WO2025189068A1 - Mrna-lipid nanoparticle immune modulators against allergic and inflammatory diseases - Google Patents

Abstract

Provided are compositions comprising mRNA molecules encoding antigens, such as allergens and autoantigens, and methods of use thereof, optionally in combination with an mTOR inhibitor, to prevent or reduce allergic responses or to promote tolerance to the encoded antigens.

Description

MRNA-LIPID NANOPARTICLE IMMUNE MODULATORS AGAINST

ALLERGIC AND INFLAMMATORY DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/563,127, filed March 8, 2024, and U.S. Provisional Application No. 63/677,814, filed July 31, 2024, which are hereby incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under DK078392 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “046483-6282- OOWO_Sequence_Listing.xml” having a creation date of March 7, 2025, and having a size of 28,538 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0004] Allergic diseases have reached epidemic proportions, affecting nearly 30% of the worldwide population (Edwards et al., 2012, Nat Rev Microbiol, 10:459-471; Agache et al., 2022, Allergy, 77: 1389-1407; Ozdemir et al., 2023, Ann Allergy Asthma Immunol, 6:703-712). Current therapeutic approaches focus primarily on relieving symptoms through administration of bronchodilators, or managing inflammation by employing glucocorticoids, inhibitors of pro- inflammatory mediators, or biological agents such as anti-IgE, anti-cytokines, and cytokine receptors (Holgate et al., 2008, Nat Rev Immunol, 8:218-230; Pavord et al., 2018, Lancet, 391 :350-400; Papi et al., 2018, Lancet, 391 :783-800). Despite substantial advances, unchecked allergic inflammation remains a leading cause for hospital admission, morbidity of children and adults, and cause for mortality.

[0005] Allergic inflammation is characterized by interactions among various cell populations, including epithelial cells, dendritic cells (DCs), innate lymphoid cells (ILCs), and macrophages, which initially respond to antigens and promote T and B cell activation and differentiation (Papi et al., 2018, Lancet, 391 :783-800; Hammad et al., 2021, Cell, 184: 1469- 1485; Kopp et al., 2023, Immunity, 56:687-694; Molofsky et al., 2023, Immunity, 56:704-722). Naive CD4+ T cells transform into T helper type 2 (Th2) cells upon antigen recognition and have a pivotal role in promoting allergic disease through the production of type 2 cytokines, and by interacting with other innate and structural cells (Walker et al., 2018, Nat Rev Immunol, 18: 121- 133; Hammad et al., 2022, Annu Rev Immunol, 40:443-467).

[0006] The application of nucleoside-modified mRNA vaccines has exhibited remarkable efficacy in curtailing viral and bacterial infections (Chaudhary et al., 2021, Nat Rev Drug Discov, 20:817-838; Alameh et al., 2022, Curr Top Microbiol Immunol, 440: 111-145; Arevalo et al., 2022, Science, 378:899-904; Whitaker et al., 2023, Curr Opin Infect Dis, 36:385-393) and is promising for other diseases ranging from cancer to autoimmunity (Lorentzen et al., 2022, Lancet Oncol, 23:e450-e458; Kon et al., 2023, Nat Rev Clin Oncol, 20:739-754; Krienke et al., 2021, Science, 371 : 145-153; Xu et al., 2023, ACS Nano, 17:4942-4957). mRNA vaccines possess several advantages over conventional counterparts: they exhibit strong immunogenicity, induce rapid and highly specific immune responses, display limited persistence in the body to preclude chronic effects, can be manufactured with high quality under controlled conditions, are safe due to the absence of viral components and integration into host DNA, and are cost-effective (Pardi et al., 2020, Curr Opin Immunology, 65: 14-20). Importantly, recent findings have demonstrated that encapsulation of the nucleoside-modified mRNA within lipid nanoparticle (LNP) formulations removes the necessity for additional adjuvants to provoke immune responses, given that LNPs intrinsically exhibit adjuvant properties (Alameh et al., 2021, Immunity, 54:2877-2892; Ndeupen et al., 2021, iScience, 24: 103479). LNPs provide protection against mRNA degradation, enabling administration of lower mRNA doses while maintaining robust efficacy. Studies have demonstrated the uptake of nucleoside-modified mRNA encapsulated into LNP by neutrophils, monocytes, macrophages, and DCs, its successful translation into protein, and subsequent presentation of epitopes of coded protein through the major histocompatibility complexes (MHCs) to both CD4+ and CD8+ T cells, the cells crucial for the establishment of immune memory against pathogens (Chaudhary et al., 2021, Nat Rev Drug Discov, 20:817-838; Teijaro et al., 2021, Nat Rev Immunol, 21 : 195-197; Verbeke et al., 2022, Immunity, 55:1993-2005; Li et al., 2022, Nat Immunol, 23:543-555).

[0007] Engineered against infectious diseases and cancer, mRNA-LNP vaccines stimulate an immune response by fostering the generation of Th1, T follicular helper (Tfh), and cytotoxic CD8+ T cells, accompanied by IgG antibodies (Chaudhary et al., 2021, Nat Rev Drug Discov, 20:817-838; Teijaro et al., 2021, Nat Rev Immunol, 21 : 195-197; Pardi et al., 2018, J Exp Med, 215: 1571-1588; Laczko et al., 2020, Immunity, 53:724-732). It has been demonstrated that the antigen-specific mRNA-LPX (liposome) vaccine stimulates Treg cells, offering protection against experimental autoimmune encephalomyelitis (Krienke et al., 2021, Science, 371: 145- 153).

[0008] There remains a need in the art for prophylactic and therapeutic compositions for treating and preventing inflammatory and allergic responses.

SUMMARY OF THE INVENTION

[0009] In some embodiments, the invention relates to a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen.

[0010] In some embodiments, the nucleoside-modified RNA molecule comprises pseudouridine. In some embodiments, the nucleoside-modified RNA molecule comprises 1- m ethy 1 -p seudouri dine .

[0011] In some embodiments, the allergen is a food allergen or an aero allergen. In some embodiments, the allergen is OVA, Ara h 1, Ara h 2, Ara h 3, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 12, Ara h 13, Derpl , Derp2, or Derp 23, or a fragment or variant thereof. In some embodiments, the allergen comprises an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a fragment or variant thereof. In some embodiments, the nucleoside-modified RNA molecule encodes an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a fragment or variant thereof.

[0012] In some embodiments, the composition further comprises at least one antiinflammatory agent. In some embodiments, the anti-inflammatory agent comprises an mTOR inhibitor. In some embodiments, the mTOR inhibitor is a small molecule. In some embodiments, the mTOR inhibitor is everolimus, rapamycin. Sirolimus, temsirolimus, ridaforolimus, Torin-1, or a non-rapalog derived inhibitor or an analog or derivative thereof.

[0013] In some embodiments, the composition further comprises an adjuvant.

[0014] In some embodiments, the composition further comprises a lipid nanoparticle

(LNP). In some embodiments, the nucleoside-modified RNA is encapsulated within the LNP.

[0015] In some embodiments, the invention relates to a method of reducing a T helper type 2 (Th2) response against one or more antigens in a subject comprising administering to the subject an effective amount of a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[0016] In some embodiments, the invention relates to a method of stimulating the production of allergen specific regulatory T cells (Tregs) in a subject comprising administering to the subject an effective amount of a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition. [0017] In some embodiments, the invention relates to a method of promoting tolerance to one or more antigen in a subject comprising administering to the subject an effective amount of a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[0018] In some embodiments, the invention relates to a method of increasing allergenspecific IgG production in a subject comprising administering to the subject an effective amount of a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[0019] In some embodiments, the invention relates to a method of reducing pro-allergic IgE production in a subject comprising administering to the subject an effective amount of a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[0020] In some embodiments, the invention relates to a method of treating, preventing, or decreasing the risk of an allergic response against one or more antigen in a subject comprising administering to the subject an effective amount of a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[0021] In some embodiments, the allergic response is abdominal pain, allergic rhinitis, anaphylaxis, colonic inflammation, diarrhea, eczema, hives, itching, nausea, vomiting, or any combination thereof.

[0022] In some embodiments, the invention relates to a method of treating, preventing, or decreasing the risk of developing an allergic disease in a subject comprising administering to the subject an effective amount of a composition for modulating an immune response against at least one antigen in a subject. In some embodiments, the composition comprises at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the allergic disease is rhinitis, atopy, asthma, COPD, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria, anaphylactic shock, eosinophilic gastrointestinal diseases including eosinophilic esophagitis, food-protein induced allergic proctocolitis, or hay fever. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[0023] In some embodiments, the invention relates to a composition for increasing the antigen specific regulatory T cells (Tregs) in a subject, the composition comprising at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the nucleoside-modified RNA molecule comprises pseudouridine. In some embodiments, the nucleoside-modified RNA molecule comprises 1 -methyl -pseudouridine.

[0024] In some embodiments, the composition further comprises at least one antiinflammatory agent. In some embodiments, the anti-inflammatory agent comprises an mTOR inhibitor. In some embodiments, the mTOR inhibitor is a small molecule. In some embodiments, the mTOR inhibitor is everolimus, rapamycin, sirolimus, temsirolimus, ridaforolimus, Torin-1, non-rapalog derived inhibitors or an analog or derivative thereof. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition further comprises a lipid nanoparticle (LNP). In some embodiments, the nucleoside-modified RNA is encapsulated within the LNP.

[0025] In some embodiments, the invention relates to a method of treating or preventing an inflammatory or autoimmune disease in a subject, comprising administering to the subject an effective amount of a composition for increasing the antigen specific regulatory T cells (Tregs) in a subject, the composition comprising at least one nucleoside-modified RNA molecule encoding an antigen. In some embodiments, the antigen is an allergen. In some embodiments, the antigen is an autoantigen. In some embodiments, the nucleoside-modified RNA molecule comprises pseudouridine. In some embodiments, the nucleoside-modified RNA molecule comprises 1-methyl-pseudouridine. In some embodiments, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[0026] In some embodiments, the invention relates to a composition for modulating an immune response in a subject, the composition comprising at least one nucleoside-modified RNA molecule encoding an antigen and at least one mTOR inhibitor. In some embodiments, the nucleoside-modified RNA molecule comprises pseudouridine. In some embodiments, the nucleoside-modified RNA molecule comprises 1-methyl-pseudouridine. In some embodiments, the mTOR inhibitor is a small molecule. In some embodiments, the mTOR inhibitor is everolimus (e.g. Afinitor, Afmitor Disperz, Zortress), rapamycin, sirolimus (e.g. Rapamune, Hyftor, Fyarro), temsirolimus (Torisel), ridaforolimus, Torin-1, or non-rapalog derived inhibitors or an analog or derivative thereof.

[0027] In some embodiments, the composition further comprises an adjuvant.

[0028] In some embodiments, the composition further comprises a lipid nanoparticle

(LNP). In some embodiments, the nucleoside-modified RNA is encapsulated within the LNP.

[0029] In some embodiments, the invention relates to a method of modulating the response of a subject to a composition comprising at least one nucleoside-modified RNA molecule encoding an antigen comprising administering an mTOR inhibitor to the subject. In some embodiments, the method reduces adverse effects when compared to administration of the same composition comprising at least one nucleoside-modified RNA molecule encoding an antigen without an mTOR inhibitor. In some embodiments, the method increases immunological memory to the antigen when compared to administration of the same composition comprising at least one nucleoside-modified RNA molecule encoding an antigen without an mTOR inhibitor.

BRIEF DESCRIPTION OF DRAWINGS

[0030] The following detailed description of embodiments provided herein will be better understood when read in conjunction with the appended drawings. It should be understood that embodiments provided in the present disclosure are not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0031] Figure 1 A- Figure IE depict the results of example experiments demonstrating that OVA encoded mRNA-LNP vaccine induces antigen-specific CD4 T cell expansion and differentiation toward Th1 and Tfh phenotypes. Figure 1A depicts a schematic of the experimental design. Naive OTII cells expressing CD45.1 were injected into CD45.2 WT mice three days prior to empty LNP or OVA-mRNA vaccine administration (day -3). The vaccine was delivered intramuscularly (i.m.) on days 0 and 7 (5 μg per mouse). Local lymph nodes (LNs) were collected on day 7 or 11. Figure IB depicts the results of expansion of donor CD45.1+ OTII cells analyzed by flow cytometry on day 11, and the quantification of number of donor cells within the local LNs of recipients. The data represents individual values along with their mean. Figure 1C depicts the frequencies of donor cells from Figure IB expressing specific markers or producing cytokines after short ex vivo stimulation. The data are displayed as Box and Whiskers plot. Figures 1B-1C present data from three independent experiments (n= 6 to 10). ***P ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA with FDR correction. Figures 1D-1E depict a dose-response analysis of donor CD45.1+ OTII cells following a single OVA-mRNA vaccine dose on day 7 (n= 3 to 4). One representative experiment is displayed, out of three repetitions. Data are mean ± SEM. **P ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA, ns = not significant.

[0032] Figure 2A- Figure 2G depict the results of example experiments demonstrating immunization with OVA- mRNA vaccine reduces allergic responses in airways. Figure 2A depicts the experimental workflow. Mice were subjected to a single intramuscular (i.m.) injection of different doses of OVA-mRNA vaccine. Sensitization with OVA+Alum was administrated intraperitoneally (i.p.) at days 24 and 36. Subsequently, daily challenges with 50 μg of OVA were applied to induce an allergic asthma response, two intratracheal (i.t.) followed by two intranasal (i.n.) injections. BALF and lungs were collected on day 2 following the final challenge. Figure 2B depicts the number of indicated cells in the BALF. Figure 2C depicts the frequency of GATA3+, FOXP3+, and cytokine producing cells among CD4+ T cells in the lungs. In Figures 2B and 2C, dashed lines indicate cell values in naive mice that have not undergone immunization and asthma induction. One of three replicated experiments is shown (n= 5 to 6), and data are mean ± SEM. Figures 2D-2G depict example experiments wherein mice received either a single or two doses of the OVA-mRNA vaccine or LNP administered one week apart (2 μg per injection), followed by a sensitization and airway challenge protocol. Figure 2D depicts the indicated cell count in the BALF (n= 4 to 7). Figure 2E depicts histological staining of inflammatory cell infiltration in the lungs: hematoxylin and eosin (H&E) and anti -major basic protein (a-MBP) staining. Shown are representative panels. Figures 2F and 2G depict frequencies of indicated CD4 (Figure 2F) and CD8 (Figure 2G) T cells in the lungs (n= 4 to 7). In Figures 2D, 2F, and 2G, the data is presented as the mean, with each circle representing an individual sample. Shown is one of the three replicated experiments. *P ≤ 0.05, **P ≤ 0.01, ***p ≤ 0.001, ****P ≤ 0.0001 by one-way ANOVA with FDR correction, ns = not significant.

[0033] Figure 3A- Figure 3H depict the results of example experiments demonstrating mRNA vaccine in combination with mTOR inhibitor suppresses inflammatory T cell generation. Naive CD45.1 -expressing OTII cells were transferred into CD45.2 WT mice three days prior to i.m. LNP or OVA-mRNA vaccine administration. The vaccine was delivered intramuscularly (i.m.) on days 0 and 7. Everolimus (EVL) was administered daily (5mg/kg, i.p.) starting two days prior to the LNP or vaccine injection and continued until the endpoint or a maximum of 14 days. Figure 3A depicts the experimental design, wherein local lymph nodes (LNs) were harvested at the indicated time points. Figure 3B depicts the kinetics of expansion of donor cells and their activation status (frequency of CD44+ cells among donor cells) were evaluated. The graphs provide a summary of findings of four independent experiments (n= 11 to 15). Figure 3C depicts representative flow cytometry analysis of IFNγ and TNFα production, or CD25 and FOXP3 expression in donor OTII cells across various treatment conditions on day 7 is presented. Numbers in the plots indicate frequencies of gated cells. Figures 3D and 3E depict summary graphs of IFNg+ and TNFa+ (Figure 3D) or FOXP3+ CD25+ and FOXP3-CD25+ donor OTII cells (Figure 3E). The graphs provide a summary of three independent experiments (n= 7 to 15). Figure 3F depicts kinetics of the emergence of Treg or CD25+ cells at the indicated time points. The graphs provide a summary of findings of four independent experiments (n= 11 to 15). Figures 3G and 3H depict a Treg functional assay. OVA-specific Treg and non-Treg CD4 cells were isolated from the local lymph nodes of mice treated with 0VA-mRNA+ everolimus (suppressors) on day 7 and co-cultured at indicated ratios with naive CFSE-labelled OVA- specific OTII cells (responders) in the presence of 323-339 OVA peptide-loaded irradiated splenocytes. The proliferation of CFSE-labelled OVA-specific cells was evaluated after 72 hours of co-culture. Figure 3G depicts a representative flow cytometry histogram displaying divided cells. Figure 3H depicts a summary of findings from six combined experiments. The indicated percentage reflects the frequency of divided responder cells. Data in Figures 3B, 3D-3F, and 3H are mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by multiple unpaired t tests (Figures 3B, 3F, and 3H) or one-way ANOVA (Figures 3D and 3E) with FDR correction, ns = not significant.

[0034] Figure 4A- Figure 4J depict the results of example experiments demonstrating mRNA vaccine in combination with mTOR inhibitor averts allergic and inflammatory responses in asthmatic mice. Mice received two doses of LNP or OVA-mRNA vaccine a week apart, with parallel everolimus administration for 14 days. After two weeks, all mice were OVA+Alum sensitized and subsequently OVA challenged for four days. BALF and lung tissue were collected two days after the final challenge. Naive mice served as unmanipulated controls. Figure 4A depicts the experimental design. Figure 4B depicts a heat map of indicated secreted cytokines and chemokines in the BALF and Figure 4C depicts the ELISA of Eotaxin 2 (n= 4 to 10). In Figures 4D and 4E depict the results of Rapsody scRNA-Seq of lung tissue harvested two days after the final challenge. “Naive” is unmanipulated mice. Figure 4D depicts uniform manifold approximation and projection (UMAP) of T cells from the lung tissue illustrates 5 cell clusters identified by unsupervised clustering, encompassing 4 different conditions (n=2). Figure 4E depicts selected gene expression in different T cell populations. Figure 4F depicts frequencies of cell subsets based on treatment conditions. Results are shown as parts of whole plots. Figure 4G depicts flow cytometry analysis of T cell populations in the lungs. FoxP3-CD4+ or CD8+ cells were distinguished as naive CD44- or activated CD44+ cells. A summary of findings from three combined experiments (n= 8 to 14) is shown. Figure 4H depicts frequency of indicated CD8+ T cells in the lungs, one of three experiments is shown (n= 6 to 7). Figure 41 depicts frequencies of CD4 cells expressing indicated transcriptional factors and cytokines in the lungs, one of three experiments is shown (n= 6 to 7). Figure 4J depicts analysis of the airway resistance in the indicated mice in response to increasing concentrations of methacholine (n = 7 to 12). Figure 4K depicts representative PAS-stained sections in bronchi and bronchiole for mucus production. A mucus score based on the percentage of staining. Arrows indicate goblet cells producing mucin. Graph represents a summary of four experiments (n= 9 to 26). Figures 4C, 41, and 4K depict Box and Whiskers plots. In figures 4G and 4J, data are mean ± SEM. Certain graphs depict data from combined experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by one-way ANOVA with FDR correction, ns = not significant.

[0035] Figure 5 A- Figure 5C depict the results of example experiments demonstrating an

OVA-mRNA vaccine induces antigen-specific immune responses by T and B cells. CD45.2 WT mice were injected with naive OTII cells expressing CD45.1, followed by administration with LNP or OVA-mRNA vaccine i.m. as shown in Figure 1A. Figure 5A depicts frequencies of host cells expressing indicated markers or cytokines (n= 6 to 10). The data are displayed as Box and Whiskers plot. Figure 5B depicts representative flow cytometry plots of donor OTII cells (CD45.1+) and host CD4+ (CD45.2+) T cells producing indicated cytokines after short ex vivo stimulation and expressing CD44 and FOXP3. Figure 5C depicts OVA-specific IgG levels in the serum of the mice 18 days after first immunization with different concentrations of OVA-mRNA vaccine (n= 4 to 5). The experiment was repeated more than five times. Data are mean ± SEM. In Figures 5A and 5C, **P ≤ 0.01 **P ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA, ns = not significant.

[0036] Figure 6A- Figure 6D depict the results of example experiments demonstrating an OVA-mRNA vaccine booster increases anti-allergic responses. Figure 6A depicts the experimental workflow. Mice received either one or two doses of the OVA-mRNA vaccine or LNP administered one week apart (2 μg per injection), followed by a sensitization and airway challenge protocol. Mice who received a single dose were immunized on day 7 for consistency. Figure 6B depicts frequencies of eosinophils, neutrophils, and CD8 T cells in the BALF (n= 4 to 7). Figure 6C depicts frequency of CD4 cells producing indicated cytokines and Figure 6D depicts frequency of Treg cells among total CD4 cells in the lung (n= 4 to 7). In Figures 6B-6D, the data is presented as the mean, with each circle representing an individual sample. Shown is one of the three replicated experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA with FDR correction, ns = not significant.

[0037] Figure 7A and 7B depict the kinetic response of antigen-specific cells to mRNA ± mTOR inhibitor. Naive CD45.1 -expressing OTII cells were transferred into CD45.2 WT mice three days prior to i.m. LNP or OVA-mRNA vaccine administration. The vaccine was delivered intramuscularly (i.m.) on days 0 and 7. Everolimus (EVL) was administered daily (5mg/kg, i.p.) starting two days prior to the LNP or vaccine injection and continued until the endpoint or a maximum of 14 days. Figure 7A depicts IFNg and TNFa production by host CD4+ and CD8+ T cells on day 7. Box and Whiskers plots provide a summary of findings from three combined experiments (n= 6 to 10). *P ≤ 0.05, ****p ≤ 0.0001by one-way ANOVA with FDR correction, ns, not significant. Figure 7B depicts kinetics of IFNg and TNFa production by donor OTII cells at the indicated time points. Data are mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by multiple unpaired t tests with FDR correction, ns = not significant.

[0038] Figure 8A- Figure 8B depict the results of example experiments demonstrating cytokine expression in the lungs. Figure 8A depicts protein level of indicated molecules in the BALF. Figure 8B depicts quantitative PCR (qPCR) analysis of the specified transcripts expressed in lung tissue, displayed as Box and Whiskers plots (n= 6 to 15). **P ≤ 0.01, ***P ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA with FDR correction, ns = not significant.

