar to TLR7 / 8 agonist 1 ( Figures 7A-7F This indicates that this modification has minimal interference (if any) with agonist-receptor interactions. The TLR7 agonist activity of C12-TLRa was further validated on HEK Blue reporter cells stably expressing the TLR7 receptor. As a positive control, free TLR7 / 8 agonist 1 exhibited a bimodal dose-response curve for TLR7 agonist activity, characterized by an initial dose-dependent increase followed by a decrease in analyte concentration. Figure 8 However, the TLR7 agonist activity of C12-TLRa was observed to be monotonic and dose-dependent within the same dose range, suggesting that it has more predictable pharmacological properties.

[0355] Next, using microfluidic mixing, adjuvant lipids, DOPE, cholesterol, and DMG-PEG were formulated with mRNA in a molar ratio of 35:16:46.5:2.5 (lipids:DOPE:chol:DMG-PEG) to prepare C12-TLRa LNPs. The mRNA encapsulation efficiency was approximately 75%, indicating that this adjuvant lipid, like other ionizable lipids, can complex and encapsulate negatively charged mRNA into LNPs. C12-TLRa LNPs encapsulated with m1ψ-modified firefly luciferase mRNA (mLuc) were subsequently used to treat DC2.4 dendritic cells. Although free mLuc could not transfect cells, C12-TLRa LNPs exhibited dose-dependent mRNA transfection (…). Figure 2B This indicates that adjuvant lipids can successfully deliver mRNA into cells.

[0356] Table 3. Composition of an exemplary LNP To further evaluate the potency of C12-TLRa, LNP was compared with a series of polyamine-derived lipids synthesized using a similar method. Figure 2C In vitro mLuc transfection results showed that C12 TLRa had relatively low transfection capacity, especially compared to C12-113, which performed best. Figure 2D ).

[0357] Example 2: Optimization and characterization of adjuvant lipid-substituted LNPs Because abundant antigen expression is beneficial for mRNA vaccines, some lipids in C12-113 LNPs (i.e., C12-113) are replaced by C12-TLRa, thereby providing the LNP with TLR7 / 8 agonist properties without affecting its efficacy in mRNA delivery. Therefore, a series of LNPs with increased C12-113 to TLRRa LNP C12-113 ratios (e.g., 1 to 17.5 mol%) were formulated and in vitro mLuc delivery was performed. Interestingly, with increasing C12-TLRa substitution, the mRNA transfection efficiency of C12-113 / TLRa LNPs first increased and then decreased. Figure 2E C12-113 / TLRa LNP (5 mol% C12-TLRa) achieved the highest transfection rate and was selected as the subject of subsequent research.

[0358] The adjuvant activity of the mRNA-loaded C12-113 / TLRa LNP was then verified in HEK-blue reporter cells. Although C12-113 LNP did not possess TLR7 agonist activity, C12-113 / TLRa LNP exhibited dose-dependent TLR7 agonist activity. Figure 2F Therefore, C12-113 / TLRa LNP stimulated the production of the pro-inflammatory cytokine TNF-α in DC2.4 cells in a dose-dependent manner, which was significantly superior to C12-113 LNP. Figure 9 To further confirm the general applicability of this strategy, DLin-MC3-DMA (MC3) LNPs and adjuvant lipid-substituted MC3-LNPs (i.e., MC3 / TLRa LNPs) were formulated and tested. Consistently, MC3 / TLRa LNPs exhibited TLR7 agonist activity, but MC3 LNPs did not. Figure 10 These results indicate that replacing part of the lipids with C12-TLRa provides adjuvant activity for LNP.

[0359] Next, the physicochemical properties of the C12-113 / TLRa LNP were characterized. The hydrodynamic diameter of the LNP was found to be approximately 52 nm, with a polydispersity index (PDI) of 0.127. Furthermore, this LNP possesses a neutral surface charge and a p-value of 6.42. Ka This meets the criteria for effective mRNA delivery in vivo. The encapsulation efficiency of mRNA in C12-113 / TLRa LNP is above 93%. Transmission electron microscopy (TEM) images show that C12-113 / TLRa LNP has a uniform spherical structure. Figure 2G The C12-113 LNP without C12-TLRa replacement has very similar parameters. Figure 11 This indicates that replacing LNP with 5% C12-TLRa does not change its physicochemical properties.

[0360] Table 4. Characterization data of exemplary LNPs Example 3: In vitro mRNA delivery and DC activation Next, the in vitro mRNA delivery and adjuvant activity of C12-113 / TLRa LNP were systematically investigated. Both C12-113LNP and C12-113 / TLRa LNP showed dose-dependent mLuc transfection in DC2.4 cells (…). Figure 3A However, with Figure 2E Consistently, at any tested mRNA dose, C12-113 / TLRa LNP consistently outperformed C12-113 LN. No significant cytotoxicity was observed in either LNP. Figure 12A-12B ).

[0361] Similarly, in primary bone marrow-derived dendritic cells (BMDCs), C12-113 / TLRa LNP consistently showed higher mRNA transfection efficiency than C12-113 LNP. Figure 3B Correspondingly, MC3 / TLRa also significantly outperforms MC3 LNP in mLuc delivery. Figure 13 These results strongly confirm that C12-TLRa substitution can increase LNP-mediated mRNA delivery in vitro.

