Universal nucleic acid-based vaccines and methods of use thereof

Abstract

Described herein are compositions comprising a nucleic acid sequence (e.g., mRNA) encoding an infectious antigen antigen polypeptide and a nucleic acid sequence (e.g., mRNA) encoding at least one universal T-cell epitope (UTE), as well as compositions comprising a nucleic acid sequence (e.g., mRNA) encoding an infectious antigen antigen polypeptide and at least one universal T-cell epitope, and methods for using the compositions.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 376,908, filed on September 23, 2022, the content of which is hereby incorporated by reference in its entirety.

[0003] Reference to Sequence Listing

[0004] The Sequence Listing, created on September 22, 2022 and having a size of 1,353,272 bytes, submitted as an XML file named "11538-003WO1_Sequence_Listing.xml" on September 25, 2023 is hereby incorporated by reference in accordance with 37 C.F.R. § 1.834. BACKGROUND OF THE INVENTION

[0005] Vaccine-induced neutralizing antibodies are considered the gold standard for preventing pathogen entry or causing disease. However, many pathogens have developed mechanisms to evade antibody protection, such as some RNA viruses (e.g., SARS-CoV-2), which typically mutate viral structural proteins, thereby reducing the protective efficacy over time. Thus, there is a need to develop a universal vaccine that ensures broad efficacy against multiple strains or different variants of a pathogen.

[0006] To date, the importance of T cell immunity in vaccination may have been relatively underestimated. T cells, particularly cytotoxic CD8 T cells, play a central role in the control of infections, including the prevention of severe COVID-19 and HSV-1 / 2-related diseases. These cytotoxic T cells can kill any infected cell by recognizing foreign peptides presented on the cell surface in association with major histocompatibility complex class I molecules (MHC I). In cases where the pathogen-derived peptides are selected from conserved regions of different strains or variants, the activated T cells will be able to provide broad-spectrum protection. Thus, a vaccine platform that can induce sufficient high-quality antibodies and memory T cells will greatly enhance the efficacy of protection, particularly against infections that cannot be effectively controlled by antibodies alone.

[0007] Therefore, there is a need for a platform for generating universal vaccines. SUMMARY OF THE INVENTION

[0008] This document describes compositions, nanoparticles, and / or vaccines that include nucleic acid sequences encoding an infectious agent antigen polypeptide, such as DNA or RNA (e.g., mRNA), and nucleic acids encoding at least one universal T cell epitope, as well as compositions, nanoparticles, and / or vaccines that include nucleic acid sequences encoding an infectious agent antigen polypeptide and at least one universal T cell epitope. The universal T cell epitopes (UTEs) described herein are epitopes that are highly conserved across various strains / variants / serotypes of a virus. In some embodiments, the compositions, the nanoparticles, and / or the vaccines may further include a pharmaceutically acceptable carrier.

[0009] In some embodiments, the composition that includes a nucleic acid sequence encoding an infectious agent antigen polypeptide (e.g., mRNA) and a nucleic acid sequence encoding a universal T cell epitope can elicit an antibody response in a subject. In some embodiments, the composition that includes a nucleic acid sequence encoding the infectious agent antigen polypeptide and a nucleic acid sequence encoding a universal T cell epitope can elicit both an antibody response and a T cell response in a subject. In some embodiments, the composition that includes a nucleic acid sequence encoding the infectious agent antigen polypeptide and a nucleic acid sequence encoding a universal T cell epitope can elicit a broad T cell response in a subject. The broad T cell response described herein refers to a T cell response against at least one universal T cell epitope such that the composition or vaccine is protected against different variants of the same virus.

[0010] In some embodiments, the composition that includes a nucleic acid sequence encoding the infectious agent antigen polypeptide (e.g., mRNA) and a universal T cell epitope can elicit an antibody response in a subject. In some embodiments, the composition that includes a nucleic acid sequence encoding the infectious agent antigen polypeptide and a universal T cell epitope can elicit both an antibody response and a T cell response in a subject. In some embodiments, the composition that includes a nucleic acid sequence encoding the infectious agent antigen polypeptide and the universal T cell epitope can elicit a broad T cell response in a subject. For example, in some embodiments, the nucleic acid sequence encoding the infectious agent antigen polypeptide and at least one universal T cell epitope can be recognized by the immune system of a subject to elicit a CD4+ T cell response or / and a CD8+ T cell response.