[0039] Figure 9A- Figure 9C depict the results of scRNA-Seq analysis of lung cells. Lung tissue was harvested two days after the final challenge and subjected to Rapsody scRNA- Seq. “Naive” is unmanipulated mice. Figure 9A depicts uniform manifold approximation and projection (UMAP) plot illustrating 15 cell clusters identified by unsupervised clustering, encompassing 4 different conditions (n=2). Figure 9B depicts a heatmap of genes representing each cell population (adjusted P < 0.05, shown is the top of highly expressed genes). Figure 9C depicts the frequency of various cell subsets. Colors denote comparisons of cell percentages across conditions, the size of circles identifies the relative frequency of different cell type within the sample.

[0040] Figure 10A- Figure 10C depict the results of scRNA-Seq analysis of T cells in the lung. Figure 10A depicts representative, highly expressed genes of activated CD8 T cells in different treatment conditions (p value <0.05, fold change compared to other T cell population >0.5). Asterisks represent genes found in CD38+KLRG1- CD8+ T cell population after SARS- CoV-2 mRNA immunization in human. Figure 10B depicts a Venn diagram of genes highly expressed in human CD38+KLRG1- CD8+ T cell population after SARS-CoV-2 mRNA immunization and mouse activated CD8 cells compared to other T cell populations. Figure 10C depicts the expression of selected genes exhibiting differential expression in activated CD4 T cells (not expressing Foxp3 mRNA) according to treatment conditions (p value <0.05).

[0041] Figure 11 depicts the results of example experiments demonstrating OVA mRNA- LNP increases antigen-specific anti-allergic IgG and reduces pro-allergic IgE.

[0042] Figure 12 depicts the results of example experiments demonstrating OVA mRNA- LNP reduces gastrointestinal allergic symptoms.

[0043] Figure 13 depicts the results of example experiments demonstrating Ara h2 mRNA-LNP induces peanut specific IgG production. Mice were vaccinated by Ara h2-mRNA- LNP or LNP (5 ug) on days 0 and 7. Blood was collected on days 0 and 22.

[0044] Figure 14 depicts the house dust mite (HDM) allergy model and results of example experiments to test whether allergen-specific mRNA vaccination protects against HDM- induced asthma. The results demonstrate that the allergen-specific mRNA-LNP vaccine can induce protection against multi -protein allergens. Derpl and Derp2 immunization increases allergen-specific IgG1 and IgG2a antibodies for Derpl and Derp2, respectively.

[0045] Figure 15 depicts the results of example experiments demonstrating that Derpl + Derp2 mRNA-LNP immunization increases IFNg production (Th1) and decreases frequencies of GATA3⁺(Th2), IL-5/IL-13⁺(Th2), and IL-17A⁺(Th17) cells after HDM challenge.

[0046] Figure 16A- Figure 16C depict the results of example experiments demonstrating that Derpl mRNA-LNP vaccination protects against asthma induced by the HDM allergen, Derpl. Figure 16A depicts the experimental workflow. Figure 16B depicts Derpl mRNA-LNP vaccine reduces eosinophilia and mucus production in the lungs upon Derp1 allergen challenge. Figure 16C depicts Derpl mRNA-LNP vaccinee reduces frequency of pro-allergic Th2 and Th17 cells and increases anti-allergic Th1 cells in the lungs upon Derpl allergen challenge.

[0047] Figure 17 depicts research models for preventive care and immunotherapy with an allergen-specific vaccine.

[0048] Figure 18 depicts the results of example experiments to test an allergen-specific mRNA vaccine administered in the presence or absence of an mTOR inhibitor for preventing development of chronic allergy, reducing Th2 responses, and increasing frequency of Th1 and CD8 cells in the lung. These results demonstrate that pre-treatment with allergen-specific mRNA-LNP vaccine protects against chronic allergy and asthma and reduces eosinophilia in the lungs.

[0049] Figure 19 depicts the results of example experiments demonstrating that pretreatment with allergen-specific mRNA-LNP vaccine produces pro-allergic Th2 cells (CD4⁺GATA3⁺) and increases anti-allergic Th1 (CD4⁺IFNg⁺) and cytotoxic (CD8⁺Perforin⁺) cells in the lungs after induction of chronic disease.

[0050] Figure 20 depicts the results of example experiments demonstrating that the preventive care model pre-treatment with OVA-mRNA reduces eosinophil count in the lung.

[0051] Figure 21 depicts the results of example experiments demonstrating that the preventive care model pre-treatment with OVA-mRNA reduces pro-allergic cytokine levels.

[0052] Figure 22 depicts the results of example experiments demonstrating that the preventive care model pre-treatment with OVA-mRNA reduces mucus production.

[0053] Figure 23 depicts the results of example experiments demonstrating that the preventive care model pre-treatment with OVA mRNA reduces airway hyperresponsiveness.

[0054] Figure 24 depicts the results of example experiments demonstrating that the preventive care model mRNA immunization provides protection against chronic inflammation.

[0055] Figure 25 depicts the results of example experiments demonstrating that the preventive care model mRNA immunization alters distribution of T cell populations in allergic asthma.

[0056] Figure 26 depicts the results of example experiments demonstrating that the preventive care model mRNA-LNP immunization alters CD8 T cell phenotype in allergic asthma.

[0057] Figure 27 depicts the results of example experiments demonstrating allergenspecific IgG production for OVA, Ara h2, Derp1, and Derp2 mRNA-LNP vaccine compared with an mRNA-LNP control.

[0058] Figure 28 depicts the results of example experiments demonstrating that the preventive care model mRNA-LNP immunization switches CD4 T cell phenotype to protect against allergy. Allergen specific mRNA-LNP vaccine protects against acute and chronic allergic airway responses by reducing frequencies of Th2 cells, eosinophilia, mucus production, and airway hypersensitivity, and increases anti-allergic Th1 and CD8 responses.

[0059] Figure 29 depicts the results of example experiments of allergy immunotherapy with Ag-sensitization, Ag-specific mRNA vaccination, and Ag-challenge steps. The experiments test whether allergen-specific mRNA vaccines can be used as immunotherapy against allergic responses by induction of IgG antibodies, reduction of allergic inflammation, suppression of Th2 cell activity, and induction of Th1 and tolerant T cells. The results demonstrate that mRNA vaccination increases allergen-specific IgGl and IgG2, but not pro-allergic IgE.

[0060] Figure 30 depicts the results of example experiments demonstrating that allergy immunotherapy with the mRNA-LNP vaccine reduces eosinophilia in bronchial lavage fluid (BALF) and lung tissue, decreases mucus production in the lung and lowers airway hypersensitivity.

[0061] Figure 31 depicts the results of example experiments demonstrating that allergy immunotherapy with mRNA-LNP reduces pro-allergic Th2 cell response (GATA3¹ and IL- 5⁺/IL- 13⁺ cells) and increases frequencies of anti-allergic Th1 (IFNg⁺) and tolerant cells.

[0062] Figure 32 depicts the proportion of cells in CD4 of subpopulations of CD4 T cells in the lung in response to allergy immunotherapy with the mRNA-LNP vaccine.

[0063] Figure 33 depicts the results of example experiments demonstrating that allergy immunotherapy with the mRNA-LNP vaccine increases frequency of activated CD8 T cells in the lung and particularly boosts CD38⁺KLRG1 CD8 T cell population.

[0064] Figure 34 depicts the results of example experiments demonstrating that the immunotherapy model treatment with mRNA reduces allergic responses.

[0065] Figure 35 depicts the results of example experiments demonstrating that the immunotherapy model treatment with mRNA reduces mucus production and airway hypersensitivity.

[0066] Figure 36 depicts the results of example experiments demonstrating that the immunotherapy model treatment with mRNA elevates allergen-specific IgGl but not IgE antibody production. The allergen-specific mRNA treatment effectively reduces the major symptoms of allergic asthma, lowers levels of pro-allergic cytokines, and increases the production of anti-allergic IgG antibodies, demonstrating a viable immunotherapy for allergies. [0067] Figure 37 depicts the results of example experiments demonstrating that allergen immunotherapy with an allergen-specific mRNA-LNP vaccine is safe and does not induce anaphylactic response compared to a subcutaneously (s.c.) injected allergen. Mice were sensitized with OVA protein through ear skin and on day 30 injected with an OVA mRNA-LNP vaccine (intramuscular, i.m.) or a high dose of endotoxin free OVA protein (subcutaneous s.c.) as a standard immunotherapy. Mice without sensitization were used as a control for OVA s.c. injection.

[0068] Figure 38A- Figure 38C depict the results of example experiments demonstrating immunization with a non-specific mRNA-LNP vaccine increases the immunotherapeutic effect of allergen-specific mRNA-LNP vaccine. Figure 38A depicts the experimental workflow. Figure 39B depicts co-administration of allergen-specific (Derpl+Derp2 mRNA-LNP) and non-specific (OVA mRNA-LNP) vaccines reduces eosinophilia in the lungs. Figure 38C depicts coadministration of allergen-specific (Derpl+Derp2 mRNA-LNP) and non-specific (OVA mRNA- LNP) vaccines reduces the frequency of pro-allergic Th2 cells and increases the percentage of anti-allergic Th1 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention relates to compositions and methods for treating, preventing or reducing inflammatory, autoimmune, or allergic responses in a subject. In some embodiments, the compositions and methods modulate the immune response in a subject. In some embodiments, the compositions and methods reduce the production of inflammatory cytokines in a subject in response to an allergen or autoantigen. In some embodiments, the compositions and methods promote the differentiation of antigen- or allergen-directed Tregs in a subject. In some embodiments, the compositions and methods promote tolerance to an allergen in a subject.

[0070] In some embodiments, the invention provides a composition comprising at least one lipid nanoparticle (LNP) comprising at least one nucleoside-modified RNA molecule encoding at least one allergen. In one embodiment, the allergen is an egg allergen. In one embodiment, the egg allergen is ovalbumin (OVA). In one embodiment, the allergen is a peanut allergen. In one embodiment, the peanut allergen is selected from the group consisting of Ara h 1, Ara h 2, Ara h 3, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 12 and Ara h 13. In one embodiment, the peanut allergen is Ara h 2. In one embodiment, the allergen is a house dust mite (HDM) allergen. In one embodiment, the HDM allergen is selected from the group consisting of Derp1, Derp 2, and Derp23.

[0071] In some embodiments, the composition further comprises an anti-inflammatory agent. In some embodiments, the agent is a mechanistic target of rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is everolimus (e.g., Afinitor, Afinitor Disperz, Zortress), rapamycin, sirolimus (e.g., Rapamune, Hyftor, Fyarro), temsirolimus (Torisel), ridaforolimus, Torin-1, or non-rapalog derived inhibitors or an analog or derivative thereof. In some embodiments, the mTOR inhibitor is everolimus.

[0072] The present invention also relates to methods of treating or preventing allergic, autoimmune and inflammatory diseases or disorders using the compositions of the invention. In some embodiments, the invention relates to methods of treating or preventing an allergic reaction or allergic disease using the compositions of the invention. In some embodiments, the method comprises administering the compositions of the invention to a subject before exposure to an allergen. In some embodiments, the method comprises administering the compositions of the invention to a subject during exposure to an allergen. In some embodiments, the method comprises administering the compositions of the invention to a subject after exposure to an allergen. In some embodiments, the method comprises administering the compositions of the invention to a subject before the development of an allergic disease or disorder. In some embodiments, the method comprises administering the compositions of the invention to a subject after the development of an allergic disease or disorder. In some embodiments, the invention relates to methods of treating or preventing an inflammatory reaction using the compositions of the invention. In some embodiments, the inflammatory reaction is asthma.

Definitions:

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0074] As used herein, each of the following terms has the meaning associated with it in this section. [0075] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0076] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0077] The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Natural antibodies are typically tetramers of immunoglobulin molecules. The term “antibody” as used herein encompasses antibody fragments, which refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Antibodies or antibody fragments as described herein may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fab, F(ab)2, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0078] The term “antigen” or “Ag” as used herein is defined as a molecule that binds to an antibody or a T cell receptor. 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. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. The present disclosure provides, but is not limited to, the use of partial nucleotide sequences. Moreover, an antigen need not be encoded by a “gene” at all. An antigen can be generated, synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0079] The term “allergen” as used herein refers to any molecule that has been reported to induce allergic, including but not limited to IgE mediated, reactions upon their exposure to an individual. Any macromolecule, including proteins or peptides, can serve as an allergen. Furthermore, allergens can be derived from recombinant or genomic DNA or RNA.

Furthermore, one skilled in the art will understand that an allergen need not be encoded solely by a full-length nucleotide sequence of a gene. The present disclosure provides, but is not limited to, the use of partial nucleotide sequences. Moreover, an allergen need not be encoded by a “gene” at all. An allergen can be generated, synthesized, or can be derived from a biological sample.

[0080] The term “immunogen” as used herein, is intended to denote a substance of matter, which is capable of inducing an immune response in an individual. This immune response may involve either antibody production, or the activation of specific immunogenically- competent cells, or both. In some embodiments, an immunogen elicits a humoral response. In some embodiments, an immunogen elicits a cellular response. In some embodiments, the immune response is an adaptive immune response. Any DNA or RNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “immunogen” as that term is used herein. In some embodiments, the immune response significantly engages pathogenic agents that share immunological features with the immunogen. “Immunogen” refers to any substance introduced into the body in order to generate an immune response. That substance can a physical molecule, such as a protein, or can be encoded by a vector, such as DNA, RNA, or a virus. In some embodiments, a composition described herein is an immunogen. In some embodiments, a RNA molecule described herein is an immunogen. In some embodiments, an RNA molecule described herein encodes an immunogen. In some embodiments, a composition (e.g., a pharmaceutical composition, immunogenic compsition, vaccine) described herein comprises an immunogen.

[0081] “Immune response,” as the term is used herein, means a process involving the activation and/or induction of an effector function in, by way of non-limiting examples, a T cell, B cell, natural killer (NK) cell, and/or an antigen-presenting cell (APC). Thus, an immune response, as would be understood by the skilled artisan, includes, but is not limited to, any detectable antigen-specific activation and/or induction of a helper T cell or cytotoxic T cell activity or response, production of antibodies, antigen presenting cell activity or infiltration, macrophage activity or infiltration, neutrophil activity or infiltration, and the like.

[0082] As used herein, an “immunogenic composition” is any molecule that induces an immune response upon administration. An immunogenic composition may comprise an antigen (e g., a peptide or polypeptide), a nucleic acid encoding an antigen, a cell expressing or presenting an antigen or cellular component, a virus expressing or presenting an antigen or cellular component, or any combination thereof.

[0083] As used herein, the term “vaccine” refers to an immunogenic composition that provides protective immunity upon inoculation into a subject.

[0084] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (e.g., mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of RNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0085] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0086] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. [0087] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0088] As used herein, a nucleotide sequence is “substantially homologous” or “substantially identical” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the original nucleotide sequence at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0089] As used herein, an amino acid sequence is “substantially homologous” or “substantially identical” to any of the amino acid sequences described herein when its amino acid sequence has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least

90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least

96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%. The identity between two sequences can be determined by using the BLASTP algorithm for amino acid sequences or the BLASTN algorithm for nucleotide sequences (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0090] The term “variant” as used herein with respect to a nucleic acid refers (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequence substantially identical thereto. A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof.

[0091] The term “variant” as used with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also refer to a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. (Kyte et al., 1982, J. Mol. Biol. 157: 105-132). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0092] As used herein, the terms “fragment” or “functional fragment” refer to a fragment of an antigen or a nucleic acid sequence encoding an antigen that, when administered to a subject, provides an increased immune response. Fragments are generally 10 or more amino acids or nucleic acids in length. “Fragment” may mean a polypeptide fragment of an antigen that is capable of eliciting an immune response in a subject. A fragment of an antigen may be 100% identical to the full length except missing at least one amino acid from the N and/or C terminal, in each case with or without signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length antigen, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.

[0093] A fragment of a nucleic acid sequence that encodes an antigen may be 100% identical to the full length except missing at least one nucleotide from the 5’ and/or 3’ end, in each case with or without sequences encoding signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added. The fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. [0094] “Isolated” as used herein means (1) altered or removed from the natural state and/or (2) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (3) designed, produced, prepared, and/or manufactured by the hand of man. In some embodiments, a nucleic acid or a peptide naturally present in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0095] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N- glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0096] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell. Exemplary modified nucleosides are described elsewhere herein. For example, an RNA (e.g., an IVT mRNA) where some or all of the uridines have been replaced with pseudouridine, 1 -methyl pseudouridine, 5-methyl-uridine or another modified nucleoside, such as those described elsewhere herein. In some embodiments, the nucleotide sequence may contain a sequence where some or all cytodines are replaced with methylated cytidine, or another modified nucleoside, such as those described elsewhere herein.

[0097] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0098] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0099] The term “polyribonucleotide” as used herein is defined as a chain of ribonucleotides. Furthermore, nucleic acids are polymers of ribonucleotides. Thus, nucleic acids and polyribonucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polyribonucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[00100] In some embodiments, an RNA molecule, polyribonucleotide, polynucleotide or nucleic acid of the present disclosure is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[00101] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[00102] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. By way of one non-limiting example, a promoter that is recognized by bacteriophage RNA polymerase and is used to generate the RNA by in vitro transcription.

[00103] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response.

[00104] In some embodiments, “pseudouridine” refers to m1acp³Y (l-methyl-3-(3-amino- 3 -carboxypropyl) pseudouridine). In another embodiment, the term refers to m¹Y (1- methylpseudouridine). In another embodiment, the term refers to Ym (2’-O- methylpseudouridine. In another embodiment, the term refers to m⁵D (5-methyldihydrouridine). In another embodiment, the term refers to m³Y (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment provided in the present disclosure.

[00105] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm), which includes one or more lipids.

[00106] The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. [00107] As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[00108] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[00109] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH.

[00110] The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid.

[00111] The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.

[00112] “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[00113] The terms “subject,” “patient,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some non-limiting embodiments, the patient, subject or individual is a mammal, bird, poultry, cattle, pig, horse, sheep, ferret, primate, dog, cat, guinea pig, rabbit, bat, or human.

[00114] A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

[00115] In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

[00116] By the term “modulating,” as used herein, means mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as a human.

[00117] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[00118] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[00119] The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, prevention, or eradication of at least one sign or symptom of a disease or disorder.

[00120] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. [00121] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[00122] The phrase “under transcriptional control” or “operatively linked” with reference to a promoter as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[00123] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fdlers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the present disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[00124] Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description

[00125] The present invention relates to compositions and methods for preventing, reducing, and/or treating an inflammatory, autoimmune, or allergic response and/or disease in a subject. In some embodiments, the invention provides a composition comprising at least one nucleic acid molecule encoding at least one antigen. In some embodiments, the antigen is an autoantigen. In some embodiments, the antigen is an allergen. In some embodiments, the allergen is a food allergen. In some embodiments, the allergen is an aero allergen. In one embodiment, the allergen is an egg allergen. In one embodiment, the egg allergen is ovalbumin (OVA). In one embodiment, the allergen is a peanut allergen. In one embodiment, the peanut allergen is selected from the group consisting of Ara h 1, Ara h 2, Ara h 3, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 12, and Ara h 13. In one embodiment, the peanut allergen is Ara h 2. In one embodiment, the peanut allergen is wild type Ara h 2. In one embodiment, the peanut allergen is hypoallergenic Ara h 2. In one embodiment, the allergen is a house dust mite (HDM) allergen. In one embodiment, the HDM allergen is selected from the group consisting of Derp1, Derp 2, and Derp 23. In some embodiments, the HDM allergen is a mutant of Derp 1, Derp 2, or Derp 23. In some embodiments, the HDM allergen is Derp 2 with a K96A mutation.

[00126] In some embodiments, the allergen-encoding nucleic acid of the present composition is a nucleoside-modified RNA. In some embodiments, the allergen-encoding nucleic acid of the present composition is a purified nucleoside-modified RNA.

[00127] In some embodiments, the composition comprises an adjuvant. In one embodiment, the composition comprises a nucleoside-modified RNA encoding an antigen, e.g. an allergen, and an LNP, wherein the LNP has adjuvant activity. In some embodiments, the composition comprises an RNA molecule encoding an adjuvant. In one embodiment, the composition comprises a first nucleoside-modified RNA, which encodes an antigen, e.g. an allergen, and a second nucleoside-modified RNA, which encodes an adjuvant.

[00128] In some embodiments, the composition comprises a combination of a) a delivery vehicle comprising a nucleoside-modified RNA encoding an antigen, e.g. an allergen, and b) an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is an mTOR inhibitor. In some embodiments, the mTOR inhibitor is a small molecule. In some embodiments, the mTOR inhibitor is a small molecule selected from the group consisting of everolimus (e.g. Afinitor, Afinitor Disperz, Zortress), rapamycin. Sirolimus (e.g. Rapamune, Hyftor, Fyarro), temsirolimus (Torisel), ridaforolimus, Torin-1, or non-rapalog derived inhibitors or an analog or derivative thereof. In some embodiments, the mTOR inhibitor is everolimus.