[0362] To investigate the improved mRNA transfection after C12 TLRa incorporation into LNPs, endosome escape of LNPs was assessed. LNPs labeled with the lipid-like fluorescent dye DiO were used to treat DC2.4 cells, and their subcellular distribution was observed after counterstaining of endosomes / lysosomes and nuclei. Figure 3C In C12-113 LNP-treated cells, numerous yellow spots were observed, indicating that C12-113 LN (green) was heavily embedded within endosomes / lysosomes (red). In contrast, C12-113 / TLRa LNP-treated cells exhibited a more uniform green signal in the cytoplasm, indicating more efficient escape from endosomes / lysosomes. These results suggest that C12-TLRa substitution enhances endosome escape from LNPs, which could explain the improved mRNA transfection. Figures 3A-3B Based on molecular simulations, multiple interactions were observed between C12-TLRa and TLR7. Figures 7A-7E Therefore, it is reasonable to assume that the strong affinity between the incorporated C12-TLRa and endosome TLR7 / 8 can enhance the physical interaction between the LNP and the endosome membrane, thereby leading to enhanced endosome disruption. Figure 3D ).

[0363] Next, the immunostimulatory effect of C12-113 / TLRa LNP carrying m1ψ-modified SARS-CoV-2 mRNA, which encodes the diproline-modified spike glycoprotein of SARS-CoV-2, was evaluated. Notably, the coding sequence of this nucleoside-modified mRNA is identical to that of the mRNAs used in two FDA-approved vaccines (i.e., mRNA-1273 and BNT162b2). Since TLR7 / 8 agonists can stimulate DC maturation and the release of pro-inflammatory cytokines, flow cytometry was used to analyze mature DCs (CD80). + CD86 + ), and the secreted TNF-α, IL-12p70, and IL-1β were analyzed using enzyme-linked immunosorbent assay (ELISA) 24 hours after treatment. C12-113 / TLRa LNP significantly increased the percentage of mature DCs in DC2.4 cells and BMDCs ( Figure 3E-3F and Figures 14A-14B In contrast, C12-113 LNP moderately induced DC maturation. Furthermore, compared to C12-113 LNP, C12-113 / TLRa LNP significantly increased TNF-α, IL-12p70, and IL-1β levels in DC2.4 cells and BMDCs. Figure 3G-3H In summary, these results indicate that C12-TLRa substitution significantly enhances the adjuvant effect and DC activation of LNPs.

[0364] Example 4: In vivo mRNA transfection and innate immune response Next, we investigated whether adjuvant lipid-substituted LNPs could reproduce the increased mRNA transfection and innate immune response in vivo. C57BL / 6 mice were subcutaneously (sc) immunized at the tail root with mLuc-loaded C12-113 LNPs or C12-113 / TLRa LNPs, and luciferase expression was observed in vivo by bioluminescence imaging at 6 and 24 hours post-treatment. Figure 4A Both LNPs primarily transfect the injection site, but can also transfect inguinal lymph nodes (iLN). Consistent with in vitro transfection results, the mRNA transfection rate of C12-113 / TLRa-LNP at both the injection site and iLN was higher than that of C12-113 LNP. Figure 4A ).

[0365] To avoid systemic toxicity, it is important to confine adjuvant activity and mRNA transfection to the sites of vaccine administration and drainage lymph nodes. Therefore, the distribution and transfection of LNPs in major organs and iLNs were investigated 24 hours after injection of lipid-like fluorescent dye DiR-labeled mLuc-loaded LNPs. In vitro fluorescence and luminescence imaging confirmed that no significant accumulation and transfection of either LNP was observed in major organs (i.e., liver, heart, spleen, lung, and kidney). Figure 4B ).

[0366] Interestingly, despite being based on fluorescence quantification, both LNPs achieved considerable accumulation in iLN. Figure 4C However, C12-113 / TLRa LNP showed stronger mRNA transfection ability in iLN than C12-113 LNP, consistent with in vivo ventral luminescence imaging. Figure 4A These results are also closely related to in vitro mRNA transfection. Figures 3A-3B Furthermore, the enhanced mRNA expression at the injection site and iLN mediated by C12-113 / TLRa LNP was highly reproducible across different LNP batches. Figure 15 ).

[0367] Finally, the duration and translation kinetics of LNP-prepared mRNAs were investigated. Compared to C12-113 LNPs, C12-113 / TLRa LNPs achieved more persistent (over 14 days) and sustained stronger mRNA expression at the injection site. Figures 16A-16B This sustained expression is attributed to nucleoside modification and LNP formulation. In summary, these results indicate that C12-TLRa substitution can significantly enhance the expression of LNP-formulated mRNA at the injection site and drainage LN, while minimizing systemic off-target distribution or expression.

[0368] To assess in vivo innate immune responses, intracellular lymphocytes (iLNs) were collected from mice 24 hours after immunization with LNPs loaded with SARS-CoV-2 mRNA, and intracellular dendritic cell (DC) maturation was analyzed by flow cytometry. C12-113 / TLRa LNPs significantly increased the percentage of mature DCs ( Figure 4D-4E and Figure 17 This was significantly higher than that of C12-113 LNP. Next, iLN and serum cytokines (TNF-α, IL-12p70, and IL-1β) were analyzed by ELISA at 6 and 24 hours post-vaccination. While C12-113 LNP moderately and transiently induced intralymphatic cytokine production, C12-113 / TLRa LNP elicited a higher and more durable cytokine response. Figure 4F-4H As expected, due to the small systemic exposure of LNP (). Figure 4B-4CThe serum cytokine levels of neither of the two LNPs increased. Figures 18A-18C These results indicate that C12-TLRa substitution can significantly enhance the strength and duration of the local innate immune response induced by SARS-CoV-2 mRNA-LNP vaccines without causing systemic inflammation.