[0011] In some embodiments, the universal T cell epitope can provide greater protection against infectious agent variants.

[0012] In some embodiments, the nucleic acid sequence can encode a pathogen antigen polypeptide. In some embodiments, the pathogen can be a virus. For example, negative-sense single-stranded RNA viruses of the family Paramyxoviridae, such as human metapneumovirus (hMPV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), measles virus (MeV), varicella-zoster, influenza virus (e.g., influenza A and influenza B), herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus (e.g., smallpox, monkeypox), HIV, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, coronaviruses (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), African swine fever (ASF), foot-and-mouth disease virus (FMDV), feline herpesvirus-1 / feline viral rhinotracheitis, canine distemper, feline coronavirus (FCoV), or any combination thereof. In some embodiments, the pathogen can be monkeypox, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), SARS-CoV-2, or any combination thereof.

[0013] Methods for treating, inhibiting, reducing, ameliorating, and / or preventing viral infections caused by a pathogen in a subject are described herein, the methods comprising administering to the subject a composition described herein, a nanoparticle described herein, or a vaccine described herein.

[0014] In some embodiments, methods for activating T cells, B cells, or any combination thereof, stimulating the proliferation of T cells, B cells, or any combination thereof, eliciting an immune response to a pathogen in a subject, and / or enhancing an immune response generated by a nucleic acid-based vaccine are also described, the methods comprising introducing a nucleic acid sequence encoding at least one universal T cell epitope into the nucleic acid-based vaccine. In some embodiments, the methods can include administering to the subject a composition described herein, a nanoparticle described herein, or a vaccine described herein.

[0015] In some embodiments, the nucleic acid may include DNA, RNA, any combination thereof (e.g., plasmid DNA, minicircle DNA, minimally immunogenic defined gene expression (MIDGE) and Doggybone, messenger RNA (mRNA), circular (cirRNA), self-amplifying RNA (saRNA, also known as SAM) or DNA-initiated SAM (DLSAM)). The nucleic acid may encode a bicistronic or polycistronic construct, e.g., a DNA comprising at least two antigens.

[0016] In some embodiments, the subject may be a human. In some embodiments, the subject may be a non-human vertebrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings incorporated in and constituting a part of this specification illustrate several aspects described hereinafter.

[0018] Figures 1A - 1H : Naming and validation of PanCoVAX mRNA. (1A) Scheme of PanCoVAX mRNA. (1B) IVT mRNA of DVS, OVS and MTE. (1C-1G) Expression levels of DVS and OVS mRNA in 293T cells were determined by Western blot (1C), flow cytometry (1D-1E) DVS (1D) and OVS (1E), and immunostaining (1F-1G) DVS (1F) and OVS (1G). (1H) Expression level of MTE mRNA in 293T cells.

[0019] Figures 2A - 2C : Physicochemical characterization of lipid nanoparticle formulations. (2A) Size and polydispersity index (PDI) of ARV-L002 (“ARV-T1”) and ARV-L001 (“SM102”) LNP formulations using DVS or DVS / MTE. (2B) Surface charge (ζ potential) and mRNA encapsulation efficiency of ARV-L001 and ARV-L002 LNP formulations using DVS or DVS / MTE. (2C) Western blot analysis showing in vitro expression of spike protein over time in 293T cells after transfection with 2.5 μg mRNA.

[0020] Figures 3A - 3F: ELISA and pseudovirus neutralization assays. (3A) Immunization protocol: BALB / c mice were immunized at weeks 0 and 3. Serum samples were collected on days 14 and 35 after the primary immunization. Convalescent serum derived from COVID-positive human serum was used as a control. Spike-specific antibody responses total IgG (3B) and (3C) and neutralizing antibodies (3D-3F) against delta pseudovirus (3D), omicron pseudovirus (3E), and omicron-BA2 pseudovirus (3F) were evaluated using the serum by ELISA and pseudovirus neutralization assays.