Immunomodulatory composition

[00129] In one embodiment, the present invention provides an immunomodulatory composition for modulating an immune response against an antigen, e.g. an allergen, or an autoantigen in a subject. In some embodiments, the immunomodulatory composition may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen, or a combination thereof. In some embodiments, the composition comprises or encodes all or part of any antigen (e.g. allergen or autoantigen) described herein, or an immunogenically functional equivalent thereof. In other embodiments, the composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell, LNP, or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination. An immunomodulatory composition of the present invention, and its various components, may be prepared and/or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

[00130] In some embodiments, the composition of the invention increases Treg levels and/or function. In some embodiments, the composition of the invention promotes tolerance- induced signals in T cells, and facilitates the differentiation of CD4⁺ T cells toward Treg cells. In some embodiments, the composition of the invention increases allergen specific IgG production. In some embodiments, the composition of the invention reduces pro-allergic IgE. In some embodiments, the composition of the invention modulates Th2 cell responses. In some embodiments, the composition of the invention reduces Th2 cell responses against an allergen by inducing allergen tolerance. In some embodiments, the composition of the invention reduces eosinophil count in the lung. Modulation of immunity (e.g., an increase in allergen directed T reg cells, or reduction in type 2 inflammatory cytokines) by the expression of the allergen can be detected by observing in vivo or in vitro the response of all or any part of the immune system in the host against the allergen. [00131 ] The therapeutic compounds or compositions of the invention may be administered preventively (i.e., to prevent an inflammatory, autoimmune, or allergic response, disease, or disorder) or therapeutically (i.e., to treat an inflammatory, autoimmune, or allergic response, disease, or disorder) to subjects suffering from, or at risk of (or susceptible to) developing the inflammatory, autoimmune, or allergic response, disease, or disorder. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of an inflammatory, autoimmune, or allergic response, disease, or disease, such that an inflammatory, autoimmune, or allergic response, disease, or disorder is prevented or alternatively delayed in its progression. Prophylactic administration may also occur prior to a subject’s exposure to an allergen. In the context of the field of medicine, the term “prevent” encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

Antigen

[00132] The present invention relates to compositions and methods for preventing or reducing inflammatory, autoimmune, or allergic responses in a subject. In some embodiments, the invention provides a composition comprising at least one nucleic acid molecule encoding at least one antigen. In some embodiments, the antigen is an autoantigen. In some embodiments, the antigen is an allergen. In some embodiments, the allergen is a food allergen. In some embodiments, the allergen is an aero allergen.

[00133] Examples of allergens include, but are not limited to, egg, peanut, milk and dairy, tree nuts, fish, shellfish, wheat, gluten, soy, sesame, apple, apricot, carrot, celery, cherry, peach, pear, plum, potato, anise, carraway seed, coriander, fennel, parsley, banana, cucumber, melons, zucchini, kiwi, citrus, tomato, peppers, broccoli, cabbage, cauliflower, garlic, onion, black pepper, mustard, color additives, corn, meat, gelatin, sunflower seed, poppy seed, avocado, mango, acacia gum, allspice, amaranth, annatto,, Balsam of Peru, barley, beans, beer, buckwheat, cardamom, cassia, celeriac, chamomile, chocolate, cocoa, cinnamon, clove, coconut, coffee, cottonseed, cumin, curry, dill, ethanol, flax seed, ginger, grapes, guava, honey, royal jelly, hops, karaya gum, lentils, lupine, mace, maple syrup, millet seed, mushrooms, mycoprotein, nutmeg, oats, papaya, paprika, pea, cayenne pepper, white pepper, pineapple, pomegranate, psyllium, quinine, rape seed, rice, rye, spinach, squash, strawberry, beet, tragacanth gum, turnip, vanilla, vitamin A, vitamin E, wine, yeast, pharmaceuticals, tree allergens (e.g. birch (Betula), alder (Alnus), cedar (Cedrus), hazel (Corylus), hornbeam (Carpinus), horse chestnut (Aesculus), willow (Salix), poplar (Populus), plane (Platanus), linden/lime (Tilia) and olive (Olea)); plant allergens (e.g. rye, ragweed (Ambrosia), plantain (Plantago), sorrel-dock (Rumex), fat hen (Chenopodium), mugwort (Artemisia) and pigweed), plant contact allergens (e.g. poison oak, poison ivy and nettles/parietaria (Urticaceae)), grass allergens (e.g. ryegrass (Lolium sp), Timothy (Phleum pratense), Johnson, Bermuda, fescue and bluegrass allergens), molds or fungi (e.g. Alternaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor, and thermophilic actinomycetes), house mites (e.g. dermatophagoides pterosinyssis), feathers, animal hair and dander (e.g. from cats and dogs), insects (e.g. bee, wasp and ant venom, and cockroach calyx allergens), and fragments thereof.

[00134] In certain embodiments, the allergen is an egg allergen. In one embodiment, the egg allergen is ovalbumin (OVA). In one embodiment, the allergen comprises an amino acid sequence of SEQ ID NO: 1, or a fragment or variant thereof.

[00135] In some embodiments, the allergen is a peanut allergen. In one embodiment, the peanut allergen is selected from the group consisting of Ara h 1, Ara h 2, Ara h 3, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 12, and Ara h 13 . In one embodiment, the peanut allergen is Ara h 2. In one embodiment, the peanut allergen is the wild-type version of Ara h 2. In one embodiment, the peanut allergen is a hypoallergenic version of Ara h 2. In one embodiment In one embodiment, the peanut allergen comprises an amino acid sequence of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, or a fragment or variant thereof

[00136] In one embodiment, the composition comprises a nucleic acid sequence encoding an egg allergen. In one embodiment, the egg allergen is OVA. In one embodiment, the composition comprises a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO: 1, or a fragment or variant thereof. In one embodiment, the composition comprises a nucleoside-modified mRNA nucleotide sequence encoding SEQ ID NO: 1, or a fragment or variant thereof, wherein one or more residues are modified nucleosides as described elsewhere herein.

[00137] In one embodiment, the composition comprises a nucleic acid sequence encoding a peanut allergen. In one embodiment, the peanut allergen is selected from the group consisting of Ara h 1, Ara h 2, Ara h 3, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 12, and Ara h 13. In one embodiment, the nucleic acid sequence encoding a peanut allergen comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or a fragment or variant thereof.

[00138] In one embodiment, the peanut allergen is Ara h 2. In some embodiments, the composition comprises a nucleic acid encoding an amino acid sequence of SEQ ID NO:2, or a fragment or variant thereof. In one embodiment, the composition comprises a nucleoside- modified mRNA nucleotide sequence encoding SEQ ID NO:2, or a fragment or variant thereof, wherein one or more residues are replaced with a modified nucleoside as described elsewhere herein.

[00139] In one embodiment, the composition comprises a nucleic acid sequence encoding a HDM allergen. In one embodiment, the HDM allergen is selected from Derp1, Derp2, and Derp 23. In one embodiment, the composition comprises a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a fragment or variant thereof. In one embodiment, the composition comprises a nucleoside-modified mRNA nucleotide sequence encoding SEQ ID NO:3, or a fragment or variant thereof, wherein one or more residues are modified nucleosides as described elsewhere herein. In one embodiment, the composition comprises a nucleoside-modified mRNA nucleotide sequence encoding SEQ ID NO:4, or a fragment or variant thereof, wherein one or more residues are modified nucleosides as described elsewhere herein. In one embodiment, the nucleic acid sequence encoding an HDM allergen comprises SEQ ID NO: 21.

[00140] In certain embodiments, the allergen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an allergen described herein and retains the immunogenic function of the original amino acid sequence. For example, in certain embodiments, the amino acid sequence of the allergen has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[00141] In some embodiments, the allergen is a wild-type protein. In some embodiments, the amino acid sequence of the allergen is a variant of a wild-type protein with mutations in the amino acid sequence. In some embodiments, a mutation in the amino acid sequence of the allergen is outside of the IgE binding site. In some embodiments, a mutation in the amino acid sequence of the allergen is within the IgE binding site.

[00142] In one embodiment, the allergen is encoded by a nucleic acid sequence of a nucleic acid molecule. In certain embodiments, the nucleic acid sequence comprises DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. In one embodiment, the nucleic acid sequence comprises a modified nucleic acid sequence. For example, in one embodiment the allergen-encoding nucleic acid sequence comprises nucleoside-modified RNA, as described in detail elsewhere herein. In certain instances, the nucleic acid sequence comprises additional sequences that encode linker or tag sequences that are linked to the allergen by a peptide bond.

[00143] In some embodiments, the antigen is an autoantigen. In some embodiments, the autoantigen is associated with an autoimmune disorder.

[00144] Examples of autoimmune disorders include, but are not limited to, rheumatoid arthritis/seronegative arthropathies, osteoarthritis, inflammatory bowel disease, systemic lupus erythematosis, iridoeyelitis/uveitistoptic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's gramilornatosis, sarcoidosis, including, but not limited to, rheumatoid arthritis/seronegative arthropathies, osteoarthritis, inflammatory bowel disease, systemic lupus erythematosis, iridoeyelitis/uveitistoptic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's gramilornatosis, sarcoidosis, myocarditis, postmyocardial infarction syndrome, postpericardiotomy syndrome, subacute bacterial endocarditis (SBE), anti-glomerular basement membrane nephritis, interstitial cystitis, lupus nephritis, autoimmune hepatitis, primary biliary cholangitis(PBC), primary sclerosing cholangitis, anti synthetase syndrome, alopecia areata, autoimmune angioedema, autoimmune progesterone dermatitis, autoimmune urticaria, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, discoid lupus erythematosus, epidermolysis bullosa acquisita, erythema nodosum, gestational pemphigoid, hidradenitis suppurativa, lichen planus, lichen sclerosus, linear IgA disease (LAD), morphea, pemphigus vulgaris, pityriasis lichenoides et varioliformis acuta, Mucha-Habermann disease, psoriasis, systemic scleroderma, vitiligo, Addison's disease, autoimmune polyendocrine syndrome (APS) type 1, autoimmune polyendocrine syndrome (APS) type 2, autoimmune polyendocrine syndrome (APS) type 3, autoimmune pancreatitis (AIP), diabetes mellitus type 1, autoimmune thyroiditis, Ord's thyroiditis, Graves' disease, autoimmune oophoritis, endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmune enteropathy, Coeliac disease, Crohn's disease, microscopic colitis, ulcerative colitis, antiphospholipid syndrome(APS, APLS), aplastic anemia, autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune thrombocytopenic purpura, cold agglutinin disease, essential mixed cryoglobulinemia, Evans syndrome, pernicious anemia, pure red cell aplasia, thrombocytopenia, adiposis dolorosa, adult-onset Still's disease, ankylosing spondylitis, CREST syndrome, drug-induced lupus, enthesitis-related arthritis, eosinophilic fasciitis Felty syndrome, IgG4-related diseasejuvenile arthritis, Lyme disease (chronic), mixed connective tissue disease (MCTD), palindromic rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, Schnitzler syndrome, undifferentiated connective tissue disease (UCTD), dermatomyositis, fibromyalgia, inclusion body myositis, myositis, myasthenia gravis, neuromyotonia, paraneoplastic cerebellar degeneration, polymyositis, acute disseminated encephalomyelitis (ADEM), acute motor axonal neuropathy, anti-N-methyl-D-aspartate (Anti- NMDA) receptor encephalitis, balo concentric sclerosis, Bickerstaffs encephalitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Hashimoto's encephalopathy, idiopathic inflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome, multiple sclerosis, pattern II, Oshtoran Syndrome, pediatric autoimmune neuropsychiatric disorder associated with streptococcus (PANDAS), progressive inflammatory neuropathy, restless leg syndrome, stiff person syndrome, sydenham chorea, transverse myelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome, Graves ophthalmopathy, intermediate uveitis, ligneous conjunctivitis, Mooren's ulcer, neuromyelitis optica, opsoclonus myoclonus syndrome, optic neuritis, scleritis, Susac's syndrome, sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune inner ear disease(AIED), Meniere's disease, Behcet's disease, eosinophilic granulomatosis with polyangiitis (EGPA), giant cell arteritis, granulomatosis with polyangiitis (GPA), IgA vasculitis (IgAV), Kawasaki's disease, leukocytoclastic vasculitis, lupus vasculitis, rheumatoid vasculitis, microscopic polyangiitis (MPA), polyarteritis nodosa (PAN), polymyalgia rheumatic, urticarial vasculitis, vasculitis, and primary immune deficiency.

[00145] In one embodiment, the autoantigen is encoded by a nucleic acid sequence of a nucleic acid molecule. In certain embodiments, the nucleic acid sequence comprises DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. In one embodiment, the nucleic acid sequence comprises a modified nucleic acid sequence. For example, in one embodiment the autoantigen-encoding nucleic acid sequence comprises nucleoside-modified RNA, as described in detail elsewhere herein. In certain instances, the nucleic acid sequence comprises additional sequences that encode linker or tag sequences that are linked to the autoantigen by a peptide bond.

Adjuvant

[00146] In one embodiment, the composition comprises an adjuvant. In one embodiment, the composition comprises a nucleic acid molecule encoding an adjuvant. In one embodiment, the adjuvant-encoding nucleic acid molecule is an IVT RNA molecule. In one embodiment, the adjuvant-encoding nucleic acid molecule is a nucleoside-modified RNA molecule.

[00147] Exemplary adjuvants include, but are not limited to, TLR ligands (e.g., TLR 2-9, flagellin, monophosphoryl lipid A, dsRNA etc.), alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL- 12, IL- 15, MHC, CD80, CD86, helicase adjuvants, NOD and inflammasome adjuvants, inorganic compounds (potassium alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide), oils (paraffin oil, propolis, adjuvant 65 based on peanut oil, squalene, MF59), bacterial products (killed bacteria Bordatella pertussis, Myobacterium boyis, toxoids, MPL), plant saponins (from Quillaia, soybean, polygala senega), GpG oligonucleotides, and combinations (Freund's complete adjuvant, Freund's incomplete adjuvant, AS01, Matrix-M). Other genes which may be useful adjuvants include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P- selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3- Ig, and functional fragments thereof.

[00148] In certain embodiments, the composition comprises an LNP. In some embodiments the LNP acts as an adjuvant.

Nucleic Acids

[00149] In one embodiment, the invention includes a nucleoside-modified nucleic acid molecule. In one embodiment, the nucleoside-modified nucleic acid molecule encodes an allergen. In one embodiment, the invention includes a nucleoside-modified nucleic acid molecule encoding an adjuvant.

[00150] The nucleotide sequences encoding an allergen or adjuvant, as described herein, can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and/or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a peptide or polypeptide according to the invention. Therefore, the scope of the present invention includes nucleotide sequences that are substantially homologous to the nucleotide sequences recited herein and encode an allergen or adjuvant of interest.

[00151] As used herein, a nucleotide sequence is “substantially homologous” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, of at least 65%, of at least 70%, of at least 65%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, or of at least 99%. A nucleotide sequence that is substantially homologous to a nucleotide sequence encoding an allergen can typically be isolated from a producer organism of the allergen based on the information contained in the nucleotide sequence by means of introducing conservative or non-conservative substitutions, for example. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

[00152] Further, the scope of the invention includes nucleotide sequences that encode amino acid sequences that are substantially homologous to the amino acid sequences recited herein and preserve the function of the original amino acid sequence.

[00153] As used herein, an amino acid sequence is “substantially homologous” to any of the amino acid sequences described herein when its amino acid sequence has a degree of identity with respect to the amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 65%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, or of at least 99%. The identity between two amino acid sequences can be determined by using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[00154] In one embodiment, the invention relates to a construct comprising a nucleotide sequence encoding an allergen. In one embodiment, the construct comprises a plurality of nucleotide sequences encoding a plurality of antigens (e.g., allergens or self-antigens or a combination thereof). For example, in certain embodiments, the construct encodes 1 or more, 2 or more, 5 or more, or more antigens (e.g., allergens or self-antigens or a combination thereof). In one embodiment, the invention relates to a construct comprising a nucleotide sequence encoding an adjuvant.

[00155] In one embodiment, the composition comprises a plurality of constructs, each construct encoding one or more antigen (e.g., allergen or self-antigen). In certain embodiments, the composition comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more constructs.

[00156] In another embodiment, the construct is operatively bound to a translational control element. The construct can incorporate an operatively bound regulatory sequence for the expression of the nucleotide sequence of the invention, thus forming an expression cassette. Vectors

[00157] The nucleic acid sequences coding for the antigen (e.g., allergen or self-antigen) can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[00158] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, a PCR-generated linear DNA sequence, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors and vectors optimized for in vitro transcription.

[00159] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, carbohydrates, peptides, cationic polymers, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[00160] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/RNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[00161] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[00162] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the allergen of the present invention, in order to confirm the presence of the mRNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Northern blotting and RT-PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunogenic means (ELIS As and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

In vitro transcribed RNA

[00163] In one embodiment, the composition of the invention comprises in vitro transcribed (IVT) RNA encoding an antigen (e.g., allergen or self-antigen). In one embodiment, the composition of the invention comprises IVT RNA encoding a plurality of antigens (e.g., allergens or self-antigens, or a combination thereof). In one embodiment, the composition of the invention comprises IVT RNA encoding an adjuvant. In one embodiment, the composition of the invention comprises IVT RNA encoding one or more antigen (e.g., allergen or self-antigen) and one or more adjuvants.

[00164] In one embodiment, an IVT RNA can be introduced to a cell as a form of transient transfection. IVT RNA is generally produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. In one embodiment, the desired template for in vitro transcription is an allergen. In one embodiment, the desired template for in vitro transcription is an adjuvant capable of enhancing an adaptive immune response.

[00165] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest of a portion of a gene. The gene can include some or all of the 5' and/or 3' untranslated regions (UTRs). The gene can include exons and introns. In another embodiment, the DNA to be used for PCR is a gene from a plant or animal. In another embodiment, the DNA to be used for PCR is from a plant or animal and includes the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[00166] Genes that can be used as sources of DNA for PCR include genes that encode polypeptides that induce or enhance an allergic response in an organism. In certain instances, the genes are useful for a short-term treatment. In certain instances, the genes have limited safety concerns regarding dosage of the expressed gene.

[00167] In various embodiments, a plasmid is used to generate a template for in vitro transcription of mRNA, which is used for transfection. [00168] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. In certain embodiments, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[00169] The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[00170] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.

[00171] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[00172] In one embodiment, the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product, which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA, which is effective in eukaryotic transfection when it is polyadenylated after transcription.

[00173] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[00174] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination in competent bacterial cells for plasmid propagation.

[00175] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase. In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[00176] 5' caps also provide stability to mRNA molecules. In one embodiment, RNAs produced by the methods to include a 5' cap! structure. Such cap! structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase enzymes (CellScript, Madison, WI). Alternatively, 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[00177] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001). In certain embodiments RNA of the invention is introduced to a cell with a method comprising the use of TransIT®-mRNA transfection Kit (Minis, Madison WI), which, in some instances, provides high efficiency, low toxicity, transfection.

Nucleoside-modified RNA

[00178] In one embodiment, the composition of the present invention comprises a nucleoside-modified nucleic acid encoding an antigen (e.g., allergen or self-antigen) as described herein. In one embodiment, the composition of the present invention comprises a nucleoside- modified nucleic acid encoding an adjuvant as described herein. In one embodiment, the composition of the present invention comprises a nucleoside-modified nucleic acid encoding one or more antigen (e.g., allergen or self-antigen) and one or more adjuvants.

[00179] For example, in one embodiment, the composition comprises a nucleoside- modified RNA. In one embodiment, the composition comprises a nucleoside-modified mRNA. Nucleoside-modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent Nos. 8,278,036, 8,691,966, and 8,835,108, each of which is incorporated by reference herein in its entirety.

[00180] In some embodiments, nucleoside-modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days (Kariko et al., 2008, Mol Ther 16: 1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small and that makes it applicable for human therapy.

[00181] In some instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA. In certain embodiments, using mRNA rather than protein also has many advantages. Half-lives of proteins in the circulation are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days. Purification of proteins is problematic, and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[00182] In certain embodiments, the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In certain embodiments, inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16: 1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23: 165-175).

[00183] It has been demonstrated that the presence of modified nucleosides, including pseudouridines in RNA suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23: 165-175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it is shown that the presence of pseudouridine improves the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892).

[00184] In some embodiments, the nucleoside-modified nucleic acid molecule is a purified nucleoside-modified nucleic acid molecule. For example, in certain embodiments, the composition is purified to remove double-stranded contaminants. In certain instances, a preparative high performance liquid chromatography (HPLC) purification procedure is used to obtain pseudouridine-containing RNA that has superior translational potential and no innate immunogenicity (Kariko et al., 2011 , Nucleic Acids Research 39:el42). Administering HPLC- purified, pseudourine-containing RNA coding for erythropoietin into mice and macaques resulted in a significant increase of serum EPO levels (Kariko et al., 2012, Mol Ther 20:948- 953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy. In certain embodiments, the nucleoside-modified nucleic acid molecule is purified using non-HPLC methods. In certain instances, the nucleoside-modified nucleic acid molecule is purified using chromatography methods, including but not limited to HPLC and fast protein liquid chromatography (FPLC). An exemplary FPLC -based purification procedure is described in Weissman et al., 2013, Methods Mol Biol, 969: 43-54. Exemplary purification procedures are also described in U.S. Patent Application Publication No. US2016/0032316, which is hereby incorporated by reference in its entirety.

[00185] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises an isolated nucleic acid encoding an allergen, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises a vector, comprising an isolated nucleic acid encoding an allergen, adjuvant, or combination thereof, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.

[00186] In one embodiment, the nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein. For example, in some embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In one embodiment, the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In one embodiment, the nucleoside- modified RNA is synthesized by T3 phage RNA polymerase.

[00187] In one embodiment, the modified nucleoside is mlacp3'P (l-methyl-3-(3-amino- 3 -carboxypropyl) pseudouridine. In one embodiment, the modified nucleoside is m1Ψ (1- methylpseudouridine). In one embodiment, the modified nucleoside is Ψm (2'-O- methylpseudouridine). In one embodiment, the modified nucleoside is m5D (5- methyldihydrouridine). In one embodiment, the modified nucleoside is m3Ψ (3- methylpseudouridine). In one embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In one embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In one embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[00188] In another embodiment, the nucleoside that is modified in the nucleoside- modified RNA the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

[00189] In another embodiment, the modified nucleoside of the present invention is m5C (5-methylcytidine). In another embodiment, the modified nucleoside is m5U (5 -methyluridine). In another embodiment, the modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the modified nucleoside is s2U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[00190] In other embodiments, the modified nucleoside is mlA (1 -methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6- hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1- methylinosine); ml Im (1,2'-O-dimethylinosine); m3C (3 -methylcytidine); Cm (2'-O- methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm (5,2'-O-dimethylcytidine); ac4Cm (N4-acetyl-2'-O-methylcytidine); k2C (lysidine); mlG (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O- methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQO (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G+ (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5- methoxy carbonylmethyluridine); mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cmnm5Um (5-carboxymethylaminomethyl-2'-O- methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6- dimethyladenosine); Im (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2'-O- dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3 -methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,2'-O-dimethyladenosine); m62Am (N6,N6,O-2'- trimethyladenosine); m2,7G (N2,7-dimethylguanosine); m2,2,7G (N2,N2,7-trimethylguanosine); m3Um (3,2'-O-dimethyluridine); m5D (5 -methyldihydrouridine); f5Cm (5-formyl-2'-O- methylcytidine); ml Gm (1,2'-O-dimethylguanosine); ml Am (1,2'-O-dimethyladenosine); xm5U (5-taurinomethyluridine); (5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2 (isowyosine); or ac6A (N6-acetyladenosine).