[0369] Example 5: Enhancing the cellular immune response to SARS-CoV-2 Further investigation was conducted into the adaptive immune responses elicited by the original and adjuvant lipid-substituted SARS-CoV-2 mRNA-LNP vaccines. Mice were vaccinated twice using a prime-boost strategy at three-week intervals. Figure 5A It is noteworthy that no skin abnormalities were observed at the injection site after immunization. Figure 19 No weight loss was observed. Figure 20 Two weeks after a booster dose, spleens were collected from immunized mice, and splenocytes were stimulated with a SARS-CoV-2 receptor-binding domain (RBD) peptide pool. Antigen-specific CD4 counts were measured by flow cytometry. + and CD8 + T cells ( Figures 5B-5C and Figures 21A-21C Both LNPs induce RBD-specific CD4 expression of Th1 cytokines (interferon [IFN]-γ, IL-2, and TNF-α). + T cells, however, showed a significantly higher amplitude of C12-113 / TLRa LNP compared to C12-113 LNP. Figure 5B Furthermore, neither of these vaccine formulations induced the expression of type 2 (Th2) cytokines (IL-4, IL-5, and IL-17) on CD4+. + T cells, thereby supporting Th1-biased T cell immune responses. C12-113 / TLRa LNP also induced increased expression of RBD-specific CD8+ cytokines expressing Th1 immune response cytokines (IFN-γ, IL-2, and TNF-α) and cytotoxic markers (CD107α). + T cells ( Figure 5C ).

[0370] These results indicate that the C12-TLRa-substituted SARS-CoV-2 mRNA-LNP vaccine can induce stronger RBD-specific Th1 and CD8 responses. + T cell immune responses. Multifunctional T cells are considered better immune-related agents for fighting pathogens. Next, the multifunctionality of these RBD-specific T cell responses was evaluated. Compared to C12-113 LNPs, C12-113 / TLRaLNPs induced a significantly higher proportion of double- and triple-positive CD4+.+ and CD8 + T cells ( Figure 5D CD4 + (IL-2 + TNF-α + ) and CD8 + (INF-γ + TNF-α + The cytokine secretion patterns of double-positive cells differ between the two groups of cells and are consistent with the helper effects and cytotoxic activities of these two different lymphocyte populations.

[0371] Example 6: Enhancing the humoral immune response to SARS-CoV-2 Next, the humoral immune response induced by the SARS-CoV-2 mRNA-LNP vaccine was analyzed. Figure 6A The total titer of RBD-specific IgG binding antibodies was determined by endpoint dilution ELISA of the serum from vaccinated mice. Both mRNA-LNP vaccines induced high levels of RBD-specific IgG (…). Figure 6B However, the IgG titer of C12-113 / TLRa LNP was an order of magnitude higher than that of C12-113 LNP (1.22 x 10⁻⁶). 7 Compared to 1.35 x 10 6 Furthermore, ELISA analysis of RBD-specific IgG subsets showed that, compared to C12-113 LNP, C12-113 / TLRa LNP induced similar IgG1 titers and higher IgG2c titers, with a higher IgG2c / IgG1 ratio. Figure 22 This further highlights that adjuvant lipid-substituted LNPs tend to elicit a stronger Th1-biased immune response. Neutralizing antibody (NAb) levels were then measured using a vesicular stomatitis virus (VSV)-based pseudovirus neutralization assay. Figure 6C Both mRNA-LNP vaccines induced high levels of extensive NAb against the SARS-CoV-2 D614G mutant ancestral strains β (B.1.351) and (B.1.617.2). Specifically, the C12-113 / TLRa LNP vaccine reduced the 50% lesion-neutralizing titer (FRNT) against the SARS-CoV-2 β variant (B.1.351). 50 The FRNT ratio is close to approximately 1 / 5608, significantly higher than the approximately 1 / 2640 of the C12-113 LNP vaccine. 50 .

[0372] To investigate the induction of B cell memory, fluorescent RBD probes were used to assess splenic B cell responses. Figures 23A-23CCompared to C12-113 LNP, C12-113 / TLRa LNP vaccination significantly increased the number of RBD-specific B cells. Figure 6D These cells exhibit a memory phenotype (CD38). + GL7 - ; Figure 6E In mice vaccinated with C12-113 / TLRa LNP, most RBD-specific B cells co-express the memory markers PD-L2 and CD80. Figure 6E This aligns with the potential for rapid differentiation into antibody-secreting cells (ASCs) upon re-attack. Therefore, C12-113 / TLRa LNP vaccination can generate a larger but phenotypically similar pool of RBD-specific memory B cells.

[0373] Finally, three months after the second vaccination, long-lived plasma cells (LLPCs) in the bone marrow were analyzed; these cells can mediate durable immunity to infection by continuously producing antigen-specific antibodies. Bone marrow was collected from immunized mice, and various subsets of RBD-specific ASCs were characterized by enzyme-linked immunospot assay (ELISPOT). Although C12-113LNP moderately induced the expression of RBD-specific IgG1 and IgG... 2a and IgG 2b The generation of ASC ( Figure 6F-6H However, C12-113 / TLRaLNP induced 6.7-fold, 2.4-fold, and 6.5-fold greater expression of IgG1 and IgG, respectively. 2a and IgG 2b The ASCs. In summary, these results indicate that adjuvant lipid-substituted mRNA-LNP vaccines can elicit stronger humoral immune responses and LLPC responses.

[0374] List of implementation methods The following exemplary implementations are provided, and their numbers should not be interpreted as indicating a level of importance: Implementation method 1 provides a compound of formula (I) or a salt, solvate, stereoisomer, or isotopic configuration thereof: (I), in: R 1 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 2 Selected from H, R 5Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 5 R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H and R 5 Halogens, CN, NO2, optionally substituted C1-C6 alkyl groups, optionally substituted C3-C8 cycloalkyl groups, optionally substituted C2-C8 heterocyclic alkyl groups, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 5 yes , Where R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d At least one of them is R 5 ,or R 3a and R 3b At least one of them is R 6a ; R6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the optionally substituted C6-C 28 Alkyl, optionally substituted C6-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C8 heterocycloalkyl, optionally substituted C6-C 28 alkenyl and optionally substituted C6-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0375] Embodiment 2 provides the compound described in Embodiment 1, wherein R1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d One of them is R 5 .

[0376] Embodiment 3 provides the compounds described in Embodiment 1 or 2, wherein the compound of formula (I) is a compound of formula (Ia), or a salt, solvate, stereoisomer, or isotopic configuration thereof: (Ia), R 2 Selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H, halogen, CN, NO2, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocyclic alkyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 6a and R 6bEach is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

[0377] Embodiment 4 provides a compound as described in any one of Embodiments 1-3, wherein R 2 It is n-butyl.

[0378] Embodiment 5 provides a compound as described in any one of Embodiments 1-4, wherein R 3 ª and R 3b Each is independently represented by H.

[0379] Embodiment 6 provides a compound according to any one of Embodiments 1-5, wherein at least one of the following is satisfied: (a) R 4a R 4b R 4c and R 4d At least one of them is H; (b) R 4a R 4b R 4c and R 4d At least two of them are H; (c) R 4a R 4b R 4c and R 4d At least three of them are H; (d) R 4a R 4b R 4c and R 4d Each is H.

[0380] Embodiment 7 provides a compound as described in any one of Embodiments 1-6, wherein L is an optionally substituted C7-C 12 Aranediyl group.

[0381] Embodiment 8 provides a compound as described in any one of Embodiments 1-7, wherein L is .

[0382] Embodiment 9 provides a compound as described in any one of Embodiments 1-8, wherein R 6a and R 6b Each independently selected from R 7a and -CH2CH2C(=O)OR 7a .

[0383] Embodiment 10 provides a compound as described in any one of Embodiments 1-9, wherein R 7a and R 7b Each is independently selected from the optionally substituted C6-C 28 Alkyl, optionally substituted C6-C 28 alkenyl and optionally substituted C6-C 28 Heteroalkyl groups.

[0384] Embodiment 11 provides a compound as described in any one of Embodiments 1-10, wherein R 6aand R 6b Each is independently selected from -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl), -CH2CH(OH) (optionally substituted C6-C) 28 alkenyl), -CH2CH(OH) (optionally substituted C6-C) 28 (heteroalkyl), -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl), -CH2CH2C(=O)O (optionally substituted C6-C) 28 alkenyl) and -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

[0385] Embodiment 12 provides a compound as described in any one of Embodiments 1-11, wherein R 6a and R 6b Each is independently selected from -CH2CH(OH)(CH2)9CH3 and -CH2CH2C(=O)O(CH2). 11 CH3.

[0386] Embodiment 13 provides a compound from any one of Embodiments 1-12, which is selected from: (C12-TLRa) and (O12-TLRa).

[0387] Embodiment 14 provides a lipid nanoparticle (LNP) comprising: (a) At least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent; (b) at least one ionizable lipid; (c) at least one accessory lipid; (d) cholesterol; and (e) At least one polymer-conjugated lipid.

[0388] Embodiment 15 provides the LNP described in Embodiment 14, wherein the hydrocarbon-substituted TLR agonist is a compound of formula (II): A(B) n (II), in: A is a toll-like receptor (TLR) agonist; Each occurrence of B is independently selected from R. 6a and ; R 6a and R 6bEach occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a , R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L represents a bond, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and n is an integer selected from 1, 2, 3, and 4.

[0389] Example 16 provides the LNP described in Example 14 or 15, wherein the TLR agonist is selected from 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (TLR7 / 8 agonist 1), 1-isobutyl-1H-imidazo[4,5-c]quinoline-4-amine (imiquimod), 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (resimod), and 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (gadmod).

[0390] Embodiment 17 provides the LNP of Embodiment 14, wherein the at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent is a compound of formula (I) of any one of Embodiments 1-13.

[0391] Embodiment 18 provides an LNP as described in any one of Embodiments 14-17, wherein the at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent is selected from: (C12-TLRa) and (O12-TLRa).

[0392] Embodiment 19 provides an LNP as described in any one of Embodiments 14-18, wherein the ionizable lipid is selected from at least one of the following: , , , , and , in: R 8a R 8b R 8c R 8d R 8e and R 8f Each is independently selected from: -(optionally substituted C1-C6 alkylene)-C(=O)OR 9a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 9a (R) 9b -(optionally substituted C1-C6 alkylene)-C(=O)R 9a -(optionally substituted C1-C6 alkylene)-(R 9a -C(=O)OR 9a -C(=O)N(R) 9a (R) 9b -C(=O)R 9a and R 9a ;and R 9b With R 9b Each occurrence of is independently selected from: optionally substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 alkenyl groups, and optionally substituted C2-C groups 28 Alkyne group.

[0393] Implementation 20 provides the LNP described in Implementation 19, wherein R 8a R 8b R 8c R 8d R 8e and R 8f Each independently selected from R 9a and -CH2CH2C(=O)OR 9a .