[00191] In one embodiment, a nucleoside-modified RNA of the present invention comprises a combination of 2 or more of the above modifications. In one embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above modifications. In one embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

[00192] In various embodiments, between 0.1% and 100% of the residues in the nucleoside-modified of the present invention are modified (e.g., either by the presence of pseudouridine or another modified nucleoside base). In one embodiment, the fraction of modified residues is 0.1%. In one embodiment, the fraction of modified residues is 0.2%. In one embodiment, the fraction is 0.3%. In one embodiment, the fraction is 0.4%. In one embodiment, the fraction is 0.5%. In one embodiment, the fraction is 0.6%. In one embodiment, the fraction is 0.7%. Tn one embodiment, the fraction is 0.8%. In one embodiment, the fraction is 0.9%. In one embodiment, the fraction is 1%. In one embodiment, the fraction is 1.5%. In one embodiment, the fraction is 2%. In one embodiment, the fraction is 2.5%. In one embodiment, the fraction is 3%. In one embodiment, the fraction is 4%. In one embodiment, the fraction is 5%. In one embodiment, the fraction is 6%. In one embodiment, the fraction is 7%. In one embodiment, the fraction is 8%. In one embodiment, the fraction is 9%. In one embodiment, the fraction is 10%. In one embodiment, the fraction is 12%. In one embodiment, the fraction is 14%. In one embodiment, the fraction is 16%. In one embodiment, the fraction is 18%. In one embodiment, the fraction is 20%. In one embodiment, the fraction is 25%. In one embodiment, the fraction is 30%. In one embodiment, the fraction is 35%. In one embodiment, the fraction is 40%. In one embodiment, the fraction is 45%. In one embodiment, the fraction is 50%. In one embodiment, the fraction is 55%. In one embodiment, the fraction is 60%. In one embodiment, the fraction is 65%. In one embodiment, the fraction is 70%. In one embodiment, the fraction is 75%. In one embodiment, the fraction is 80%. In one embodiment, the fraction is 85%. In one embodiment, the fraction is 90%. In one embodiment, the fraction is 91%. In one embodiment, the fraction is 92%. In one embodiment, the fraction is 93%. In one embodiment, the fraction is 94%. In one embodiment, the fraction is 95%. In one embodiment, the fraction is 96%. In one embodiment, the fraction is 97%. In one embodiment, the fraction is 98%. In one embodiment, the fraction is 99%. In one embodiment, the fraction is 100%.

[00193] In one embodiment, the fraction is less than 5%. In one embodiment, the fraction is less than 3%. In one embodiment, the fraction is less than 1%. In one embodiment, the fraction is less than 2%. In one embodiment, the fraction is less than 4%. In one embodiment, the fraction is less than 6%. In one embodiment, the fraction is less than 8%. In one embodiment, the fraction is less than 10%. In one embodiment, the fraction is less than 12%. In one embodiment, the fraction is less than 15%. In one embodiment, the fraction is less than 20%. In one embodiment, the fraction is less than 30%. In one embodiment, the fraction is less than 40%. In one embodiment, the fraction is less than 50%. In one embodiment, the fraction is less than 60%. In one embodiment, the fraction is less than 70%.

[00194] In one embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In one embodiment, the fraction of modified residues is 0.2%. In one embodiment, the fraction is 0.3%. In one embodiment, the fraction is 0.4%. Tn one embodiment, the fraction is 0.5%. In one embodiment, the fraction is 0.6%. In one embodiment, the fraction is 0.7%. In one embodiment, the fraction is 0.8%. In one embodiment, the fraction is 0.9%. In one embodiment, the fraction is 1%. In one embodiment, the fraction is 1.5%. In one embodiment, the fraction is 2%. In one embodiment, the fraction is 2.5%. In one embodiment, the fraction is 3%. In one embodiment, the fraction is 4%. In one embodiment, the fraction is 5%. In one embodiment, the fraction is 6%. In one embodiment, the fraction is 7%. In one embodiment, the fraction is 8%. In one embodiment, the fraction is 9%. In one embodiment, the fraction is 10%. In one embodiment, the fraction is 12%. In one embodiment, the fraction is 14%. In one embodiment, the fraction is 16%. In one embodiment, the fraction is 18%. In one embodiment, the fraction is 20%. In one embodiment, the fraction is 25%. In one embodiment, the fraction is 30%. In one embodiment, the fraction is 35%. In one embodiment, the fraction is 40%. In one embodiment, the fraction is 45%. In one embodiment, the fraction is 50%. In one embodiment, the fraction is 55%. In one embodiment, the fraction is 60%. In one embodiment, the fraction is 65%. In one embodiment, the fraction is 70%. In one embodiment, the fraction is 75%. In one embodiment, the fraction is 80%. In one embodiment, the fraction is 85%. In one embodiment, the fraction is 90%. In one embodiment, the fraction is 91%. In one embodiment, the fraction is 92%. In one embodiment, the fraction is 93%. In one embodiment, the fraction is 94%. In one embodiment, the fraction is 95%. In one embodiment, the fraction is 96%. In one embodiment, the fraction is 97%. In one embodiment, the fraction is 98%. In one embodiment, the fraction is 99%. In one embodiment, the fraction is 100%. In one embodiment, the fraction of the given nucleotide that is modified is less than 8%. In one embodiment, the fraction is less than 10%. In one embodiment, the fraction is less than 5%. In one embodiment, the fraction is less than 3%. In one embodiment, the fraction is less than 1%. In one embodiment, the fraction is less than 2%. In one embodiment, the fraction is less than 4%. In one embodiment, the fraction is less than 6%. In one embodiment, the fraction is less than 12%. In one embodiment, the fraction is less than 15%. In one embodiment, the fraction is less than 20%. In one embodiment, the fraction is less than 30%. In one embodiment, the fraction is less than 40%. In one embodiment, the fraction is less than 50%. In one embodiment, the fraction is less than 60%. In one embodiment, the fraction is less than 70%.

[00195] In one embodiment, a nucleoside-modified RNA of the present invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence. In one embodiment, the nucleoside-modified RNA exhibits enhanced ability to be translated by a target cell. In one embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In one embodiment, translation is enhanced by a 3 -fold factor. In one embodiment, translation is enhanced by a 4-fold factor. In one embodiment, translation is enhanced by a 5-fold factor. In one embodiment, translation is enhanced by a 6-fold factor. In one embodiment, translation is enhanced by a 7-fold factor. In one embodiment, translation is enhanced by an 8-fold factor. In one embodiment, translation is enhanced by a 9- fold factor. In one embodiment, translation is enhanced by a 10-fold factor. In one embodiment, translation is enhanced by a 15-fold factor. In one embodiment, translation is enhanced by a 20- fold factor. In one embodiment, translation is enhanced by a 50-fold factor. In one embodiment, translation is enhanced by a 100-fold factor. In one embodiment, translation is enhanced by a 200-fold factor. In one embodiment, translation is enhanced by a 500-fold factor. In one embodiment, translation is enhanced by a 1000-fold factor. In one embodiment, translation is enhanced by a 2000-fold factor. In one embodiment, the factor is 10-1000-fold. In one embodiment, the factor is 10-100-fold. In one embodiment, the factor is 10-200-fold. In one embodiment, the factor is 10-300-fold. In one embodiment, the factor is 10-500-fold. In one embodiment, the factor is 20-1000-fold. In one embodiment, the factor is 30-1000-fold. In one embodiment, the factor is 50-1000-fold. In one embodiment, the factor is 100-1000-fold. In one embodiment, the factor is 200-1000-fold. In one embodiment, translation is enhanced by any other significant amount or range of amounts.

Lipid Nanoparticle

[00196] In one embodiment, delivery of nucleoside-modified RNA comprises any suitable delivery method, including exemplary RNA transfection methods described elsewhere herein. In certain embodiments, delivery of a nucleoside-modified RNA to a subject comprises mixing the nucleoside-modified RNA with a transfection reagent prior to the step of contacting. In one embodiment, a method of present invention further comprises administering nucleoside-modified RNA together with the transfection reagent. In one embodiment, the transfection reagent is a cationic lipid reagent. In one embodiment, the transfection reagent is a cationic polymer reagent.

[00197] In one embodiment, the transfection reagent is a lipid-based transfection reagent. In one embodiment, the transfection reagent is a protein-based transfection reagent. In one embodiment, the transfection reagent is a carbohydrate-based transfection reagent. Tn one embodiment, the transfection reagent is a cationic lipid-based transfection reagent. In one embodiment, the transfection reagent is a cationic polymer-based transfection reagent. In one embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In one embodiment, the transfection reagent is calcium phosphate. In one embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In one embodiment, the transfection reagent is any other transfection reagent known in the art.

[00198] In one embodiment, the transfection reagent forms a liposome. Liposomes, in some embodiments, increase intracellular stability, increase uptake efficiency, and improve biological activity. In one embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids, which make up the cell membrane. They have, in some embodiments, an internal aqueous space for entrapping water-soluble compounds and range in size from 0.05 to several microns in diameter. In one embodiment, liposomes can deliver RNA to cells in a biologically active form.

[00199] In one embodiment, the composition comprises a lipid nanoparticle (LNP) and one or more nucleic acid molecules described herein. For example, in one embodiment, the composition comprises an LNP and one or more nucleoside-modified RNA molecules encoding one or more allergens, adjuvants, or a combination thereof.

[00200] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm), which includes one or more lipids, for example a lipid of Formula (I), (II) or (III). In some embodiments, LNPs are included in a formulation comprising a nucleoside-modified RNA as described herein. In some embodiments, such LNPs comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or (III)) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid such as a pegylated lipid of structure (IV), such as compound Iva). In some embodiments, the nucleoside-modified RNA is encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or all of the lipid portion of the LNP, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

[00201] In various embodiments, the LNPs have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, the nucleoside- modified RNA, when present in the LNPs, is resistant in aqueous solution to degradation with a nuclease.

[00202] The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[00203] In one embodiment, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[00204] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[00205] In some embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N- (l-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[00206] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012/016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2- dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 - morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-di oxolane (DLin-K-DMA).

[00207] Suitable amino lipids include those having the formula:

wherein R1 and R2 are either the same or different and independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted C10-C24 acyl;

[00208] R3 and R4 are either the same or different and independently optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl or R3 and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;

R5 is either absent or present and when present is hydrogen or C1-C6 alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and

Y and Z are either the same or different and independently O, S, or NH.

[00209] In one embodiment, R1 and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[00210] A representative useful dilinoleyl amino lipid has the formula: wherein n is 0, 1, 2, 3, or 4. [0021 1] In one embodiment, the cationic lipid is a DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMa (DLin-K-DMA above, wherein n is 2).

[00212] In one embodiment, the cationic lipid component of the LNPs has the structure of Formula (I): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L1 and L2 are each independently -O(C=O)-, -(C=O)O- or a carbon-carbon double bond;

R1a and R1b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R1a is H or C1-C12 alkyl, and R1b together with the carbon atom to which it is bound is taken together with an adjacent R1b and the carbon atom to which it is bound to form a carboncarbon double bond;

R2a and R2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R2a is H or C1-C12 alkyl, and R2b together with the carbon atom to which it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carboncarbon double bond;

R3a and R3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R3a is H or C1-C12 alkyl, and R3b together with the carbon atom to which it is bound is taken together with an adjacent R3b and the carbon atom to which it is bound to form a carboncarbon double bond;

R4a and R4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R4a is H or C1-C12 alkyl, and R4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carboncarbon double bond;

R5 and R6 are each independently methyl or cycloalkyl;

R7 is, at each occurrence, independently H or C1-C12 alkyl; R8 and R9 are each independently C1-C12 alkyl; or R8 and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2.

[00213] In certain embodiments of Formula (I), at least one of R1a, R2a, R3a or R4a is C1-C12 alkyl, or at least one of L1 or L2 is -O(C=O)- or -(C=O)O-. In other embodiments, R1a and R1b are not isopropyl when a is 6 or n-butyl when a is 8.

[00214] In still further embodiments of Formula (I), at least one of R1a, R2a, R3a or R4a is C1-C12 alkyl, or at least one of L1 or L2 is -O(C=O)- or -(C=O)O-; and

R1a and R1b are not isopropyl when a is 6 or n-butyl when a is 8.

[00215] In other embodiments of Formula (I), R8 and R9 are each independently unsubstituted C1-C12 alkyl; or R8 and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;

[00216] In certain embodiments of Formula (I), any one of L1 or L2 may be -O(C=O)- or a carbon-carbon double bond. L1 and L2 may each be -O(C=O)- or may each be a carboncarbon double bond.

[00217] In some embodiments of Formula (I), one of L1 or L2 is -O(C=O)-. In other embodiments, both L1 and L2 are -O(C=O)-.

[00218] In some embodiments of Formula (I), one of L1 or L2 is -(C=O)O-. In other embodiments, both L1 and L2 are -(C=O)O-

[00219] In some other embodiments of Formula (I), one of L1 or L2 is a carbon-carbon double bond. In other embodiments, both L1 and L2 are a carbon-carbon double bond.

[00220] In still other embodiments of Formula (I), one of L I or L2 is -O(C=O)- and the other of L1 or L2 is -(C=O)O-. In more embodiments, one of L1 or L2 is -O(C=O)- and the other of L1 or L2 is a carbon-carbon double bond. In yet more embodiments, one of L1 or L2 is -(C=O)O- and the other of L1 or L2 is a carbon-carbon double bond.

[00221] It is understood that “carbon-carbon” double bond, as used throughout the specification, refers to one of the following structures: wherein Ra and Rb are, at each occurrence, independently H or a substituent. For example, in some embodiments Ra and Rb are, at each occurrence, independently H, C1-C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.

[00222] In other embodiments, the lipid compounds of Formula (I) have the following structure (la):

[00223] In other embodiments, the lipid compounds of Formula (I) have the following structure (lb):

[00224] In yet other embodiments, the lipid compounds of Formula (I) have the following structure (Ic): [00225] In certain embodiments of the lipid compound of Formula (I), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[00226] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[00227] In some more embodiments of Formula (I), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[00228] In some certain other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[00229] In some other various embodiments of Formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same. [00230] The sum of a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.

[00231] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[00232] The substituents at R1a, R2a, R3a and R4a of Formula (I) are not particularly limited. In certain embodiments R1a, R2a, R3a and R4a are H at each occurrence. In certain other embodiments, at least one of R1a, R2a, R3a and R4a is C1-C12 alkyl. In certain other embodiments, at least one of R1a, R2a, R3a and R4a is C1-C8 alkyl. In certain other embodiments, at least one of R1a, R2a, R3a and R4a is C1-C6 alkyl. In some of the foregoing embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

[00233] In certain embodiments of Formula (I), R1a, R1b, R4a and R4b are C1-C12 alkyl at each occurrence.

[00234] In further embodiments of Formula (I), at least one of R1b, R2b, R3b and R4b is H or R1b, R2b, R3b and R4b are H at each occurrence.

[00235] In certain embodiments of Formula (I), R1b together with the carbon atom to which it is bound is taken together with an adjacent R1b and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[00236] The substituents at R5 and R6 of Formula (I) are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R5 or R6 is methyl. In certain other embodiments one or both of R5 or R6 is cycloalkyl for example cyclohexyl. In these embodiments, the cycloalkyl may be substituted or not substituted. In certain other embodiments, the cycloalkyl is substituted with C1-C12 alkyl, for example tert-butyl. [00237] The substituents at R7 are not particularly limited in the foregoing embodiments of Formula (I). In certain embodiments, at least one R7 is H. In some other embodiments, R7 is H at each occurrence. In certain other embodiments R7 is C1-C12 alkyl.

[00238] In certain other of the foregoing embodiments of Formula (I), one of R8 or R9 is methyl. In other embodiments, both R8 and R9 are methyl.

[00239] In some different embodiments of Formula (I), R8 and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8 and R9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.

[00240] In various different embodiments, the lipid of Formula (I) has one of the structures set forth in Table 1 below.

Table 1

Representative Lipids of Formula (I)

[00241] In some embodiments, the LNPs comprise a lipid of Formula (I), a nucleoside- modified RNA and one or more excipients selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (I) is compound 1-5. In some embodiments the lipid of Formula (I) is compound 1-6.

[00242] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (II):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L1 and L2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa,

-OC(=O)NRa-, -NRaC(=O)O-, or a direct bond;

G1 is C1-C2 alkylene, -(C=O)- , -O(C=O)-, -SC(=O)-, -NRaC(=O)- or a direct bond;

G2 is -C(=O)- , -(C=O)O-, -C(=O)S-, -C(=O)NRa or a direct bond;

G3 is C1-C6 alkylene;

Ra is H or C1-C12 alkyl;

R1a and R1b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R1a is H or C1-C12 alkyl, and R1b together with the carbon atom to which it is bound is taken together with an adjacent Rib and the carbon atom to which it is bound to form a carbon-carbon double bond;

R2a and R2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2a is H or C1-C12 alkyl, and R2b together with the carbon atom to which it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R3a and R3b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R3a is H or C1-C12 alkyl, and R3b together with the carbon atom to which it is bound is taken together with an adjacent R3b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R4a and R4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4a is H or C1-C12 alkyl, and R4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R5 and R6 are each independently H or methyl;

R7 is C4-C20 alkyl;

R8 and R9 are each independently C1-C12 alkyl; or R8 and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

[00243] In some embodiments of Formula (II), L¹ and L² are each independently -O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G1 and G2 are each independently -(C=O)- or a direct bond. In some different embodiments, L¹ and L² are each independently -O(C=O)-, -(C=O)O- or a direct bond; and G1 and G2 are each independently - (C=O)- or a direct bond.

[00244] In some different embodiments of Formula (II), L¹ and L² are each independently -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRa-, -NRaC(=O)-,

-C(=O)NR^a-, -NR^aC(=O)NR^a, -OC(=O)NR^a-, -NR^aC(=O)O-, -NR^aS(O)_xNR^a-, -NR^aS(O)_x- or -S(O)_xNR^a-.

[00245] In other of the foregoing embodiments of Formula (II), the lipid compound has one of the following structures (IIA) or (IIB):

[00246] In some embodiments of Formula (II), the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).

[00247] In any of the foregoing embodiments of Formula (II), one of L¹ or L² is -O(C=O)-. For example, in some embodiments each of L¹ and L² are -O(C=O)-. [00248] In some different embodiments of Formula (II), one of L¹ or L² is -(C=O)O- For example, in some embodiments each of L¹ and L² is -(C=O)O-.

[00249] In different embodiments of Formula (II), one of L¹ or L² is a direct bond. As used herein, a “direct bond” means the group (e.g., L¹ or L²) is absent. For example, in some embodiments each of L¹ and L² is a direct bond.

[00250] In other different embodiments of Formula (II), for at least one occurrence of R^1a and R^1b, R^1a is H or C1-C12 alkyl, and R^1b together with the carbon atom to which it is bound is taken together with an adjacent Rlb and the carbon atom to which it is bound to form a carboncarbon double bond.

[00251] In still other different embodiments of Formula (II), for at least one occurrence of R^4a and R^4b, R^4a is H or C₁-C₁₂ alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[00252] In more embodiments of Formula (II), for at least one occurrence of R^2a and R^2b, R^2a is H or C₁-C₁₂ alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[00253] In other different embodiments of Formula (II), for at least one occurrence of R^3a and R^3b, R^3a is H or C₁-C₁₂ alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carboncarbon double bond.

[00254] In various other embodiments of Formula (II), the lipid compound has one of the following structures (IIC) or (IID):

wherein e, f, g and h are each independently an integer from 1 to 12.

[00255] In some embodiments of Formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).

[00256] In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[00257] In certain embodiments of Formula (II), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[00258] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[00259] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[00260] In some certain embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[00261] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.

[00262] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

[00263] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

[00264] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12. [00265] In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.

[00266] The sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[00267] The substituents at R^1a, R^2a, R^3a and R^4a of Formula (II) are not particularly limited. In some embodiments, at least one of R^1a, R^2a, R^3a and R^4a is H. In certain embodiments R^1a, R^2a, R^3a and R^4a are H at each occurrence. In certain other embodiments, at least one of R^1a, R^2a, R^3a and R^4a is C₁-C₁₂ alkyl. In certain other embodiments, at least one of R^1a, R^2a, R^3a and R^4a is C₁-C₈ alkyl. In certain other embodiments, at least one of R^1a, R^2a, R^3a and R^4a is C₁-C₆ alkyl. In some of the foregoing embodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[00268] In certain embodiments of Formula (II), R^1a, R^2a, R^3a and R^4a are C₁-C₁₂ alkyl at each occurrence.

[00269] In further embodiments of Formula (II), at least one of R^1a, R^2a, R^3a and R^4a is H or R^1a, R^2a, R^3a and R^4a are H at each occurrence.

[00270] In certain embodiments of Formula (II), R^1b together with the carbon atom to which it is bound is taken together with an adjacent ^{R1b a}nd the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[00271] The substituents at R³ and R⁶ of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments one of R³ or R⁶ is methyl. In other embodiments, each of R⁵ or R⁶ is methyl. [00272] The substituents at R⁷ of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments R⁷ is C₆-Ci6 alkyl. In some other embodiments, R7 is C₆- C₉ alkyl. In some of these embodiments, R⁷ is substituted with -(C=O)OR^b, -O(C=O)R^b, -C(=O)R^b, -OR^b, -S(O)xR^b, -S-SR^b, -C(=O)SR^b,

[00273] -SC(=O)R^b, -NR^aR^b, -NR^aC(=O)R^b, -C(=O)NR^aR^b, -NRaC(=O)NR^aR^b,

[00274] -OC(=O)NR^aR^b, -NR^aC(=O)OR^b, -NR^aS(O)_xNR^aR^b, -NR^aS(O)_xR^b or -S(O)_xNR^aR^b, wherein: R^a is H or C1-C12 alkyl; R^b is C1-C15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R⁷ is substituted with -(C=O)OR^b or -O(C=O)R^b.

[00275] In various of the foregoing embodiments of Formula (II), R^b is branched C₁-C₁₅ alkyl. For example, in some embodiments R^b has one of the following structures:

[00276] In certain other of the foregoing embodiments of Formula (II), one of R⁸ or R⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

[00277] In some different embodiments of Formula (II), R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.

[00278] In still other embodiments of the foregoing lipids of Formula (II), G³ is C₂-C₄ alkylene, for example C₃ alkylene.

[00279] In various different embodiments, the lipid compound has one of the structures set forth in Table 2 below. Table 2

Representative Lipids of Formula (II)

[00280] In some embodiments, the LNPs comprise a lipid of Formula (II), a nucleoside- modified RNA and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (II) is compound II-9. In some embodiments, the lipid of Formula (II) is compound II- 10. In some embodiments, the lipid of Formula (II) is compound II- 11. In some embodiments, the lipid of Formula (II) is compound 11-12. In some embodiments, the lipid of Formula (II) is compound 11-32.

[00281] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (III): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_x-, -S-S-, -C(=O)S-, SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or

-NR^aC(=O)O-, and the other of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, - S(O)_x-,

-S-S-, -C(=O)S-, SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, ,NR^aC(=O)NR^a-, - OC(=O)NR^a- or -NR^aC(=O)O- or a direct bond;

G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene;

G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene;

R^a is H or C₁-C₁₂ alkyl;

R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;

R³ is H, OR⁵, CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NR⁵C(=O)R⁴;

R⁴ is C₁-C₁₂ alkyl;

R³ is H or C₁-C₆ alkyl; and x is 0, 1 or 2.

[00282] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB): wherein:

A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl; n is an integer ranging from 1 to 15.

[00283] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[00284] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID): wherein y and z are each independently integers ranging from 1 to 12.

[00285] In any of the foregoing embodiments of Formula (III), one of L¹ or L² is -O(C=O)-. For example, in some embodiments each of L¹ and L² are -O(C=O)-. In some different embodiments of any of the foregoing, L¹ and L² are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L¹ and L² is -(C=O)O-.

[00286] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

[00287] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ): [00288] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[00289] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[00290] In some of the foregoing embodiments of Formula (III), R⁶ is H. In other of the foregoing embodiments, R⁶ is C₁-C₂₄ alkyl. In other embodiments, R⁶ is OH.

[00291] In some embodiments of Formula (III), G³ is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G³ is linear C₁-C₂₄ alkylene or linear C1-C24 alkenylene.

[00292] In some other foregoing embodiments of Formula (III), R¹ or R², or both, is C₆- C₂₄ alkenyl. For example, in some embodiments, R¹ and R² each, independently have the following structure: wherein:

R^7a and R^7b are, at each occurrence, independently H or C₁-C₁₂ alkyl; and a is an integer from 2 to 12, wherein R^7a, R^7b and a are each selected such that R¹ and R² each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[00293] In some of the foregoing embodiments of Formula (III), at least one occurrence of R^7a is H. For example, in some embodiments, R^7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R^7b is C₁-C₈ alkyl. For example, in some embodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

[00294] In different embodiments of Formula (III), R¹ or R², or both, has one of the following structures:

[00295] In some of the foregoing embodiments of Formula (III), R³ is OH, CN,

-C(=O)OR⁴, -OC(=O)R⁴ or -NHC(=O)R⁴. In some embodiments, R⁴ is methyl or ethyl.

[00296] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in Table 3 below.

Table 3

Representative Compounds of Formula (III)

[00297] In some embodiments, the LNPs comprise a lipid of Formula (III), a nucleoside- modified RNA and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound III-7.

[00298] In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only cationic lipids.

[00299] In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.

[00300] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[00301] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[00302] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1- stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC).

[00303] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to the neutral lipid ranges from about 2: 1 to about 8: 1.

[00304] In various embodiments, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[00305] In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to cholesterol ranges from about 2: 1 to 1 : 1.

[00306] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[00307] In certain embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GM1). In certain embodiments, the LNP comprises a sterol, such as cholesterol.

[00308] In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monom ethoxy-poly ethyl eneglycol)-2, 3 -dimyristoylglycerol (PEG-s- DMG) and the like.

[00309] In certain embodiments, the LNP comprises an additional, stabilizing -lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol-lipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- O-(2’,3’-di(tetradecanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100:1 to about 25: 1.

[00310] In some embodiments, the LNPs comprise a pegylated lipid having the following structure (IV): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has mean value ranging from 30 to 60.

[00311] In some of the foregoing embodiments of the pegylated lipid (IV), R¹⁰ and R¹¹ are not both n-octadecyl when z is 42. In some other embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R¹⁰ is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R¹¹ is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[00312] In various embodiments, z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g/mol. In some embodiments, the average z is about 45.

[00313] In other embodiments, the pegylated lipid has one of the following structures: wherein n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g/mol.

[00314] In certain embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.

[00315] In some embodiments, the LNPs comprise a lipid of Formula (I), a nucleoside- modified RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments the lipid of Formula (I)is compound 1-6. In different embodiments, the neutral lipid is DSPC. In other embodiments, the steroid is cholesterol. In still different embodiments, the pegylated lipid is compound IVa.

[00316] In certain embodiments, the LNP comprises one or more targeting moi eties, which are capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand, which directs the LNP to a receptor found on a cell surface. [00317] In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains, which bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.

[00318] Other exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

[00319] The following Reaction Schemes illustrate methods to make lipids of Formula (I), (II) or (III).

GENERAL REACTION SCHEME 1

[00320] Embodiments of the lipid of Formula (I) (e.g., compound A-5) can be prepared according to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, compounds of structure A-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of A-1, A-2 and DMAP is treated with DCC to give the bromide A-3. A mixture of the bromide A-3, a base (e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A- 5 after any necessarily workup and or purification step.

GENERAL REACTION SCHEME 2

[00321] Other embodiments of the compound of Formula (I) (e.g., compound B-5) can be prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C₁-C₂₄ alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of B-1 (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e g., pyridinum chlorochromate) and intermediate product B-3 is recovered. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and/or purification.

[00322] It should be noted that although starting materials A-1 and B-1 are depicted above as including only saturated methylene carbons, starting materials which include carbon-carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds. GENERAL REACTION SCHEME 3

[00323] Different embodiments of the lipid of Formula (I) (e.g., compound C-7 or C9) can be prepared according to General Reaction Scheme 3 (“Method C”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.

GENERAL REACTION SCHEME 4

[00324] Embodiments of the compound of Formula (II) (e.g., compounds D-5 and D-7) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein R^1a, R^1b, R^2a, R^2b, R^3a, R^3b, R^4a, R^4b, R⁵, R⁶, R⁸, R⁹, L¹, L², G¹, G², G³, a, b, c and d are as defined herein, and R⁷ represents R⁷ or a C₃-C19 alkyl. Referring to General Reaction Scheme 1, compounds of structure D-1 and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of D-1 and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up. A solution of D-3 and a base (e.g. tri methyl amine, DMAP) is treated with acyl chloride D-4 (or carboxylic acid and DCC) to obtain D-5 after any necessary work up and/or purification. D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary work up and/or purification. GENERAL REACTION SCHEME 5

[00325] Embodiments of the lipid of Formula (II) (e.g., compound E-5) can be prepared according to General Reaction Scheme 5 (“Method E”), wherein R^1a, R^1b, R^2a, R^2b, R^3a, R^3b, R^4a, R^4b, R⁵, R⁶, R⁷, R⁸, R⁹, L¹, L², G³, a, b, c and d are as defined herein. Referring to General Reaction Scheme 2, compounds of structure E-1 and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of E-l (in excess), E-2 and a base (e.g., potassium carbonate) is heated to obtain E-3 after any necessary work up. A solution of E-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride E-4 (or carboxylic acid and DCC) to obtain E-5 after any necessary work up and/or purification.

GENERAL REACTION SCHEME 6 [00326] General Reaction Scheme 6 provides an exemplary method (Method F) for preparation of Lipids of Formula (III). G¹, G³, R¹ and R³ in General Reaction Scheme 6 are as defined herein for Formula (III), and G¹ refers to a one-carbon shorter homologue of G¹. Compounds of structure F-1 are purchased or prepared according to methods known in the art. Reaction of F-1 with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester/alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).

[00327] It should be noted that various alternative strategies for preparation of lipids of Formula (III) are available to those of ordinary skill in the art. For example, other lipids of Formula (III) wherein L¹ and L² are other than ester can be prepared according to analogous methods using the appropriate starting material. Further, General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G¹ and G² are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G¹ and G² are different.

[00328] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t- butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin. Pharmaceutical Compositions

[00329] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[00330] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated 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.

[00331] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[00332] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient, which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[00333] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

[00334] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

[00335] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[00336] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[00337] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen free water) prior to parenteral administration of the reconstituted composition. [00338] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1,3 butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[00339] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers. In certain embodiments, the formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In certain embodiments, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In certain embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In certain embodiments, dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[00340] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally, the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (in certain instances having a particle size of the same order as particles comprising the active ingredient).

[00341] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.

[00342] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1,3 butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di-glycerides. Other parentally administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. [00343] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

Methods of Prevention and Treatment

[00344] The present invention provides methods of preventing or reducing an inflammatory, autoimmune, or allergic response in a subject comprising administering an effective amount of a composition comprising at least one nucleic acid molecule encoding at least one antigen. In some embodiments, the composition comprises an LNP comprising at least one nucleoside-modified RNA molecule encoding at least one antigen. In some embodiments, the antigen is an autoantigen or an allergen. In some embodiments, the method comprises administering the composition to a subject before exposure to an antigen (e.g. preventively). In some embodiments, the method comprises administering the composition to a subject during exposure to an antigen. In some embodiments, the method comprises administering the composition to a subject after exposure to an antigen (e.g. as a treatment or therapy). In some embodiments, the method comprises administering the composition to a subject before the development of an allergic, autoimmune, or inflammatory disease or disorder. In some embodiments, the method comprises administering the composition to a subject after the development of an allergic, autoimmune, or inflammatory disease or disorder. In some embodiments, the composition further comprises one or more anti-inflammatory agents. In some embodiments, the anti-inflammatory agent is an mTOR inhibitor.

[00345] In some embodiments, the invention provides methods of promoting Treg levels and/or function. In some embodiments, the invention provides methods of promoting tolerance- induced signals in T cells, and facilitating the differentiation of CD4⁺ T cells toward Treg cells. In some embodiments, the invention provides methods of increasing allergen specific IgG production. In some embodiments, the invention provides methods of reducing pro-allergic IgE. In some embodiments, the invention provides methods for reducing Th2 cell responses against an allergen by inducing allergen tolerance. In some embodiments, the invention provides methods for increasing anti-allergic Th1 and/or CD8⁺ T cell responses. In some embodiments, the invention provides methods for reducing eosinophil count in the lung. In one embodiment, the invention provides methods of reducing pro-allergic cytokine levels.

[00346] In one embodiment, the method prevents or reduces allergic response or one or more symptoms of allergic response. Examples of symptoms of allergic response include, but are not limited to, abdominal pain, allergic rhinitis, anaphylaxis, colonic inflammation, diarrhea, eczema, hives, itching, nausea, and vomiting. In one embodiment, the method reduces mucus production. In one embodiment, the method reduces airway hyperresponsiveness.

[00347] In one embodiment, the method treats or prevents the development of an allergic disease. Examples of allergic diseases include, but are not limited to, rhinitis, atopy, asthma, COPD, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria, anaphylactic shock, eosinophilic gastrointestinal diseases including eosinophilic esophagitis, food-protein induced allergic proctocolitis, and hay fever.

[00348] In some embodiments, the invention is a method of administering to a subject a composition comprising at least one nucleoside-modified RNA encoding at least one antigen. In one embodiment, the composition is administered to a subject having an inflammatory or autoimmune disease or disorder. In one embodiment, the composition is administered to a subject at risk for developing an inflammatory or autoimmune disease or disorder.

[00349] In some embodiments, the invention is a method of administering to a subject a composition comprising at least one nucleoside-modified RNA encoding at least one allergen. In one embodiment, the composition is administered to a subject having an allergic disease or allergic disorder. In one embodiment, the composition is administered to a subject at risk for developing an allergic response, allergic disease, or allergic disorder.

[00350] In certain embodiments, the method of the invention allows for sustained expression of the antigen, described herein, for at least several days following administration. In certain embodiments, the method of the invention allows for sustained expression of the antigen or adjuvant, described herein, for at least 2 weeks following administration. In certain embodiments, the method of the invention allows for sustained expression of the antigen or adjuvant, described herein, for at least 1 month following administration. However, the method, in certain embodiments, also provides for transient expression, as in certain embodiments, the nucleic acid is not integrated into the subject genome.

[00351] In certain embodiments, the method provides sustained protection against an antigen and/or disease. For example, in certain embodiments, the method provides sustained protection against an antigen and/or disease for more than 2 weeks. In certain embodiments, the method provides sustained protection against an antigen and/or disease for 1 month or more. In certain embodiments, the method provides sustained protection against an antigen and/or disease for 2 months or more. In certain embodiments, the method provides sustained protection against an antigen and/or disease for 3 months or more. In certain embodiments, the method provides sustained protection against an antigen and/or disease for 4 months or more. In certain embodiments, the method provides sustained protection against an antigen and/or disease for 5 months or more. In certain embodiments, the method provides sustained protection against an antigen and/or disease for 6 months or more. In certain embodiments, the method provides sustained protection against an antigen and/or disease for 1 year or more.

[00352] In some embodiments, a single immunization of the composition induces sustained protection against an antigen and/or disease for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, or 1 year or more.

[00353] In some embodiments, the method comprises administering the composition of the invention to an infant or child and protects against adolescent and/or adult development of an inflammatory, autoimmune, or allergic response or disease.

[00354] Administration of the compositions of the invention in a method of treatment or prevention can be achieved in a number of different ways, using methods known in the art. In one embodiment, the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration. In one embodiment, the method comprises intradermal delivery of the composition. In one embodiment, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the method comprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition.

[00355] The therapeutic treatment and preventive methods of the invention thus encompass the use of pharmaceutical compositions comprising an LNP encapsulating a nucleic acid encoding at least one antigen.

[00356] It will be appreciated that the composition of the invention may be administered to a subject in conjunction with another agent. In some embodiment, the composition may be administered in conjunction with at least one anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is an mTOR inhibitor. In some embodiments, the composition and additional agent are administered by the same method of delivery. For example, in some embodiments, the composition and additional agent are both delivered intramuscularly. In some embodiments, the composition and additional agent are administered by distinct methods of delivery.

[00357] In some embodiments, the composition comprising an LNP encapsulating a nucleic acid encoding at least one antigen further comprises at least one anti-inflammatory agent. In some embodiments, the composition further comprises at least one mTOR inhibitor. In some embodiments, the mTOR inhibitor is everolimus (e.g. Afinitor, Afinitor Disperz, Zortress), rapamycin, sirolimus (e.g. Rapamune, Hyftor, Fyarro), temsirolimus (Torisel), ridaforolimus, Torin-1, or non-rapalog derived inhibitors or an analog or derivative thereof. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from 0.1 ng/kg/day and 100 mg/kg/day. In one embodiment, the invention envisions administration of a dose, which results in a concentration of the compound of the present invention from 1 nM and 10 pM in a mammal.

[00358] Typically, dosages which may be administered in a method of the invention to a mammal, such as a human, range in amount from 0.01 μg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. In certain embodiments, the dosage of the composition will vary from about 0.1 μg to about 10 mg per kilogram of body weight of the mammal. In certain embodiments, the dosage will vary from about 1 μg to about 1 mg per kilogram of body weight of the mammal. [00359] The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the potency and duration of protection to the antigen and/or disease, the type and severity of the disease being treated, the type and age of the mammal, etc.

[00360] In some embodiments, administration of an immunomodulatory composition of the present invention may be performed by single administration or boosted by multiple administrations.

[00361] In some embodiments, the present invention provides a method of administering a composition comprising at least one nucleic acid molecule encoding at least one antigen to a subject having or at risk of developing an inflammatory response or disease, an autoimmune response or disease, or an allergic response or disease.

EXPERIMENTAL EXAMPLES

[00362] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[00363] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present disclosure, and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLE 1: mRNA-Lipid Nanoparticle Vaccine Protects Against Allergy

[00364] mRNA vaccines have been successful in inducing protective immunity against SARS-CoV-2 and can a variety of diseases. The ability of vaccination with an allergen-encoding mRNA lipid nanoparticle (LNP) to protect against allergic responses was tested. Allergen- specific mRNA-LNP immunization restrained allergic airway inflammation by shifting T-helper cell responses from type 2 to type 1. Additionally, mirroring the response to SARS CoV-2 mRNA vaccination in humans, allergen mRNA-LNP vaccine prompted the expansion of cytotoxic CD8+CD38+KLRG- cells, underscoring a conserved response across species, regardless of the mRNA-encoded protein. To mitigate non-allergic lung inflammation following allergen exposure, the allergen mRNA-LNP vaccine was co-administrated with an antiinflammatory mTOR inhibitor. This combined treatment ameliorated the allergic and inflammatory responses by diminishing Th1, Th2, and cytotoxic cells while promoting long- lasting regulatory T cells. Alongside, allergen mRNA-LNP vaccination and its combination with an mTOR inhibitor demonstrated efficacy in blocking key features of experimental asthma, including mucus production and airway hypersensitivity, thereby emerging as a preventive strategy for allergy treatment.

Antigen-Specific response to OVA mRNA-LNP'.

[00365] To confirm the efficiency and specificity of the nucleoside-modified OVA- mRNA vaccine to induce T cell responses, mice were injected with 3*10⁶ purified naive OTII cells expressing CD45.1 marker and treated intramuscularly with m1Ψ-mRNA-LNPs encoding OVA protein (OVA-mRNA), empty LNP or PBS twice on days 0 and 7. A response of antigenspecific CD4⁺ T cells in lymph nodes was analyzed 4 days later (Figure 1A). The administration of OVA-mRNA vaccine induced activation (CD44^hlgh) and robust expansion of donor OTII cells in the local lymph nodes (LNs), indicating an efficient and antigen-specific T cell response to the vaccine (Figures IB and 1C). There was an increase in the proportion of donor OTII cells producing IFNg and expressing T-bet, which are key characteristics of Th1 cell populations, and an elevation in percentages of Bcl6⁺PD1⁺ cells, corresponding to Tfh cells (Figure 1C). A similar increase was detected in the proportion of Th1 and Tfh cells within the host CD4⁺ T cell population (Figure 5A) that was in line with previous reports (Pardi et al., 2018, J Exp Med., 215: 1571-1588; Bettini and Locci, 2021, Vaccines (Basel) 9). A large percent of OTII cells also produced TNFa and IL-2 (Figure 1C), but not IL-17A and IL-4, or FOXP3 (Figure 5B) in response to OVA-mRNA. Activation of host or OTII T cells was not observed following treatment with PBS or empty LNP. [00366] To determine the optimal concentration of the vaccine for inducing T cell activation, mice receiving donor OTII cells were intramuscularly treated with a single injection of increasing amounts of the OVA-mRNA vaccine. On day 7, the local LNs were analyzed to assess the activation and expansion of donor cells. The highest response of OTII cells to the OVA-mRNA vaccine was observed within the range of 2 to 5 μg of OVA-mRNA. At these concentrations, the donor cells exhibited robust expansion, with over 80% showing elevated expression of the activation marker CD44 (Figure ID). Moreover, there was a direct correlation between the injected dose of the OVA-mRNA and the frequencies of effector OTII cells producing TNFα, IFNγ, or exhibiting Bcl6+PD1+ phenotype (Figure IE). Concurrently with the T cell response, a dose-dependent increase in the levels of OVA-specific IgG1, IgG2a, and IgG2b was observed in the serum of the mice 18 days after treatment (Figure 5C). Taken together, these findings demonstrate the specificity and high potency of the OVA-mRNA vaccine in inducing activation and differentiation of Th1 and Tfh cells. mRNA immunization reduces allergic responses:

[00367] To test whether the ability of OVA-mRNA vaccine to induce a Th1 response could be utilized for protection against allergic responses, mice were immunized with different doses of OVA-mRNA, sensitized with OVA+Alum, and subsequently challenged with OVA protein via intratracheal (i.t.) and intranasal (i.n.) routes (Figure 2A). The vaccine efficacy in preventing allergic airway responses was assessed in bronchoalveolar lavage fluid (BALF) and lungs. Notably, mice receiving a higher dose of the OVA-mRNA vaccine exhibited a significant reduction in the eosinophil count in BALF (Figure 2B) and a decreased percentage of GAT A3 ⁺ and IL-5⁺IL-13⁺ CD4⁺ T cells in the lung upon OVA challenge (Figure 2C), indicating a dosedependent effect of the OVA-mRNA vaccine against Th2 responses in the airways. Concurrently, the number of CD8⁺ T cells and neutrophils in BALF, as well as the frequency of IFNg⁺ CD4⁺ T cells in the lungs, increased sequentially with rising amounts of the OVA-mRNA vaccine (Figures 2B and 2C). The number of CD4⁺ T cells in BALF and the percentage of FOXP3⁺ Treg cells in the lungs did not show significant changes.

[00368] To further improve the anti-allergic outcomes, a boost of OVA-mRNA was administered a week after the first immunization, followed by OVA+ALUM sensitization and airway challenge with OVA protein (Figure 6A). Mice immunized with two doses of the OVA- mRNA vaccine exhibited a dramatic decrease in eosinophils in the BALF as shown by flow cytometric analyses (Figure 2D and Figure 6B). This observation was further substantiated through histological staining of lung tissue using hematoxylin and eosin (H&E) and anti-major basic protein (anti-MBP) staining, revealing eosinophilic infiltration in pulmonary vessels, alveolar ducts, and peribronchial regions in the allergen-challenged LNP group (Figure 2E). Conversely, the OVA-mRNA single dose group, and even more notably, the vaccine boosted group, exhibited a pronounced reduction in eosinophil presence throughout the BALF (Figure 2D) and lung tissue (Figure 2E). This reduction corresponded with the decrease in Th2 cell responses observed in the lung. Although the proportion of CD4⁺ T cells remained constant between mice treated with either one or two doses of the mRNA vaccine, those boosted exhibited a more notable decline in the CD4⁺GATA3⁺ cell frequency in the lungs upon OVA challenge (Figure 2F). They also showed reduced percentage of IL-4, IL-5, and IL- 13 producing CD4⁺ T cells (Figure 6C) compared to single-dose vaccinated mice.