[0394] Implementation 21 provides the LNP described in Implementation 19 or 20, wherein R 9a and R 9b Each is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C1-C 28 Alkenyl and optionally substituted C1-C 28 Heteroalkyl groups.

[0395] Implementation 22 provides an LNP as described in any one of Implementations 19-21, wherein R 7a and R 7b Each is independently selected from -CH2CH(OH) (optionally substituted C1-C) 28 Alkyl), -CH2CH(OH) (optionally substituted C1-C) 28 alkenyl), -CH2CH(OH) (optionally substituted C2-C) 28 (heteroalkyl), -CH2CH2C(=O)O (optionally substituted C1-C) 28 Alkyl), -CH2CH2C(=O)O (optionally substituted C1-C) 28 alkenyl) and -CH2CH2C(=O)O (optionally substituted C2-C) 28 (heteroalkyl groups).

[0396] Implementation 23 provides an LNP as described in any one of Implementations 19-22, wherein R 7a and R 7b Each is independently selected from -CH2CH(OH)(CH2)9CH3 and -CH2CH2C(=O)O(CH2). 11 CH3.

[0397] Embodiment 24 provides an LNP as described in any one of Embodiments 14-23, wherein the at least one ionizable lipid comprises: (C12-113).

[0398] Embodiment 25 provides an LNP as described in any one of Embodiments 14-24, wherein the at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1 mol% to about 17.5 mol% of the LNP.

[0399] Embodiment 26 provides an LNP as described in any one of Embodiments 14-25, wherein the at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1, 2.5, 5, 10, or about 17.5 mol of the LNP.

[0400] Embodiment 27 provides an LNP as described in any one of Embodiments 14-26, wherein the at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent comprises about 5 mol of the LNP.

[0401] Embodiment 28 provides an LNP as described in any one of Embodiments 14-27, wherein the at least one ionizable lipid comprises about 10 mol% to about 60 mol% of the LNP.

[0402] Embodiment 29 provides an LNP as described in any one of Embodiments 14-28, wherein the at least one ionizable lipid comprises about 34, 32.5, 30, 25 or about 17.5 mol of the LNP.

[0403] Embodiment 30 provides an LNP as described in any one of Embodiments 14-29, wherein the at least one ionizable lipid comprises about 30 mol of the LNP.

[0404] Embodiment 31 provides an LNP as described in any one of Embodiments 14-30, wherein the at least one auxiliary lipid comprises at least one selected from dioleoylphosphatidylethanolamine (DOPE) and distearate phosphatidylcholine (DSPC).

[0405] Embodiment 32 provides an LNP as described in any one of Embodiments 14-31, wherein the at least one auxiliary lipid accounts for about 1 mol% to about 50 mol% of the LNP.

[0406] Embodiment 33 provides an LNP as described in any one of Embodiments 14-32, wherein the at least one auxiliary lipid comprises about 16 mol of the LNP.

[0407] Embodiment 34 provides an LNP as described in any one of Embodiments 14-33, wherein the cholesterol accounts for about 5 mol% to about 60 mol% of the LNP.

[0408] Embodiment 35 provides an LNP as described in any one of Embodiments 14-34, wherein the cholesterol accounts for about 46.5 mol of the LNP.

[0409] Embodiment 36 provides an LNP as described in any one of Embodiments 14-35, wherein the at least one polymer-conjugated lipid comprises 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000).

[0410] Embodiment 37 provides an LNP as described in any one of Embodiments 14-36, wherein the at least one polymer-conjugated lipid accounts for about 0.1 mol% to about 20 mol% of the LNP.

[0411] Embodiment 38 provides an LNP as described in any one of Embodiments 14-37, wherein the at least one polymer-conjugated lipid comprises about 2.5 mol of the LNP.

[0412] Embodiment 39 provides an LNP as described in any one of Embodiments 14-38, wherein the molar ratio of the LNP is (a): (b): (c): (d): (e) approximately 5: 30: 16: 46.5: 2.5.

[0413] Embodiment 40 provides an LNP as described in any one of Embodiments 14-39, wherein the LNP further comprises at least one cargo molecule.

[0414] Embodiment 41 provides the LNP described in Embodiment 40, wherein the cargo is at least one selected from nucleic acids, small molecules, proteins, therapeutic agents, antibodies, and any combination thereof.

[0415] Implementation 42 provides the LNP described in Implementation 40 or 41, wherein the cargo is a nucleic acid.

[0416] Implementation 43 provides the LNP described in Implementation 41 or 42, wherein the nucleic acid is DNA or RNA.

[0417] Embodiment 44 provides an LNP as described in any one of Embodiments 41-43, wherein the nucleic acid is selected from mRNA, cDNA, pDNA, microRNA, siRNA, modified RNA, antagonism, antisense molecules, and any combination thereof.

[0418] Embodiment 45 provides an LNP as described in any one of Embodiments 40-44, wherein the cargo is at least partially encapsulated in the LNP.

[0419] Embodiment 46 provides an LNP as described in any one of Embodiments 40-45, wherein the cargo is mRNA.

[0420] Embodiment 47 provides the LNP described in Embodiment 46, wherein the total lipids (i.e., hydrocarbon-substituted Toll-like receptor agonists and ionizable lipids) of the LNP are in a weight ratio of about 5:1 to about 20:1 to mRNA (i.e., the weight ratio of (a) + (b):mRNA).

[0421] Implementation 48 provides the LNP described in Implementation 46 or 47, wherein the total lipids of the LNP (i.e., hydrocarbon-substituted Toll-like receptor agonists and ionizable lipids) to mRNA are in a weight ratio of approximately 10:1 (i.e., (a) + (b) : mRNA weight ratio).