[00369] Notably, allergen-challenged mice vaccinated twice exhibited an increased proportion of CD4⁺ T cells producing IFNg, reflecting intensified Th1 responses (Figure 2F), and a decreased frequency of Treg cells compared to OVA-mRNA single-dose or LNP-treated mice (Figure 6D). Additionally, there was an enrichment in CD8⁺ T cells and neutrophils in the BALF (Figure 2D and Figure 6B). The increased count of CD8⁺ T cells was accompanied by an elevated frequency of CCR5^high and Perforin⁺ CD8⁺ T cells in the lungs (Figure 2G), underscoring enhanced migratory ability of CD8⁺ T cells and their heightened cytotoxic potential. H&E histological examination corroborated the presence of accumulated lymphoid cells within the lung tissue of allergen-challenged OVA-mRNA immunized and boosted mice (Figure 2E).

[00370] Taken together, these results demonstrate that immunization with the modified allergen-specific mRNA-LNP vaccine reduces Th2 effectors but induces heightened Th l and CD8 responses after allergen challenge.

Combination of mRNA and ml'OR inhibitor regulates T cell activation'.

[00371] Several studies have suggested that mTOR inhibitors, such as rapamycin and its derivative everolimus (EVL), impact DC maturation, inhibit T cell activation and proliferation, promote tolerance-induced signals in T cells, and facilitate the differentiation of CD4⁺ T cell toward Treg cells when coupled with a potent antigenic signal (Thomson et al., 2009, Nat Rev Immunol, 9:324-337; Daniel et al., 2010, Proc Natl Acad Sci USA, 107:16246-16251; Pinheiro et al., 2020, J Immunol, 205:2577-2582; Liu et al., 2021, ACS Nano, 15: 1608-1626; Chi, 2012, Nat Rev Immunol, 12:325-338). Thus, concurrent administration of the OVA-mRNA vaccine with mTOR inhibition was evaluated to determine if concurrent administration modulates T cell activation and differentiation, supporting a tolerogenic rather than an inflammatory response; mitigating inflammation induced by the mRNA-LNP vaccine while maintaining anti-allergic responses.

[00372] A kinetic analysis was conducted of the activation and expansion of adoptively transferred OTII cells, harvested from the local lymph nodes of the host following OVA-mRNA immunization with and without EVL (Figure 3A). While donor OTII cells exhibited accelerated expansion with the OVA-mRNA vaccine alone (Figure 3B), cells activated in the presence of OVA-mRNA + EVL displayed a temporal delay in activation, as indicated by the CD44 marker, and a weaker growth. By day 7, the majority of cells in both groups demonstrated sustained CD44 level, indicating the development of a memory phenotype (Figure 3B). Notably, the frequency of OTII cells activated in the presence of EVL retained higher compared to cells activated with the OVA-mRNA alone at later time points, suggesting their increased survival rate.

[00373] Effector functions of T cells in local lymph nodes on day 7 post the first OVA- mRNA immunization was assessed. EVL in combination with OVA-mRNA significantly decreased IFNg and TNFa production by OTII (Figures 3C and 3D) and host CD4⁺ and CD8⁺ T cells (Figure 7A) compared to OVA-mRNA treatment, indicating its anti-inflammatory effect. Furthermore, OVA-mRNA+EVL increased the frequency of CD25⁺ high cells and induced Treg cell differentiation, as evidenced by an elevated proportion of FOXP3⁺CD25⁺ cells (Figures 3C and 3E). EVL alone had no impact on donor OTII or host T cells, highlighting the specificity of the effect of EVL when combined with the OVA-mRNA vaccine (Figures 3C-3E and Figure 7A). EVL extended the persistence of Treg and FOXP3-CD25^high cells over time (Figure 3F), while maintaining low frequencies of IFNg⁺ cells, coupled with restored capacity to produce TNFa at later time points (Figure 7B).

[00374] To examine the suppression capability of Treg cells generated in the presence of the OVA-mRNA vaccine and EVL, an ex vivo Treg functional assay was conducted. CD25⁺ and CD25- CD4⁺ T cells from OVA-mRNA+ EVL vaccinated OTII (CD45.1⁺) mice were isolated and cultured for 3 days with CFSE-labeled naive OTII cells (CD45.1⁺ CD45.2-) in the presence of irradiated splenocytes (CD45.2⁺) loaded with OVA peptide 323-339. CD4⁺ CD25⁺ (Treg) cells effectively inhibited the proliferation of naive CFSE-labeled OTII cells in a dose-dependent manner (Figures 3G and 3H), while purified CD4⁺ CD25- (non-Treg) cells, used as an internal control, did not suppress the proliferation of CFSE-labeled responder cells (Figures 3G and 3H). These results further confirmed the intrinsic suppressive function of Treg cells induced by the combination of OVA-mRNA and EVL. Collectively, these results demonstrate that OVA-mRNA immunization in the presence of an mTOR inhibitor reduces Th1 and CD8 responses and promotes the generation of Treg cells with suppressive activity and extended survival.

Anti-allergic response of vaccine-modulated T cells:

[00375] To evaluate the impact of OVA-mRNA immunization combined with antiinflammatory treatment on airway allergy development, mice received two doses of OVA- mRNA or control LNP, with or without EVL. Subsequently, they were sensitized with OVA+Alum and then challenged with OVA (Figure 4A). An analysis of pro-allergic and inflammatory cytokines/chemokines from the BALF fluid was conducted. Th2 cytokines, such as IL-4, IL-5, and IL-13, and chemokine Eotaxin- 2 (CCL24) were reduced by both OVA-mRNA and OVA-mRNA+EVL immunizations, while inflammatory derivatives like MIP-la/b, MIG, IP- 10, RANTES, TNFa, and IFNg were elevated in OVA-mRNA immunized group but reduced in OVA-mRNA+EVL treated animals (Figures 4B, 4C, Figure 8A, and Table 4). These results were substantiated by q-PCR analysis of the lung tissue, highlighting anti-inflammatory and antiallergic effects of OVA-mRNA+EVL immunizations (Figure 8B).

[00376] To further mechanistically dissect the response of OVA-mRNA modified T cells in allergic airway inflammation, single-cell RNA-sequencing (scRNA-seq) analysis was conducted on lung samples. The samples were obtained from naive mice and those pre-treated with LNP, OVA-mRNA, or OVA-mRNA+EVL, followed by OVA+Alum sensitization and OVA airway challenge. Lung cells were collected, barcoded, and subjected to BD Rapsody gravity -based single cell RNA sequencing. Clustering analysis identified 15 distinct subpopulations of cells with canonical markers for each subset (Figure 9A and 9B) (Han et al., 2018, Cell, 172; 1091-1107; Grieshaber-Bouyer et al., 2021, Nat commun, 12:2856; Bain et al., 2022, Mucosal Immunol, 15:223-234; Fei et al., 2022, Nat Genet, 54: 1051-1061 ; Wang et al., 2023, Nucleic Acids Res, 51:501-516). Applying this method, differences in the elevated frequency of ab-T cells were observed, with declines in monocyte and B cell proportions in OVA-mRNA and OVA-mRNA+EVL treated groups compared to naive and LNP -treated animals (Figure 9C). mRNA+EVL immunization normalized the frequency of neutrophils, which had increased in the OVA-mRNA group, further underscoring the anti-inflammatory effect of OVA-mRNA+EVL pre-treatment.

[00377] Focusing on αβ-T cells, 5 populations of cells were identified, including naive and activated CD4, CD8, and T reg cells (Figure 8D). The identity of each cell subset was confirmed using canonical T cell markers (Figure 8E). Distinctly separating T cell subsets based on the various conditions, we observed a reduction in the frequencies of naive T cells and an elevation in activated CD4⁺ T cells across different conditions when compared to naive mice (Figure 8F). The scRNA-seq analysis demonstrated an enrichment of activated CD8⁺ T cells in the allergen-challenged OVA-mRNA treatment and Treg cells within the allergen-challenged OVA-mRNA+EVL condition (Figure 8F, which was further supported by the flow cytometry results of Figure 4G).

[00378] Differential analysis of transcriptomics of activated CD8 cells revealed their heightened activation status in the OVA-mRNA condition, illustrated by an elevated expression of effector and cytotoxic genes and genes related to actin reorganization (Figure 10A). Additionally, activated CD8⁺ T cells from the allergen-challenged OVA-mRNA group exhibited reduced Klrgl expression while expressing high level of Cd38 that resembled a population of CD38⁺KLRG1- CD8⁺ T cells identified in humans vaccinated with the SARS-CoV-2 mRNA vaccine and enriched in virus-protective antigen-specific cytotoxic T cells (Zhang et al., 2023, Nat Immunol, 24: 1725-1734). Using flow cytometry analysis, expansion of CD38⁺KLRG1- CD8⁺ T cells in allergen-challenge OVA-mRNA immunized mice compared to other experimental groups was demonstrated (Figure 4H). Notably, 33 genes elevated in human CD38⁺KLRG1 CD8⁺ T cell from SARS-CoV-2 mRNA vaccinated individuals were highly expressed in activated CD8 cells from allergen-challenge OVA-mRNA mice (Figures 10A (with asterisks), and 10B and Table 5 Like human cells, murine CD38⁺KLRGL CD8⁺ T cells exhibited elevated levels of perforin production, underscoring their cytotoxic activity, which was reduced in allergen-challenged OVA-mRNA+EVL mice (Figure 4H). This finding suggests that mRNA vaccination induces a partially conserved CD8 cellular responses across two species (humans and mice), regardless of the mRNA-encoded protein.

[00379] To substantiate CD4 cell differences during allergen challenge, transcriptional analysis of activated FoxP3^negative CD4 T cells was performed. scRNA-seq revealed that LNP- treated (asthmatic) mice exhibited a Th2 phenotype, allergen-challenged OVA-mRNA- immunized mice displayed a Th1 phenotype, and allergen-challenged OVA-mRNA+EVL mice exhibited characteristics reminiscent of Treg cells with reduced proportion of both Th1 and Th2 cells (Figuer 10C). Flow cytometry analysis corroborated these findings, showing a reduction of IL-5⁺IL-13⁺ and GATA3⁺ CD4⁺ T cells in both OVA-mRNA immunized mice with or without EVL, while IFNγ producing CD4⁺ T cells were decreased significantly in OVA-mRNA+EVL conditions (Figure 41). Collectively, the results indicate that the allergen-specific mRNA vaccine counteracts allergic responses by eliciting robust Th1 and cytotoxic CD8⁺ T cell responses following allergen challenge. Concurrently, mRNA immunization in the presence of mTOR inhibition reduces Th1, Th2, and cytotoxic reactions and favors generation of long-lasting Treg cells.

Table 4 :

Table 5:

Clinical effects of allergen mRNA vaccination:

[00380] To substantiate the anti-allergic effects of OVA-mRNA and OVA-mRNA+EVL immunizations, clinical symptoms in allergen-challenged mice were evaluated. Both OVA- mRNA and OVA-mRNA+EVL immunized mice maintained normal airway resistance measured through challenge with inhaled methacholine compared to asthmatic LNP and LNP+EVL mice (Figure 4J). Furthermore, these mice showed significant reduction in mucus production in the lungs, as analyzed by periodic acid-Schiff (PAS) staining (Figure 4K). These results indicate that the allergen-specific mRNA vaccine, both in the presence and absence of mTOR inhibition, provides protection against allergic responses.

[00381] Allergen-specific immunotherapy (AIT) is a recognized strategy for treating allergic disorders. However, with the increasing prevalence of allergic diseases, prophylaxis for individuals predisposed to allergies is gaining importance. Various anti-allergic vaccine approaches, primarily utilizing recombinant and synthetic proteins, aim to induce a Th2 counteractive response by promoting Th1 cell generation and elevating allergen-specific IgG, or inducing allergen-tolerant and T regulatory cells. While the concept of developing allergen prophylactic vaccines is promising, limited formulations have advanced to pre-clinical studies and clinical trial consideration (Tulaeva et al., 2020, Front Immunol, 11 : 1368). Herein, the effectiveness of an allergen-specific mRNA-LNP vaccine (OVA-mRNA) in preventing type 2 immunity by directing the differentiation of CD4+ and CD8+ T cells towards inflammatory Th1 and cytotoxic cells is demonstrated, mirroring effects seen in mRNA vaccines encoding viral antigens (Chaudhary et al., 2021, Nat Rev Drug Discov, 20:817-838; Pardi et al., 2018, J Exp Med, 215: 1571-1588; Laczko et al., 2020, Immunity, 53:724-732). Moreover, upon allergen challenge, the majority of OVA-mRNA-modified CD8+ T cells exhibited a CD38+KLRG1- phenotype, resembling the protective CD8+ T cells identified in SARS-CoV-2 vaccinated individuals (Zhang et al., 2023, Nat Immunol, 24:1725-1734), suggesting shared features in CD8 responses against viral infections and allergen exposure across two species (humans and mice).

[00382] Given the heightened inflammatory responses of OVA-mRNA-modulated T cells after allergen challenge, a protective strategy was implemented leveraging the capacity mTOR inhibition to limit T cell activation and induce CD4+ T cell differentiation toward Treg cells (Thomson et al., 2009, Nat Rev Immunol, 9:324-337; Daniel et al., 2010, Proc Natl Acad Sci USA, 107: 16246-16251; Pinheiro et al., 2020, J Immunol, 205:2577-2582; Liu et al., 2021, ACS Nano, 15: 1608-1626; Chi, 2012, Nat Rev Immunol, 12:325-338). Indeed, OVA-mRNA immunization in the presence of an mTOR inhibitor reduced inflammatory T cell generation, promoted suppressive Treg cell development, and extended the survival of antigen-specific memory T cells. Importantly, an increased proportion of Treg cells and a decreased frequencies of Th1, Th2, and cytotoxic CD8+ T cells were further observed in the lungs of OVA- mRNA+EVL mice upon allergen exposure. These results indicate that while both OVA-mRNA and OVA-mRNA+mTOR inhibitor treatments offer protection against the development of experimental allergic airway inflammation, noted by reduction in mucus production and improved lung resistance to methacholine, they operated through distinct cellular mechanisms altering the phenotype and proportions of antigen-specific T cells. These data reveal that the robust allergy-counteractive Th1 response induced by the mRNA-LNP vaccine can be modulated to elicit sufficient anti-inflammatory and anti-allergic effects when coupled with an immunomodulator like an mTOR inhibitor. Further studies will clarify the complex interaction of these factors and their integration into the therapeutic approaches. These strategies can be applied to address not only airway inflammation but also other allergic disease involving adaptive immune responses, such as food allergy, atopic dermatitis, rhinitis, and more. Moreover, this strategy will be beneficial for autoimmune diseases where Treg cells have a protective role.

The materials and methods are now described.

Mice:

[00383] C57BL/6 mice (B6J, stock no. 000664) were purchased from The Jackson

Laboratory. OTII mice (stock no. 004194) were bred with CD45.1 mice (stock no. 002014). Both female and male mice aged 6 to 10 weeks were included in this study. Mice were bred and housed under specific pathogen-free conditions. All experiments complied with protocols approved by the Cincinnati Children’s Hospital Medical Center (CCHMC) Animal Use and Care Committee. mRNA design and production:

[00384] The amino acid sequence of the ovalbumin (OVA) was obtained from GenBank accession number MF321513.1. The sequence underwent codon optimization and GC enrichment using our proprietary algorithm to improve expression and reduce potential immunogenicity of the in vitro transcribed mRNA. The codon-optimized OVA sequence was gene synthesized by Genscript and cloned into our proprietary in vitro transcription template containing an optimized T7 promoter, 3'UTR, 5'UTR, and a 100-adenine tail. The OVA nucleoside modified mRNA was prepared using the MegaScript transcription kit (ThermoFisher Scientific), co-transcriptionally capped using the CleanCap™ system (TriLink Biotechnologies), and purified using a modified cellulose base chromatography method (Baiersdorfer et al., 2019, Mol Ther Nucleic Acids, 15:26-35) precipitated, eluted in nuclease-free water, and quantified using a microvolume spectrophotometer NanoDrop One system. Length and integrity were determined using agarose gel electrophoresis. Endotoxin content was measured using the GenScript Toxisensor chromogenic assay, and values were below detection levels (0.1 EU/mL). mRNA was frozen at -20°C until formulation.

LNP production and characterization.

[00385] Cellulose-purified N1 -methylpseudouridine (m1Ψ)-containing RNAs were encapsulated in LNPs using a self-assembly process as previously described wherein an ethanolic lipid mixture of ionizable cationic lipid, phosphatidylcholine, cholesterol, and polyethylene glycol-lipid was rapidly mixed with an aqueous solution containing mRNA at acidic pH (Maier et al., 2013, Mol Ther, 21 : 1570-1578). The LNP formulation used in this study is proprietary to Acuitas Therapeutics; the proprietary lipid and LNP composition are described in US patent US 10,221,127. The hydrodynamic size, poly dispersity index (PDI), and zeta potential of mRNA-LNPs were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The mRNA encapsulation efficiency was determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen). Particles had a size of 80nm, a poly dispersity index of 0.02, and an encapsulation efficiency above 95%.

Adoptive transfer:

[00386] Cells were collected from lymph nodes and spleens of OTII mice expressing CD45.1. Cells were forced through 70mm cell strainer and treated with ACK lysing buffer to remove red blood cells. Naive CD4⁺ T cells were isolated using the EasySep™ Mouse Naive CD4+ T Cell Isolation Kit. A total 3*10⁶ cells per mouse were intravenously injected in 200 μl PBS into sex-matched C57BL/6 recipients, which were immunized 3 days after cell injection. Lymph nodes were collected at the specified time points.

OVA-mRNA, LNP immunization and everolimus treatment:

[00387] Wild type (wt) C57BL/6 mice were injected i.m. into the lower left leg with increasing doses (0.1, 0.3, 1, 2, and 5 μg) or a consistent amount (2 μg) of the m1Ψ-mRNA- LNPs encoding an ovalbumin protein or empty LNP on days 0 and 7, as indicated.

Intraperitoneal treatment with everolimus (5 mg/kg of body weight) commenced two days before the first immunization and continued daily for 14 days. LNP, OVA-mRNA, and everolimus were injected with 28 1/2-gauge insulin syringes.

Sensitization and allergen challenge protocol for allergic asthma.

[00388] LNP or OVA-mRNA immunized mice were sensitized through intraperitoneal injection with 100 μg ovalbumin (OVA, Sigma) emulsified in 100μl of Imject ALUM (Thermo Scientific) in a total volume of 200 ml per mouse on days 24 and 36 after first immunization, or as indicated in specific experiments. Subsequently, on days 48 and 49, mice were intratracheally challenged, followed by intranasal challenge on days 50 and 51, with 50 μg of OVA. Forty-eight hours after the final OVA challenge, airway hyperresponsiveness (AHR) was assessed, or mice were euthanized with pentobarbital and BALF and lungs were collected for further assessments. Naive mice served as control.

BALF and Lung preparation .

[00389] For bronchoalveolar lavage fluid (BALF) collection, lungs were lavaged once with 0.8 ml of PBS through cannulation of the tracheal tube. Total cell numbers in the collected fluid were counted using a hemocytometer. BALFs were centrifuged, and supernatants were stored at -80°C for ELISA or cytokine/chemokine multiplex assays. Cells were resuspended in PBS with 2% FBS and subjected for live flow cytometry staining on the same day.

[00390] One lobe of the lungs was minced and subjected to Liberase TL digestion (0.25 mg/ml) in the presence of DNasel (0.5 mg/ml) at 37°C for 1 hour. The resulting suspension was forced through 70-micron cell strainers, washed, and resuspended in RPMI 10% FBS with DNasel (0.5 mg/ml). These cells were used for live or intracellular staining or subjected to scRNA-seq.

Lung histopathology:

[00391] Two days after the final OVA challenge, mice were euthanized with pentobarbital for histological examination. Lungs were inflated through the tracheal tube with 0.7 ml of 10% neutralized buffered formalin, removed, fixed overnight in 10% formalin, and then dehydrated in 70% ethanol. Lung tissues were embedded in paraffin and cut into 5-pm thick sections, which were subsequently deparaffinized. The sections were stained with hematoxylin and eosin (H&E), acid-Schiff (PAS), or subjected to immunohistochemical stain against murine eosinophilic major basic protein (MBP) as reported (Zuo et al., 2010, J Immunol, 185:660-669).

[00392] Mucus-containing goblet cells were detected by PAS staining. PAS-stained goblet cells in the airway epithelium were quantified using a scoring system (0: ,5% goblet cells; 1 : 5 to 25%; 2: 25 to 50%; 3: 50 to 75%; 4: .75%), as described (Townsend et al., 2000, Immunity, 13:573-583). Twenty to fifty airways per mouse were examined, and the average score was calculated. Histologic analyses were performed in a blinded manner by the same person.

Airway hyperresponsiveness measurements:

[00393] The FlexiVent system (SCIREQ Scientific Respirator Equipment, Inc, Montreal, Quebec, Canada) was utilized to evaluate airway hyperresponsiveness 48 hours after final OVA exposure. Mice were anesthetized with a mixture of ketamine (90-120 mg/kg), xylzine (10-20 mg/kg) and paralyzed with pancuronium bromide (0.8-1.2 mg/kg). Tracheas were cannulated with an 18-gauge blunt cannula. Mice were ventilated at 150 breaths/min, 3.0cm water positive and expiratory pressure, and allowed to stabilize on the machine for 2 minutes. Mice were then exposed to methacholine (0, 6.25, 12.5, 25, and 50 mg/ml) aerosolized in PBS for 15 seconds and ventilated for an additional 10 seconds. Ventilation cycle measurements were taken until resistance peaked. Airways were then re-recruited by deep inflation and the next methacholine dose was administered.

Flow cytometry staining:

[00394] Freshly obtained cells from BALF and lungs of naive or allergen-challenged mice were stained for surface markers, including anti -mouse CD45, CD11c, CD64, CD1 lb, CD3, CD4, CD8, Grl or Ly6G, and SiglecF at 4°C for 60 minutes.

[00395] For surface and intracellular staining of T cells, cells from the lungs or LNs were incubated in PBS at 4°C for 10 minutes with the fixable viability dye eFluor 780. After washing with PBS containing 2% FBS, cells were fixed and permeabilized using Foxp3/Transcri ption Factor Fixation/Permeabilization kit according to the manufacturer’s instructions. Subsequently, cells were stained with anti-mouse fluorescent antibody cocktails. For lungs, anti-mouse CD44, CD4, CD8, GATA3, FoxP3, Helios, KLRG1, CD38, and CCR5 antibodies were used. For LNs, anti-mouse CD44, CD4, CD45.1, CD45.2, GATA3, FoxP3, CD25, T bet, Bcl6, and PD1 antibodies were utilized. The staining process occurred at room temperature for 60 minutes.

[00396] For intracellular cytokine detection, cells from lungs or LNs were stimulated with phorbol 12, 13 -dibutyrate (PDBU) (500 ng/ml) and 1 pM ionomycin in the presence of Brefeldin A at 37 °C incubator for 4 hours. Cells were stained in PBS for 10 minutes with the fixable viability dye eFluor 780 in the presence of Brefeldin A and then were fixed and permeabilized with the FoxP3 fixation kit. Lung T cells were stained with fluorescent antibodies against mouse CD4, CD8, CD44, GATA3, FoxP3, IL-4, IL-5, IL-13, IFNg, and Perforin.

[00397] All staining was performed in the presence of Fc Block (anti-mouse CD16/CD32). Data acquired with a BD LSR Fortessa flow cytometer (BD Biosciences) were analyzed by FlowJo software (Tree Star Inc.). The absolute number of cells in each population in the BALF was calculated by multiplying the total cell numbers in the collected BALF by the percentage of flow cytometry gated cells.

Treg immunosuppression assay:

[00398] Naive OTII cells expressing CD45.1 were isolated from spleens and LNs using the EasySep™ Mouse Naive CD4⁺ T Cell Isolation Kit and labeled with 5 mM of CFSE (CarboxyFluoroscein Succinimidyl Ester) for 8 minutes. Treg and non-Treg CD4⁺ T cells were purified from local lymph nodes of OTII mice expressing CD45.1 and CD45.2 markers using the EasySep™ Mouse CD4⁺CD25⁺ Regulatory T Cell Isolation Kit II. These mice were immunized i.m. with 4 mg of OVA-mRNA vaccine 7 days prior to the assay, along with daily everolimus treatment. Irradiated (300 RAD) splenocytes from wild type C57BL/6 mice expressing CD45.2 were loaded with 2mg/ml of OVA peptide (323-329) and used as antigen-presenting cells for CFSE-labeled responders and Treg suppressors. Non-Treg CD4 T cells from the same immunized mice served as control cells. Different ratios of suppressor and responder T cells (1: 1, 2:1, 4:1, 8: 1) were employed, and proliferation of CD45.1⁺CD45.2- CFSE-labeled cells was analyzed by flow cytometry on day 3 of in vitro co-culture. From the distribution of the proportion of cells in each CFSE peak, the cell yield of dividing cells was calculated. Quantitative PCR:

[00399] Mouse lungs were collected and homogenized in Trizol with Quagen beads. mRNA was isolated following Trizol protocol and purified using Quick-RNA Miniprep Kit (Zymo Research). For real-time polymerase chain reaction (PCR) analysis, mRNA was reverse- transcribed with the ProtoScript cDNA synthesis kit (New England BioLabs). Primers specific for mouse 114, 115, 1113, Ifng, Cxcl9, Cxcl10, Ccl5, Cel11, Ccl24, and Eif3k were obtained from Integrated DNATechnol ogies . SYBR Green Real-Time PCR was performed and analyzed using the ABI Quant-Studio 7 Flex Real-Time PCR system (Thermo Fisher Scientific). The transcripts of interest were normalized to Eif3k cDNA.

Multiplex and ELISA CCL24 and Abs:

[00400] BALF was collected 2 days after last challenged and analyzed with the Mouse Cytokine/ 32 -Plex Discovery Assay (Eve Technologies Corp., Canada). The Mouse CCL24/Eotaxin-2/MPIF-2 DuoSet enzyme linked immunosorbent assay (ELISA) kit was used to measure the concentration of Eotaxin-2 in the BALF.

[00401] To measure OVA-specific antibody secretion, blood was collected from the tail 2 weeks after the last dose of OVA-mRNA immunization and 10 days after the last OVA+ALUM sensitization. Serum was separated by centrifugation at 5000 rpm for 5 minutes at room temperature. 96-well plates were coated with 10mg/ml of ovalbumin overnight at 4°C. Serum samples were added in different dilutions. Detection of OVA-specific antibodies from serum was performed using HRP-conjugated anti-IgG1, anti-IgG2a, or biotinylated anti-IgG2b followed by HRP accordingly to manufacture’s instructions. 3, 3', 5, 5' tetramethylbenzidine (TMB) was used as a substrate. The colorimetric reaction was stopped with 10% H₃PO₄, and the optical density was quantified using an ELISA plate reader at 450 nm, with subtraction of background absorbance at 570 nm. OVA-specific monoclonal antibodies IgG1, IgG2a, and IgG2b were used as standards. To prevent non-specific binding, SuperBlock™ Blocking Buffer (ThermoFisher) was used for blocking and dilutions.

Single cell RNA-seq collection:

[00402] Single cell transcriptome analysis of the murine esophagus was performed using the BD Rhapsody Single-Cell Analysis System (BD, Bioscience). To obtain a single cell suspension, the left lobe of the lungs was minced and incubated with Liberase TL (0.25 mg/ml) in the presence of DNasel (0.5 mg/ml) for enzymatic digestion. Cells from two mice per group were pooled, treated with ACK lysing buffer for 1 minute, washed, passed through a 70-mm cell strainer, and counted using the TC20 Automated Cell Counter (BioRad). Subsequently, cells were processed on the Rhapsody platform following the manufacturer's protocol. Cells from each treatment group were labeled with individual sample tags (BD® Mouse Single-Cell Multiplexing Kit (Cat. No. 633793)). Before loading into the cartridges, cells were quantified on the Rhapsody scanner following staining with Vybrant® DyeCycle™ Green (Invitrogen, V35004) for 5 min at room temperature. Sixty thousand cells, barcoded samples were pooled in equal amounts from four treatment groups (Naive, LNP, mRNA, mRNA+EVL), were loaded into each cartridge, with two cartridges used for sequencing. Single cells were isolated with the BD Rhapsody Express Single-Cell Analysis System according to the manufacturer’s recommendations, after the final bead wash step, forty thousand cells were detected as singlets.

[00403] Libraries were prepared following the BD Rhapsody System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol (BD Biosciences; 633801) and sequenced at CCHMC DNA sequencing and genotyping core on NovaSeq 6000 using PE (paired-end) 100 sequencing format. Following sequencing and pre-processing by the Rhapsody WTA analysis pipeline using exact cell count, 28,000 cells per cartridge were used for the downstream analysis.

Single-cell RNA sequencing data analysis:

[00404] Raw single-cell sequencing data from four experimental conditions (Naive, LNP, mRNA, mRNA+Ev.) were analyzed with vl.12.1 BD Rhapsody WTA Analysis Pipeline (www. scomix.bd.com/hc/en-us/articles/360047408451-BD-Rhapsody -Analysis-Pipeline-Updates) on Seven Bridges Platform (sevenbridges.com/platform). Sequencing reads in FASTQ files were aligned to GRCm38.p6 reference genome (gencodegenes.org/mouse/release_M19.html). Cell calling was performed with the disabled “Refined Putative Cell Calling” parameter. Instead, the “Exact Cell Count” input was set to 28,000 cells. Generated feature-barcode matrices from two batches were merged, preserving the experimental condition and the batch information of each cell within its barcode. Merged feature-barcode matrix was then uploaded to SciDAP (scidap.com) for all subsequent data analysis steps. [00405] Low-quality cells were removed with Single-cell RNA-Seq Filtering Analysis pipeline (github.com/datirium/workflows/blob/master/workflows/sc-rna-filter.cwl). The workflow was run with the following QC (quality control) thresholds: minimum 500 transcripts per cell, at least 500 but not more than 5,000 genes per cell, maximum 5 percent of transcripts mapped to mitochondrial genes. Doublets were identified and removed by scDblFinder R package (fl000research.com/articles/10-979/v2) as part of the above-mentioned pipeline.

[00406] The remaining high-quality cells were processed by Single-cell RNA-Seq Dimensionality Reduction Analysis pipeline (github.com/datirium/workflows/blob/master/workflows/sc-rna-reduce.cwl). During the analysis molecular count data were first corrected for technical variability, then integrated by using the pairs of cells sharing a matched biological state as integration anchors. Heterogeneity associated with the cell cycle stage was completely removed. Other configuration parameters included: 1) setting normalization method to “sctglm” which resulted in using glmGamPoi R package within the SCTransform function (genomebiology. biomedcentral.com/articles/10.1186/sl3059-021- 02584-9 ; 2) limiting the number of the most variable genes for both normalization and integration procedures to 3,000; 3) selecting the first 40 PCs (principal components) for PCA (principal component analysis) and UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction algorithms.

[00407] Then, dimensionally reduced single-cell data were clustered with Single-cell RNA-Seq Cluster Analysis pipeline (github.com/datirium/workflows/blob/master/workflows/sc- rna-cluster.cwl). The workflow was run with 40 PCs and 0.5 clustering resolution. For each of the 24 obtained clusters the gene markers were identified by calling FindAllMarkers function (satijalab.org/seurat/reference/fmdallmarkers_with the default parameters. Cluster with damaged cells (low transcripts per cell counts and not specific gene markers) was removed, and both Single-cell RNA-Seq Dimensionality Reduction Analysis and Single-cell RNA-Seq Cluster Analysis pipelines were rerun with 20 PCs and increased to 1.0 clustering resolution. The resulted clusters produced cell types which were used in the following steps of the analyses.

[00408] Cells belonging to the T cell cluster, were separated and re-clustered by applying Single-cell RNA-Seq Dimensionality Reduction Analysis and Single-cell RNA-Seq Cluster Analysis pipelines. Dimensionality was reduced to 15 PCs and the number of highly variable genes was limited to 1000. The clustering resolution was set to 0.5. [00409] Differentially expressed genes between experimental conditions were identified with Single-cell RNA-Seq Differential Expression Analysis pipeline (www. github.com/datirium/workflows/blob/master/workflows/sc-rna-de-pseudobulk.cwl) run on the selected subsets of cells using Wilcoxon Rank Sum test.

Statistical analyses'.

[00410] The results are presented as means ±SEM or displayed with box and whiskers plots. Normality tests were applied to validate Gaussian distribution. Multiple-group comparisons were conducted using one-way analysis of variance (ANOVA), with FDR correction applied. All statistical analyses, except scRNA-seq analyses, were performed with Prism 9 software (GraphPad Software Inc.). P < 0.05 was considered statistically significant.

EXAMPLE 2: OVA mRNA-LNP reduces allergic symptoms.

[00411] OVA mRNA-LNP increases antigen-specific anti-allergic IgG and reduces pro- allergic IgE (Figure 11). OVA mRNA-LNP reduces diarrhea and intestine permeability (Figure 12).

EXAMPLE 3 : Ara h2 mRNA-LNP induces peanut specific IgG production.

[00412] Ara h2 mRNA-LNP induces peanut specific IgG production (Figure 13). Mice were vaccinated by Ara h2 -mRNA-LNP or LNP (5 ug) on days 0 and 7. Blood was collected on days 0 and 22.

EXAMPLE 4: Derpl and Derp2 mRNA-LNP induces house dust mite (HDM) specific IgG production.

[00413] Figure 14 depicts the house dust mite (HDM) model and results of experiments demonstrating that the allergen-specific mRNA-LNP vaccine induces protection against multiprotein allergens. Derpl and Derp2 immunization increases allergen specific IgGl and IgG2a antibodies for Derpl and Derp2, respectively. Further, Derp1 + Derp2 mRNA-LNP immunization increases IFNg production (Th1) and decreases frequencies of GATA3⁺(Th2), IL- 5/IL-13⁺(Th2), and IL-17A⁺(Th17) cells after HDM challenge (Figure 15). [00414] Figure 16 depicts the results of experiments demonstrating that Derpl mRNA- LNP vaccination protects against asthma induced by the HDM allergen, Derpl. Figure 16A depicts the experimental workflow. Figure 16B depicts Derpl mRNA-LNP vaccine reduces eosinophilia and mucus production in the lungs upon Derpl allergen challenge. Figure 16C depicts Derpl mRNA-LNP vaccinee reduces frequency of pro-allergic Th2 and Th 17 cells and increases anti-allergic Th1 cells in the lungs upon Derpl allergen challenge.

[00415]

EXAMPLE 5: Preventive and immunotherapy models of allergy treatment.

[00416] Figure 17 depicts research models for preventive care and immunotherapy with an allergen-specific vaccine.

[00417] Preventive pre-treatment with allergen-specific mRNA-LNP vaccine protects against chronic allergy and asthma and reduces eosinophilia in the lungs (Figure 18). The allergen-specific mRNA vaccine administered in the presence or absence of an mTOR inhibitor prevents development of chronic allergy, reduces Th2 responses, and increases frequency of Th1 and CD8⁺ T cells in the lung (Figure 18). Pre-treatment with allergen-specific mRNA-LNP vaccine produces pro-allergic Th2 cells (CD4⁺GATA3⁺) and increases anti-allergic Th1 (CD4⁺IFNg⁺) and cytotoxic (CD8⁺Perforin⁺) T cells in the lungs after induction of chronic disease (Figure 19).

[00418] The preventative care model pre-treatment with OVA-mRNA reduces eosinophil count in the lung (Figure 20), reduces pro-allergic cytokine levels (Figure 21), reduces mucus production (Figure 22), reduces airway hyperresponsiveness (Figure 23), provides protection against chronic inflammation (Figure 24), alters distribution of T cell populations in allergic asthma (Figure 25), and alters CD8 T cell phenotype in allergic asthma (Figure 26).

[00419] Figure 27 depicts the results of example experiments demonstrating allergenspecific IgG production for OVA, Ara h2, Derp1, and Derp2 mRNA-LNP vaccines compared with an mRNA-LNP control.

[00420] In summary, experiments demonstrated that the preventive care model mRNA- LNP immunization switches CD4 T cell phenotype to protect against allergy. Allergen specific mRNA-LNP vaccine protects against acute and chronic allergic airway responses by reducing frequencies of Th2 cells, eosinophilia, mucus production, airway hypersensitivity, and increases anti-allergic Th1 and CD8⁺ T cell responses (Figure 28).

[00421] Experiments testing whether allergen-specific mRNA vaccines can be used as immunotherapy against allergic responses by induction of IgG antibodies, reduction of allergic inflammation, suppression of Th2 cell activity, and induction of Th1 and tolerant T cells were performed. The immunotherapy model comprises the steps of Ag-sensitization, Ag-specific mRNA vaccination, and Ag-challenge (Figure 29). The results depicted in Figure 14 demonstrate that mRNA vaccine immunotherapy increases allergen specific IgGl and IgG2, but not pro- allergic IgE. Allergy immunotherapy with the mRNA-LNP vaccine also reduces eosinophilia in bronchial lavage fluid (BALF) and lung tissue, decreases mucus production in the lung and lowers airway hypersensitivity (Figure 30). Further, allergy immunotherapy with mRNA-LNP reduces pro-allergic Th2 cell response (GATA3⁺ and IL-5⁺/IL-13⁺ cells) and increases frequencies of anti-allergic Th1 (IFNg ) and tolerant cells (Figure 31).

[00422] Figure 32 depicts the proportions of T cell populations in the lung in response to allergy immunotherapy with the mRNA-LNP vaccine. Further, allergy immunotherapy with the mRNA-LNP vaccine increases frequency of activated CD8⁺ T cells in the lung and particularly boosts CD38⁺KLRG1 CD8⁺ T cell population (Figure 33).

[00423] Immunotherapy model treatment with OVA-mRNA reduces allergic responses (Figure 34). The immunotherapy model treatment reduces mucus production and airway hypersensitivity (Figure 35). Finally, immunotherapy treatment elevates allergen-specific IgGl but not IgE antibody production (Figure 36). The allergen-specific mRNA treatment effectively reduces the major symptoms of allergic asthma, lowers levels of pro-allergic cytokines, and increases the production of anti -allergic IgG antibodies, demonstrating a viable immunotherapy for allergies.

[00424] Figure 37 depicts the results of experiments demonstrating that allergen immunotherapy with an allergen-specific mRNA-LNP vaccine is safe and does not induce anaphylactic response compared to a subcutaneously (s.c.) injected allergen. Mice were sensitized with OVA protein through ear skin and on day 30 injected with an OVA mRNA-LNP vaccine (intramuscular, i.m.) or a high dose of endotoxin free OVA protein (subcutaneous s.c.) as a standard immunotherapy. Mice without sensitization were used as a control for OVA s.c. injection. EXAMPLE 6: Immunization with a non-specific antigen increases the immunotherapeutic effect of the allergen-specific mRNA-LNP vaccine.

[00425] Figure 38 the results of experiments demonstrating immunization with a nonspecific mRNA-LNP vaccine increases the immunotherapeutic effect of allergen-specific mRNA-LNP vaccine. Figure 38A depicts the experimental workflow. Figure 39B depicts coadministration of allergen-specific (Derpl+Derp2 mRNA-LNP) and non-specific (OVA mRNA- LNP) vaccines reduces eosinophilia in the lungs. Figure 38C depicts co-administration of allergen-specific (Derp1+Derp2 mRNA-LNP) and non-specific (OVA mRNA-LNP) vaccines reduces the frequency of pro-allergic Th2 cells and increases the percentage of anti-allergic Th1 cells.

EXAMPLE 7: Sequences SEQ ID NO: 1 (amino acid sequence of OVA):

MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVR

FDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQ

CVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIV

FKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASG

TMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSV LMAMGITDVF S S S ANL SGIS S AE SLKISQ A VHAAHAEINEAGREVVGS AE AGVD AAS VSE EFRADHPFLFCIKHIATNAVLFFGRCVSP

SEQ ID NO:2 (amino acid sequence of Ara h 2):

MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDED

SYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCM CEALQQIMENQ SDRLQGRQQEQQFKRELRNLPQQC GLRAPQRCDLE VE SGGRDRY

SEQ ID NO: 3 (amino acid sequence of Derpl):

MKIVLAIASLLALSAVYARPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSN

GGAINHLSDLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSINGNAPAEIDLRQMRT

VTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRG

IEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIA

VIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWG

DNGYGYFAANIDLMMIEEYPYVVIL

SEQ ID NO:4 (amino acid sequence of Derp2):

MMYKILCLSLLVAAVARDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPFQLEAVFE

ANQNTKTAKIEIKASIDGLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIAPKS ENVVVTVKVMGDDGVLACAIATHAKIRD

SEQ ID NO:5 (Derp 2 3)

MKFNIIIVFISLAILVHSSYAANDNDDDPTTTVHPTTTEQPDDKFECPSRFGYFADPKDPH

KF YIC SNWEAVHKDCPGNTRWNEDEETCT

SEQ ID NO: 6 (amino acid sequence of Ara h l):

MRGRVSPLMLLLGILVLASVSATHAKSSPYQKKTENPCAQRCLQSCQQEPDDLKQKAC

ESRCTKLEYDPRCVYDPRGHTGTTNQRSPPGERTRGRQPGDYDDDRRQPRREEGGRWG

PAGPREREREEDWRQPREDWRRPSHQQPRKIRPEGREGEQEWGTPGSHVREETSRNNPF

YFPSRRFSTRYGNQNGRIRVLQRFDQRSRQFQNLQNHRIVQIEAKPNTLVLPKHADADNI

LVIQQGQATVTVANGNNRKSFNLDEGHALRIPSGFISYILNRHDNQNLRVAKISMPVNTP

GQFEDFFPASSRDQSSYLQGFSRNTLEAAFNAEFNEIRRVLLEENAGGEQEERGQRRWST

RSSENNEGVIVKVSKEHVEELTKHAKSVSKKGSEEEGDITNPINLREGEPDLSNNFGKLF EVKPDKKNPQLQDLDMMLTCVEIKEGALMLPHFNSKAMVIVVVNKGTGNLELVAVRK

EQQQRGRREEEEDEDEEEEGSNREVRRYTARLKEGDVFIMPAAHPVAINASSELHLLGF

GINAENNHRIFLAGDKDNVIDQIEKQAKDLAFPGSGEQVEKLIKNQKESHFVSARPQSQS

QSPSSPEKESPEKEDQEEENQGGKGPLLSILKAFN

SEQ ID N0:7 (amino acid sequence of Ara h 2 wild type):

RQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDPYSPSQDPD

RRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQ

EQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDRY

SEQ ID NO: 8 (amino acid sequence of Ara h 2 hypoallergenic):

RCQSQLERANLRPCEQHLMQKIQRDEDSAGSSQHQERCCNELNEFENNQRCMCEALQQ IMENQSDRLQGRQQEQQFKRELRNLPQQCGLRAPQRCD

SEQ ID NO: 9 (amino acid sequence of Ara h 3):

RQQPEENACQFQRLNAQRPDNRIESEGGYIETWNPNNQEFECAGVALSRLVLRRNALRR PFYSNAPQEIFIQQGRGYFGLIFPGCPRHYEEPHTQGRRSQSQRPPRRLQGEDQSQQQRDS HQKVHRFDEGDLIAVPTGVAFWLYNDHDTDVVAVSLTDTNNNDNQLDQFPRRFNLAG NTEQEFLRYQQQ SRQ SRRRSLP YSPYSPQ SQPRQEEREF SPRGQHSRRERAGQEEENEGG NIFSGFTPEFLEQAFQVDDRQIVQNLRGETESEEEGAIVTVRGGLRILSPDRKRRADEEEE YDEDEYEYDEEDRRRGRGSRGRGNGIEETICTASAKKNIGRNRSPDIYNPQAGSLKTAN DLNLLILRWLGPSAEYGNLYRNALFVAHYNTNAHSIIYRLRGRAHVQVVDSNGNRVYD EELQEGHVLVVPQNF AVAGK SQ SENFEYVAFKTD SRP SIANLAGENS VIDNLPEEVVAN SYGLQREQARQLKNNNPFKFFVPPSQQSPRAVA

SEQ ID NO: 10 (amino acid sequence of Ara h 6):