[0422] Implementation 49 provides an LNP as described in any one of Implementations 44-48, wherein the mRNA encodes SARS-CoV-2, its immunogenic fragment (e.g., spike protein), or a modified derivative thereof.

[0423] Embodiment 50 provides a pharmaceutical composition comprising any one of embodiments 14-49, lipid nanoparticles (LNPs), and a pharmaceutically acceptable carrier.

[0424] Embodiment 51 provides a method for generating an innate immune response in a subject, the method comprising administering to the subject any of the lipid nanoparticles (LNPs) described in any one of Embodiments 14-49 or the pharmaceutical composition described in Embodiment 50.

[0425] Implementation 52 provides a method for treating, preventing, and / or improving an infection, disease, or disorder in a subject, the method comprising administering lipid nanoparticles (LNPs) to the subject, the lipid nanoparticles comprising: (a) At least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent; (b) At least one ionizable lipid; (c) At least one accessory lipid; (d) Cholesterol; (e) at least one polymer-conjugated lipid; and (f) At least one cargo molecule.

[0426] Implementation 53 provides the method of implementation 52, wherein the cargo is a nucleic acid.

[0427] Implementation 54 provides the method of implementation 52 or 53, wherein the nucleic acid is DNA or RNA.

[0428] Embodiment 55 provides the method of any one of Embodiments 52-54, wherein the nucleic acid is selected from mRNA, cDNA, pDNA, microRNA, siRNA, modified RNA, anta-codone, antisense molecules, and any combination thereof.

[0429] Embodiment 56 provides the method of any one of Embodiments 52-55, wherein the cargo is at least partially encapsulated in the LNP.

[0430] Embodiment 57 provides the method of any one of Embodiments 52-56, wherein the cargo is mRNA.

[0431] Implementation 58 provides the method of implementation 57, wherein the mRNA encodes SARS-CoV-2, its immunogenic fragment (e.g., spike protein), or a modified derivative thereof.

[0432] Implementation 59 provides the method of implementation 58, wherein the infection, disease, or disorder is a SARS-CoV-2 infection.

[0433] Implementation 60 provides the method of any one of Implementations 52-59, wherein an innate immune response is promoted in the subject.

[0434] Implementation 61 provides the method of any one of Implementations 51-60, wherein the subject is a mammal.

[0435] Implementation 62 provides the method of implementation 61, wherein the mammal is a human.

[0436] The terms and expressions used herein are descriptive rather than limiting, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof. However, it should be understood that various modifications can be made within the scope of embodiments of this application. Therefore, it should be understood that although this application describes specific embodiments and optional features, modifications and variations can be made to the compositions, methods, and concepts disclosed herein by those skilled in the art, and such modifications and variations are considered to be within the scope of embodiments of this application.

Claims

1. A compound of formula (I) or a salt, solvate, stereoisomer, or isotopic configuration thereof: (I)， in: R 1 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 2 Selected from H, R 5 Optionally substituted C1-C6 alkyl, Optionally substituted C3-C8 cycloalkyl, Optionally substituted C2-C8 heterocyclic alkyl, Optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and R 5 R 6a and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H and R 5 Halogens, CN, NO2, optionally substituted C1-C6 alkyl groups, optionally substituted C3-C8 cycloalkyl groups, optionally substituted C2-C8 heterocyclic alkyl groups, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 5 yes , Where R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d At least one of them is R 5 ,or R 3a and R 3b At least one of them is R 6a ; R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the optionally substituted C6-C 28 Alkyl, optionally substituted C6-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C8 heterocycloalkyl, optionally substituted C6-C 28 alkenyl and optionally substituted C6-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

2. The compound according to claim 1, wherein R 1 R 2 R 3a R 3b R 4a R 4b R 4c and R 4d One of them is R 5 .

3. The compound according to claim 1 or 2, wherein the compound of formula (I) is a compound of formula (Ia), or a salt, solvate, stereoisomer, or isotopic configuration thereof: (I), R 2 Selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted heteroaryl groups; R 3a and R 3b Each is independently selected from H and optionally substituted C1-C6 alkyl groups; R 4a R 4b R 4c and R 4d Each is independently selected from H, halogen, CN, NO2, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocyclic alkyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 10 heteroaryl, OR A 、N(R A (R) B ), C(=O)N(R A (R) B ), C(=O)R A C(=O)OR A OC(=O)R A OC(=O)OR A SR A S(=O)R A S(=O)2R A 、N(R A )S(=O)2R B 、N(R A )C(=O)R B and S(=O)2N(R) A (R) B ); R 6a and R 6b Each is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a ; R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L is selected from bonded, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and R A and R B Each occurrence of is independently selected from H, C(=O) (optionally substituted C1-C6 alkyl), C(=O) (optionally substituted C3-C8 cycloalkyl), C(=O) (optionally substituted C2-C8 heterocyclic alkyl), C(=O) (optionally substituted C6-C 10 aryl), C(=O) (optionally substituted C2-C) 10 Heteroaryl), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 10 Aryl and optionally substituted C2-C 10 Mixed aromatic compounds.

4. The compound according to any one of claims 1-3, wherein R 2 It is n-butyl.

5. The compound according to any one of claims 1-4, wherein R 3 ª and R 3b Each is independently represented by H.

6. The compound according to any one of claims 1-5, wherein at least one of the following is satisfied: (a) R 4a R 4b R 4c and R 4d At least one of them is H; (b) R 4a R 4b R 4c and R 4d At least two of them are H; (c) R 4a R 4b R 4c and R 4d At least three of them are H; (d) R 4a R 4b R 4c and R 4d Each of them is H.