AHASAMRRERGRQGDSSSCERQVDGVNLKPCEQHIMQRIMGEQEQYDSYNFGSTRSSD QQQRCCDELNEMENTQRCMCEALQQIMENQCDGLQDRQMVQHFKRELMNLPQQCNF GAPQRCDLDVSGGRC

SEQ ID NO: 11 (amino acid sequence of Ara h 7):

MVKLSILVALLGALLVVASATRWDPDRGSRGSRWDAPSRGDDQCQRQLQRANLRPCE EHIRQRVEKEQEQEQDEYPYIQRGSRGQRPGESDEDQEQRCCNELNRFQNNQRCMCQA LQQILQNQ SFRFQQDRSQLHQMERELRNLPQNCGFRSP SRCDL S SRTP Y

SEQ ID NO: 12 (amino acid sequence of Ara h 9, LTP isoallergen 1 precursor): MASLKFAFVMLVCMAMVGAPMVNAISCGQVNSALAPCIPFLTKGGAPPPACCSGVRGL

LGALRTTADRQAACNCLKAAAGSLRGLNQGNAAALPGRCGVSIPYKISTSTNCATIKF

SEQ ID NO: 13 (Ara h 1 CDS)

AATAATCATATATATTCATCAATCATCTATATAAGTAGTAGCAGGAGCAATGAGAGG

GAGGGTTTCTCCACTGATGCTGTTGCTAGGGATCCTTGTCCTGGCTTCAGTTTCTGCA

ACGCATGCCAAGTCATCACCTTACCAGAAGAAAACAGAGAACCCCTGCGCCCAGAG

GTGCCTCCAGAGTTGTCAACAGGAACCGGATGACTTGAAGCAAAAGGCATGCGAGT

CTCGCTGCACCAAGCTCGAGTATGATCCTCGTTGTGTCTATGATCCTCGAGGACACA

CTGGCACCACCAACCAACGTTCCCCTCCAGGGGAGCGGACACGTGGCCGCCAACCC

GGAGACTACGATGATGACCGCCGTCAACCCCGAAGAGAGGAAGGAGGCCGATGGG

GACCAGCTGGACCGAGGGAGCGTGAAAGAGAAGAAGACTGGAGACAACCAAGAGA

AGATTGGAGGCGACCAAGTCATCAGCAGCCACGGAAAATAAGGCCCGAAGGAAGA

GAAGGAGAACAAGAGTGGGGAACACCAGGTAGCCATGTGAGGGAAGAAACATCTC

GGAACAACCCTTTCTACTTCCCGTCAAGGCGGTTTAGCACCCGCTACGGGAACCAAA

ACGGTAGGATCCGGGTCCTGCAGAGGTTTGACCAAAGGTCAAGGCAGTTTCAGAAT

CTCCAGAATCACCGTATTGTGCAGATCGAGGCCAAACCTAACACTCTTGTTCTTCCC

AAGCACGCTGATGCTGATAACATCCTTGTTATCCAGCAAGGGCAAGCCACCGTGAC

CGTAGCAAATGGCAATAACAGAAAGAGCTTTAATCTTGACGAGGGCCATGCACTCA

GAATCCCATCCGGTTTCATTTCCTACATCTTGAACCGCCATGACAACCAGAACCTCA

GAGTAGCTAAAATCTCCATGCCCGTTAACACACCCGGCCAGTTTGAGGATTTCTTCC

CGGCGAGCAGCCGAGACCAATCATCCTACTTGCAGGGCTTCAGCAGGAATACGTTG

GAGGCCGCCTTCAATGCGGAATTCAATGAGATACGGAGGGTGCTGTTAGAAGAGAA

TGCAGGAGGTGAGCAAGAGGAGAGAGGGCAGAGGCGATGGAGTACTCGGAGTAGT

GAGAACAATGAAGGAGTGATAGTCAAAGTGTCAAAGGAGCACGTTGAAGAACTTAC

TAAGCACGCTAAATCCGTCTCAAAGAAAGGCTCCGAAGAAGAGGGAGATATCACCA

ACCCAATCAACTTGAGAGAAGGCGAGCCCGATCTTTCTAACAACTTTGGGAAGTTAT

TTGAGGTGAAGCCAGACAAGAAGAACCCCCAGCTTCAGGACCTGGACATGATGCTC

ACCTGTGTAGAGATCAAAGAAGGAGCTTTGATGCTCCCACACTTCAACTCAAAGGCC

ATGGTTATCGTCGTCGTCAACAAAGGAACTGGAAACCTTGAACTCGTGGCTGTAAGA

AAAGAGCAACAACAGAGGGGACGGCGGGAAGAAGAGGAGGACGAAGACGAAGAA

GAGGAGGGAAGTAACAGAGAGGTGCGTAGGTACACAGCGAGGTTGAAGGAAGGCG

ATGTGTTCATCATGCCAGCAGCTCATCCAGTAGCCATCAACGCTTCCTCCGAACTCC

ATCTGCTTGGCTTCGGTATCAACGCTGAAAACAACCACAGAATCTTCCTTGCAGGTG

ATAAGGACAATGTGATAGACCAGATAGAGAAGCAAGCGAAGGATTTAGCATTCCCT

GGGTCGGGTGAACAAGTTGAGAAGCTCATCAAAAACCAGAAGGAATCTCACTTTGT

GAGTGCTCGTCCTCAATCTCAATCTCAATCTCCGTCGTCTCCTGAGAAAGAGTCTCCT

GAGAAAGAGGATCAAGAGGAGGAAAACCAAGGAGGGAAGGGTCCACTCCTTTCAA

TTTTGAAGGCTTTTAACTGAGAATGGAGGCAACTTGTTATGTATCGATAATAAGATC

ACGCTTTTGTACTCTACTATCCAAAAACTTATCAATAAATAAAAACGTTTGTGCGTT GTTTCTCC

SEQ ID NO: 14 (Ara h 2 CDS) ATGGCCAAGCTCACCATACTAGTAGCCCTCGCCCTTTTCCTCCTCGCTGCCCACGCAT CTGCGAGGCAGCAGTGGGAACTCCAAGGAGACAGAAGATGCCAGAGCCAGCTCGA GAGGGCGAACCTGAGGCCCTGCGAGCAACATCTCATGCAGAAAATCCAACGTGACG

AGGATTCATATGGACGGGACCCGTACAGCCCTAGTCAGGATCCGTACAGCCCTAGT

CAGGACCCGGACAGACGTGATCCGTACAGCCCTAGTCCATATGATCGGAGAGGCGC

TGGATCTTCTCAGCACCAAGAGAGGTGTTGCAATGAGCTGAACGAGTTTGAGAACA ACCAAAGGTGCATGTGCGAGGCATTGCAACAGATAATGGAGAACCAGAGCGATAGG

TTGCAGGGGAGGCAACAGGAGCAACAGTTCAAGAGGGAGCTCAGGAACTTGCCTCA ACAGTGCGGCCTCAGGGCACCACAGCGTTGCGACTTGGAAGTCGAAAGTGGCGGCA GAGACAGATACTAA

SEQ ID NO: 15 (Ara h 3 CDS)

CGGCAGCAACCGGAGGAGAACGCGTGCCAGTTCCAGCGCCTCAATGCGCAGAGACC

TGACAATCGCATTGAATCAGAGGGCGGTTACATTGAGACTTGGAACCCCAACAACC

AGGAGTTCGAATGCGCCGGCGTCGCCCTCTCTCGCTTAGTCCTCCGCCGCAACGCCC

TTCGTAGGCCTTTCTACTCCAATGCTCCCCAGGAGATCTTCATCCAGCAAGGAAGGG

GATACTTTGGGTTGATATTCCCTGGTTGTCCTAGACACTATGAAGAGCCTCACACAC AAGGTCGTCGATCTCAGTCCCAAAGACCACCAAGACGTCTCCAAGGAGAAGACCAA AGCCAACAGCAACGAGATAGTCACCAGAAGGTGCACCGTTTCGATGAGGGTGATCT

CATTGCAGTTCCCACCGGTGTTGCTTTCTGGCTCTACAACGACCACGACACTGATGTT

GTTGCTGTTTCTCTTACTGACACCAACAACAACGACAACCAGCTTGATCAGTTCCCC

AGGAGATTCAATTTGGCTGGGAACACGGAGCAAGAGTTCTTAAGGTACCAGCAACA

AAGCAGACAAAGCAGACGAAGAAGCTTACCATATAGCCCATACAGCCCGCAAAGTC

AGCCTAGACAAGAAGAGCGTGAATTTAGCCCTCGAGGACAGCACAGCCGCAGAGA

ACGAGCAGGACAAGAAGAAGAAAACGAAGGTGGAAACATCTTCAGCGGCTTCACG CCGGAGTTCCTGGAACAAGCCTTCCAGGTTGACGACAGACAGATAGTGCAAAACCT AAGAGGCGAGACCGAGAGTGAAGAAGAGGGAGCCATTGTGACAGTGAGGGGAGGC

CTCAGAATCTTGAGCCCAGATAGAAAGAGACGTGCCGACGAAGAAGAGGAATACG

ATGAAGATGAATATGAATACGATGAAGAGGATAGAAGGCGTGGCAGGGGAAGCAG

AGGCAGGGGGAATGGTATTGAAGAGACGATCTGCACCGCAAGTGCTAAAAAGAAC

ATTGGTAGAAACAGATCCCCTGACATCTACAACCCTCAAGCTGGTTCACTCAAAACT

GCCAACGATCTCAACCTTCTAATACTTAGGTGGCTTGGACCTAGTGCTGAATATGGA

AATCTCTACAGGAATGCATTGTTTGTCGCTCACTACAACACCAACGCACACAGCATC

ATATATCGATTGAGGGGACGGGCTCACGTGCAAGTCGTGGACAGCAACGGCAACAG

AGTGTACGACGAGGAGCTTCAAGAGGGTCACGTGCTTGTGGTGCCACAGAACTTCG

CCGTCGCTGGAAAGTCCCAGAGCGAGAACTTCGAATACGTGGCATTCAAGACAGAC

TCAAGGCCCAGCATAGCCAACCTCGCCGGTGAAAACTCCGTCATAGATAACCTGCC

GGAGGAGGTGGTTGCAAATTCATATGGCCTCCAAAGGGAGCAGGCAAGGCAGCTTA AGAACAACAACCCCTTCAAGTTCTTCGTTCCACCGTCTCAGCAGTCTCCGAGGGCTG TGGCTTAA

SEQ ID NO: 16 (Ara h 6 CDS)

GCACACGCCTCCGCAATGAGGCGCGAGAGGGGGAGACAGGGGGACTCATCAAGCT

GCGAGAGGCAGGTTGACGGGGTTAACCTCAAGCCCTGCGAGCAGCACATAATGCAG AGGATCATGGGCGAGCAAGAGCAGTACGACTCCTACAATTTTGGGAGTACTCGATC CTCCGACCAGCAACAGAGGTGCTGCGATGAGCTGAACGAGATGGAGAACACACAG AGATGCATGTGCGAGGCATTGCAGCAAATAATGGAGAACCAGTGCGATGGGTTGCA GGACAGGCAAATGGTGCAGCACTTCAAGAGAGAGCTCATGAACTTGCCCCAACAGT

GTAACTTTGGGGCACCACAGCGTTGCGATTTGGACGTGAGTGGCGGCAGATGCTAG

ACTCAAAAATTATAATCTGTGCCAAAACAAACTAGTAGGAAGTAGCTGATATTATG AGCTATTATGTATGCTTGTTTCGTTGATAATAAATATCATCACTGTATGAATGTGGTG ATAGGTTAGGTTATATGAGCACCTTCGGTGTGCTCTTATGGCTTTACCTATTTTTTGC TACTGCGAAGTTTTACCACCATGAAATAAAAGATCTTCCAGTTAAAAAAAAAAAAA

AAAAAAAAA

SEQ ID NO: 17 (Ara h 7 CDS)

ATGGTCAAGCTCAGCATCCTAGTAGCTCTCCTGGGCGCCCTTCTTGTCGTAGCCTCC

GCGACAAGATGGGATCCCGATCGAGGGTCCAGAGGGTCGAGATGGGACGCACCGA GCAGAGGGGATGACCAGTGCCAGAGGCAGTTGCAGAGGGCAAACCTGAGGCCCTGT

GAGGAACATATAAGGCAAAGGGTGGAGAAAGAGCAAGAGCAAGAGCAAGACGAGT ACCCGTACATCCAACGGGGATCCAGAGGACAACGACCCGGCGAATCTGACGAAGAC CAAGAGCAAAGGTGCTGCAACGAGCTCAACCGGTTCCAGAATAACCAAAGGTGCAT GTGCCAGGCACTTCAACAGATCCTCCAGAACCAGAGCTTTAGGTTCCAGCAGGACA

GGAGCCAGTTGCATCAGATGGAGAGGGAGCTCAGGAACTTGCCCCAGAACTGCGGG TTCAGGTCACCAAGCCGTTGCGACCTCAGTAGCCGCACGCCCTACTAA

SEQ ID NO: 18 (Ara h 9 LTP isoallergen 1 precursor)

ATGGCAAGCCTCAAGTTTGCATTTGTGATGCTTGTGTGCATGGCCATGGTGGGAGCA CCAATGGTGAATGCCATATCATGTGGCCAAGTGAACAGTGCCCTAGCACCATGCATC CCTTTCCTCACAAAGGGTGGAGCTCCTCCTCCGGCTTGTTGCAGCGGAGTTAGAGGC CTTCTCGGTGCTTTAAGAACCACCGCAGACCGCCAGGCCGCCTGTAACTGCCTCAAA

GCCGCTGCCGGTTCCCTTCGTGGCCTCAACCAAGGCAACGCCGCCGCCCTCCCTGGA AGATGCGGTGTCAGCATTCCTTACAAGATCAGCACCTCCACCAACTGTGCTACCATT AAGTTCTGA

SEQ ID NO: 19 (Ara h 12)

AGAAAACAGTTGCTGGATTCTGCATCTTCTTCCTCGTTCTCTTTCTTGCTCAGGAGGG

AGTGGTGAAAACAGAGGCAAAGCTATGCAACCACCTGGCAGATACATACAGAGGAC CATGCTTTACCAATGCAAGCTGCGATGATCATTGCAAGAACAAAGAGCACTTTGTTA GTGGAACCTGCATGAAAATGGCGTGTTGGTGTGCTCACAACTGTTGATGTAATAATA TACTTAGTAATTAAATAATGATATGAATGATACTCTGTATGTTGTCATCATATCTATC

CCTTATAATTAATAATATG

SEQ ID NO: 20 (Ara h 13)

CAGTACAAAAACGAACGATAATAATGGAGAAGAAAATGGCTGGATTCTGCATCTTT

TTCCTCATTCTCTTTCTTGCTCAGGAATATGGCGTGGAGGGAAAGGAGTGTTTGAAC CTAAGTGACAAATTCAAGGGACCGTGTTTGGGTTCAAAGAACTGCGATCATCACTGC

AGGGACATAGAGCACTTGCTCAGCGGAGTTTGCAGGGACGATTTCCGCTGCTGGTGC

AACAGAAAGTGTTAAAACTACTCCATCATCATCAAACCTCTAAAACCATATGATATA ATAATAATAATAATAATATATGAATAATAAATGCTTAGCTTGCATTATATTGGATCC CCACGATGCGTTAGACGCATGCACCTAGC

SEQ ID N0:21 (Derp 2 K96A mutant)

AUGCGCAUGCAGCUGCUGCUGCUGAUCGCCCUGUCCCUGGCCCUGGUGACCAACU

CCCGCGACCAGGUGGACGUGAAGGACUGCGCCAACCACGAGAUCAAGAAGGUGCU

GGUGCCCGGCUGCCACGGCUCCGAGCCCUGCAUCAUCCACCGCGGCAAGCCCUUC

CAGCUGGAGGCCGUGUUCGAGGCCAACCAGAACUCCAAGACCGCCAAGAUCGAGA

UCAAGGCCUCCAUCGACGGCCUGGAGGUGGACGUGCCCGGCAUCGACCCCAACGC

CUGCCACUACAUGAAGUGCCCCCUGGUGAAGGGCCAGCAGUACGACAUCAAGUAC

ACCUGGAACGUGCCCGCCAUCGCCCCCAAGUCCGAGAACGUGGUGGUGACCGUGA

AGGUGAUGGGCGACGACGGCGUGCUGGCCUGCGCCAUCGCCACCCACGCCAAGAU CCGCGACUaa

Claims

Claims

1. A composition for modulating an immune response against at least one antigen in a subject, the composition comprising at least one nucleoside-modified RNA molecule encoding an antigen, wherein the antigen is an allergen or an autoantigen.

2. The composition of claim 1, wherein the at least one nucleoside-modified RNA molecule comprises pseudouridine.

3. The composition of claim 1, wherein the at least one nucleoside-modified RNA molecule comprises 1-methyl-pseudouridine.

4. The composition of claim 1, wherein the allergen is a food allergen or an aero allergen.

5. The composition of claim 1, wherein the allergen is selected from the group consisting of OVA, Ara h 1, Ara h 2, Ara h 3, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 12, Ara h 13, Derp1, Derp2, and Derp 23, or a fragment or variant thereof.

6. The composition of claim 1, wherein the at least one allergen comprises an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a fragment or variant thereof.

7. The composition of claim 1, wherein the at least one nucleoside-modified RNA molecule encodes an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment or variant thereof.

8. The composition of claim 1, further comprising at least one anti-inflammatory agent.

9. The composition of claim 8, wherein the anti-inflammatory agent comprises an mTOR inhibitor.

10. The composition of claim 9, wherein the mTOR inhibitor is a small molecule.

11. The composition of claim 9, wherein the mTOR inhibitor is everolimus, rapamycin, sirolimus, temsirolimus, ridaforolimus, Torin-1, or non-rapalog derived inhibitors or an analog or derivative thereof.

12. The composition of claim 1, wherein the composition further comprises an adjuvant.

13. The composition of claim 1, further comprising a lipid nanoparticle (LNP).

14. The composition of claim 13, wherein the at least one nucleoside-modified RNA is encapsulated within the LNP.

15. A method of reducing a T helper type 2 (Th2) response against one or more antigen in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

16. A method of stimulating the production of allergen specific regulatory T cells (Tregs) in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

17. A method of promoting tolerance to one or more antigen in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

18. A method of increasing allergen-specific IgG production in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

19. A method of reducing pro-allergic IgE production in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

20. A method of treating, preventing or decreasing the risk of an allergic response against one or more antigen in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

21. The method of claim 20, wherein the allergic response is selected from airway hypersensitivity, abdominal pain, allergic rhinitis, anaphylaxis, colonic inflammation, diarrhea, eczema, hives, itching, nausea, and vomiting.

22. A method of treating, preventing, or decreasing the risk of developing an allergic disease in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

23. The method of claim 22, wherein the composition is administered before exposure to an allergen.

24. The method of claim 22, wherein the composition is administered after exposure to an allergen.

25. The method of any one of claims 22-24 wherein the allergic disease is selected from rhinitis, atopy, asthma, COPD, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria, anaphylactic shock, eosinophilic gastrointestinal diseases including eosinophilic esophagitis, food-protein induced allergic proctocolitis, and hay fever.

26. The method of any one of claims 15-25, wherein the composition is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

27. The method of claim 26, wherein the method comprises a single administration of the composition.

28. The method of claim 26, wherein the method comprises multiple administrations of the composition.

29. A composition for increasing the antigen specific regulatory T cells (Tregs) in a subject, the composition comprising at least one nucleoside-modified RNA molecule encoding an antigen.

30. The composition of claim 29, wherein the at least one nucleoside-modified RNA molecule comprises pseudouridine.

31. The composition of claim 29, wherein the at least one nucleoside-modified RNA molecule comprises 1-methyl-pseudouridine.

32. The composition of claim 29, wherein the antigen is an autoantigen.

33. The composition of claim 29, further comprising at least one anti-inflammatory agent.

34. The composition of claim 33, wherein the anti-inflammatory agent comprises an mTOR inhibitor.

35. The composition of claim 34, wherein the mTOR inhibitor is a small molecule.

36. The composition of claim 35, wherein the mTOR inhibitor is everolimus, rapamycin, sirolimus, temsirolimus, ridaforolimus, Torin-1, or non-rapalog derived inhibitors or an analog or derivative thereof.

37. The composition of claim 29, wherein the composition further comprises an adjuvant.

38. The composition of claim 29, further comprising a lipid nanoparticle (LNP).

39. The composition of claim 38, wherein the at least one nucleoside-modified RNA is encapsulated within the LNP.

40. A method of treating or preventing an inflammatory or autoimmune disease in a subject, comprising administering to the subject an effective amount of a composition of any one of claims 29-39.

41. The method of claim 40, wherein the composition is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

42. The method of claim 41 wherein the method comprises a single administration of the composition.

43. The method of claim 41, wherein the method comprises multiple administrations of the composition.

44. A composition for modulating an immune response in a subject, the composition comprising at least one nucleoside-modified RNA molecule encoding an antigen and at least one mTOR inhibitor.

45. The composition of claim 44, wherein the at least one nucleoside-modified RNA molecule comprises pseudouridine.

46. The composition of claim 44, wherein the at least one nucleoside-modified RNA molecule comprises 1-methyl-pseudouridine.

47. The composition of claim 44, wherein the mTOR inhibitor is a small molecule.

48. The composition of claim 47, wherein the mTOR inhibitor is everolimus, rapamycin, sirolimus, temsirolimus, ridaforolimus, Torin-1, or non-rapalog derived inhibitors or an analog or derivative thereof.

49. The composition of claim 44, wherein the composition further comprises an adjuvant.

50. The composition of claim 44, further comprising a lipid nanoparticle (LNP).

51. The composition of claim 50, wherein the at least one nucleoside-modified RNA is encapsulated within the LNP.

52. A method of modulating the response of a subject to a composition comprising at least one nucleoside-modified RNA molecule encoding an antigen comprising administering an mTOR inhibitor to the subject.

53. The method of claim 52 wherein the method reduces adverse effects when compared to administration of the same composition comprising at least one nucleoside-modified RNA molecule encoding an antigen without an mTOR inhibitor.

54. The method of claim 52 wherein the method increases immunological memory to the antigen when compared to administration of the same composition comprising at least one nucleoside-modified RNA molecule encoding an antigen without an mTOR inhibitor.