7. The compound according to any one of claims 1-6, wherein L is an optionally substituted C7-C 12 Aranediyl group.

8. The compound according to any one of claims 1-7, wherein L is .

9. The compound according to any one of claims 1-8, wherein R 6a and R 6b Each independently selected from R 7a and -CH2CH2C(=O)OR 7a .

10. The compound according to any one of claims 1-9, wherein R 7a and R 7b Each is independently selected from the optionally substituted C6-C 28 Alkyl, optionally substituted C6-C 28 alkenyl and optionally substituted C6-C 28 Heteroalkyl groups.

11. The compound according to any one of claims 1-10, wherein R 6a and R 6b Each is independently selected from -CH2CH(OH) (optionally substituted C6-C) 28 Alkyl), -CH2CH(OH) (optionally substituted C6-C) 28 alkenyl), -CH2CH(OH) (optionally substituted C6-C) 28 (heteroalkyl), -CH2CH2C(=O)O (optionally substituted C6-C) 28 Alkyl), -CH2CH2C(=O)O (optionally substituted C6-C) 28 alkenyl) and -CH2CH2C(=O)O (optionally substituted C6-C) 28 (heteroalkyl groups).

12. The compound according to any one of claims 1-11, wherein R 6a and R 6b Each is independently selected from -CH2CH(OH)(CH2)9CH3 and -CH2CH2C(=O)O(CH2). 11 CH3.

13. The compound according to any one of claims 1-12, wherein the compound is selected from: (C12-TLRa) and (O12-TLRa)。 14. A lipid nanoparticle (LNP) comprising: (a) At least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent; (b) at least one ionizable lipid; (c) at least one accessory lipid; (d) cholesterol; and (e) At least one polymer-conjugated lipid.

15. The LNP according to claim 14, wherein the hydrocarbon-substituted TLR agonist is a compound of formula (II): A(B) n (II), in: A is a toll-like receptor (TLR) agonist; Each occurrence of B is independently selected from R. 6a and ; R 6a and R 6b Each occurrence of is independently selected from -(optionally substituted C1-C6 alkylene)-C(=O)OR 7a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 7a (R) 7b -(optionally substituted C1-C6 alkylene)-C(=O)R 7a -(optionally substituted C1-C6 alkylene)-(R 7a -C(=O)OR 7a -C(=O)N(R) 7a (R) 7b -C(=O)R 7a and R 7a , R 7a and R 7b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 Alkenyl and optionally substituted C2-C 28 alkynyl group; L represents a bond, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkylene, optionally substituted C3-C8 cycloalkylene, optionally substituted C2-C8 heterocycloalkylene, optionally substituted C7-C 12 Aranediene, optionally substituted C5-C 12 Heteroalkylene compounds, optionally substituted C6-C 10 aryl and optionally substituted C2-C 10 Hybrid aryl; and n is an integer selected from 1, 2, 3, and 4.

16. The LNP according to claim 14 or 15, wherein the TLR agonist is selected from 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (TLR7 / 8 agonist 1), 1-isobutyl-1H-imidazo[4,5-c]quinoline-4-amine (imiquimod), 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (resimod), and 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylprop-2-ol (gadmod).

17. The LNP according to claim 14, wherein the at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent is a compound of formula (I) according to any one of claims 1-13.

18. The LNP according to any one of claims 14-17, wherein the at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent is selected from: (C12-TLRa) and (O12-TLRa)。 19. The LNP according to any one of claims 14-18, wherein the ionizable lipid is at least one selected from the group consisting of: , , , , and , in: R 8a R 8b R 8c R 8d R 8e and R 8f Each is independently selected from: -(optionally substituted C1-C6 alkylene)-C(=O)OR 9a -(optionally substituted C1-C6 alkylene)-C(=O)N(R) 9a (R) 9b -(optionally substituted C1-C6 alkylene)-C(=O)R 9a -(optionally substituted C1-C6 alkylene)-(R 9a -C(=O)OR 9a -C(=O)N(R) 9a (R) 9b -C(=O)R 9a and R 9a ;and R 9a and R 9b Each occurrence of is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C2-C 28 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 28 alkenyl groups, and optionally substituted C2-C groups 28 Alkyne group.

20. The LNP of claim 19, wherein R 8a R 8b R 8c R 8d R 8e and R 8f Each independently selected from R 9a and -CH2CH2C(=O)OR 9a .

21. The LNP according to claim 19 or 20, wherein R 9a and R 9b Each is independently selected from the arbitrarily substituted C1-C 28 Alkyl, optionally substituted C1-C 28 Alkenyl and optionally substituted C1-C 28 Heteroalkyl groups.

22. The LNP according to any one of claims 19-21, wherein R 7a and R 7b Each is independently selected from -CH2CH(OH) (optionally substituted C1-C) 28 Alkyl), -CH2CH(OH) (optionally substituted C1-C) 28 alkenyl), -CH2CH(OH) (optionally substituted C2-C) 28 (heteroalkyl), -CH2CH2C(=O)O (optionally substituted C1-C) 28 Alkyl), -CH2CH2C(=O)O (optionally substituted C1-C) 28 alkenyl) and -CH2CH2C(=O)O (optionally substituted C2-C) 28 (heteroalkyl groups).

23. The LNP according to any one of claims 19-22, wherein R 7a and R 7b Each is independently selected from -CH2CH(OH)(CH2)9CH3 and -CH2CH2C(=O)O(CH2). 11 CH3.

24. The LNP according to any one of claims 14-23, wherein the at least one ionizable lipid comprises: (C12-113)。 25. The LNP according to any one of claims 14-24, wherein the at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1 mol% to about 17.5 mol% of the LNP.

26. The LNP according to any one of claims 14-25, wherein the at least one Toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent accounts for about 1, 2.5, 5, 10 or about 17.5 mol of the LNP.

27. The LNP according to any one of claims 14-26, wherein the at least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent constitutes about 5 mol of the LNP.

28. The LNP according to any one of claims 14-27, wherein the at least one ionizable lipid comprises about 10 mol% to about 60 mol% of the LNP.

29. The LNP according to any one of claims 14-28, wherein the at least one ionizable lipid accounts for about 34, 32.5, 30, 25 or about 17.5 mol of the LNP.

30. The LNP according to any one of claims 14-29, wherein the at least one ionizable lipid comprises about 30 mol of the LNP.

31. The LNP according to any one of claims 14-30, wherein the at least one auxiliary lipid comprises at least one selected from dioleoylphosphatidylethanolamine (DOPE) and distearate phosphatidylcholine (DSPC).

32. The LNP according to any one of claims 14-31, wherein the at least one auxiliary lipid accounts for about 1 mol% to about 50 mol% of the LNP.

33. The LNP according to any one of claims 14-32, wherein the at least one auxiliary lipid accounts for about 16 mol of the LNP.

34. The LNP according to any one of claims 14-33, wherein the cholesterol accounts for about 5 mol% to about 60 mol% of the LNP.

35. The LNP according to any one of claims 14-34, wherein the cholesterol accounts for about 46.5 mol% of the LNP.

36. The LNP according to any one of claims 14-35, wherein the at least one polymer-conjugated lipid comprises 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000).

37. The LNP according to any one of claims 14-36, wherein the at least one polymer-conjugated lipid accounts for about 0.1 mol% to about 20 mol% of the LNP.

38. The LNP according to any one of claims 14-37, wherein the at least one polymer-conjugated lipid accounts for about 2.5 mol of the LNP.

39. The LNP according to any one of claims 14-38, wherein the molar ratio of the LNP (a): (b): (c): (d): (e) is approximately 5: 30: 16: 46.5: 2.

5.

40. The LNP according to any one of claims 14-39, wherein the LNP further comprises at least one cargo molecule.

41. The LNP of claim 40, wherein the cargo is at least one selected from nucleic acids, small molecules, proteins, therapeutic agents, antibodies, and any combination thereof.

42. The LNP according to claim 40 or 41, wherein the cargo is a nucleic acid.

43. The LNP according to claim 41 or 42, wherein the nucleic acid is DNA or RNA.

44. The LNP according to any one of claims 41-43, wherein the nucleic acid is selected from mRNA, cDNA, pDNA, microRNA, siRNA, modified RNA, anta-codone, antisense molecules, and any combination thereof.

45. The LNP according to any one of claims 40-44, wherein the goods are at least partially encapsulated in the LNP.

46. ​​The LNP according to any one of claims 40-45, wherein the cargo is mRNA.

47. The LNP of claim 46, wherein the total lipids of the LNP (i.e., hydrocarbon-substituted Toll-like receptor agonists and ionizable lipids) to mRNA are in a weight ratio of about 5:1 to about 20:1 (i.e., (a) + (b) : mRNA weight ratio).

48. The LNP according to claim 46 or 47, wherein the total lipids of the LNP (i.e., hydrocarbon-substituted Toll-like receptor agonists and ionizable lipids) to mRNA are in a weight ratio of approximately 10:1 (i.e., (a) + (b) : mRNA weight ratio).

49. The LNP according to any one of claims 44-48, wherein the mRNA encodes SARS-CoV-2, its immunogenic fragment (e.g., spike protein) or a modified derivative thereof.

50. A pharmaceutical composition comprising the lipid nanoparticles (LNPs) of any one of claims 14-49 and a pharmaceutically acceptable carrier.

51. A method for generating an innate immune response in a subject, the method comprising administering to the subject the lipid nanoparticles (LNP) of any one of claims 14-49 or the pharmaceutical composition of claim 50.

52. A method for treating, preventing, and / or improving an infection, disease, or disorder in a subject, the method comprising administering lipid nanoparticles (LNPs) to the subject, the lipid nanoparticles comprising: (a) At least one toll-like receptor (TLR) agonist substituted with at least one hydrocarbon substituent; (b) At least one ionizable lipid; (c) At least one accessory lipid; (d) Cholesterol; (e) at least one polymer-conjugated lipid; and (f) At least one cargo molecule.

53. The method of claim 52, wherein the cargo is a nucleic acid.

54. The method according to claim 52 or 53, wherein the nucleic acid is DNA or RNA.

55. The method according to any one of claims 52-54, wherein the nucleic acid is selected from mRNA, cDNA, pDNA, microRNA, siRNA, modified RNA, anta-codone, antisense molecules, and any combination thereof.

56. The method according to any one of claims 52-55, wherein the cargo is at least partially encapsulated in the LNP.

57. The method according to any one of claims 52-56, wherein the cargo is mRNA.

58. The method of claim 57, wherein the mRNA encodes SARS-CoV-2, its immunogenic fragment (e.g., spike protein), or a modified derivative thereof.

59. The method of claim 58, wherein the infection, disease, or disorder is a SARS-CoV-2 infection.

60. The method according to any one of claims 52-59, wherein an innate immune response is promoted in the subject.

61. The method according to any one of claims 51-60, wherein the subject is a mammal.

62. The method of claim 61, wherein the mammal is a human.

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