[0021] Figures 4A - 4D : Detection of T cell responses by Elispot assay. ELISPOT as described in M&M, 3 x 10^5 splenocytes was used to measure Ag-specific T cell responses when stimulated with spike (4A, 4B) overlapping peptides and MTE (4C, 4D) overlapping peptides (synthesized by GenScript and JPT (JPT), respectively).

[0022] Figures 5A - 5C : Administration studies. BALB / c mice were immunized at weeks 0 and 3 with 0.01 μg, 0.1 μg, and 1 μg of mRNA formulated with SM102 or ARV-T1 LNP. Serum samples were collected on day 14 (5A) and day 35 (5B) after the primary immunization. Spike-specific antibody responses total IgG (5A and 5B) and neutralizing antibodies (5C) on day 35 were evaluated using the serum by ELISA and pseudovirus neutralization assays.

[0023] Figures 6A - 6C : mRNA encoding MTE, OVS, or MTE / OVS efficiently protected hACE2 transgenic mice from SARS-CoV-2 delta variant infection. (6A) Immunization protocol and challenge schedule, hACE-transgenic mice (K18) were immunized twice with LNP formulated with ARV T1 containing mRNA consisting of MTE, OVS, and MTE / OVS. Fourteen days after the last immunization, the mice were infected with a lethal dose of SARS-CoV-2 delta variant. (6B) and (6C), the animal body weight (6B) and survival rate (6C) were observed daily after infection. (6D) Virus titers were determined by qRT-PCR from the lungs 4 days after infection.

[0024] Figures 7A - 7E: Detection of T cell responses by intracellular staining. Splenocytes were isolated and stimulated in the presence of different peptide pools of spike or MTE. Intracellular cytokine staining was performed after 6 hours and analyzed by flow cytometry (7A). (7B) Spike-specific CD4 T cell responses. (7C) Spike-specific CD8 T cell responses. (7D) MTE-specific CD4 T cell responses. (7E) MTE-specific CD8 T cell responses.

[0025] Figures 8A - 8F Shows the in vitro transfection efficiency of GFP mRNA (1 μg / mL) achieved after 24 hours using LNP delivered into BHK cells. (8A) Representative fluorescence images of transfected BHK cells. (8B) Analysis of transfection efficacy of GFP expression using a flow cytometer. MFI represents mean fluorescence intensity. (8C-8F) Characterization of LNP(T1)-mRNA GFP, (8C) size and PDI, (8D) Zeta potential and encapsulation efficacy, (8E) CryoEM LNP image, (8F) LNP size based on CryoEM image. Figure 9: Biodistribution of LNP(T1) in vivo and the cell types delivered. (9A) Shows the in vivo transfection efficiency of luciferase-expressing mRNA in vivo. LNPs were formulated with the indicated ionizable lipids and 1 μg of the formulated luciferase-expressing mRNA was injected intramuscularly. After administration, luciferase expression was determined by whole-body bioluminescence imaging using an IVIS Spectrum in vivo imaging system at 6 hours, 24 hours, 48 hours, and 72 hours, respectively. (9B) Biodistribution of LNP(T1)-mRNA luciferase in vivo. (9C-9D), the cell types delivered by LNP(T1)-mRNA Cre delivered intramuscularly and intravenously in Ai14 mice

[0026] Figure 10 : Evaluation of antigen-specific T cell responses by Elispot. Data are presented as mean ± SD. Statistical comparisons were analyzed using one-way ANOVA with Tukey's multiple comparison test. *p < 0.05, **p < 0.01.

[0027] Figure 11 : In vitro synthesis of HSV mRNA vaccines. HSV gB, gD, gH, gL, UL19, and MTE mRNAs were synthesized by in vitro transcription with T7 RNA polymerase and the mRNAs were run on a 0.8% MOPS agarose gel.

[0028] Figures 12A - 12F: In vitro validation of HSV mRNA vaccines. (12A - 12C) HSV mRNA gB (12A), gD (12B), and gL (12C) were transfected into 293T cells using an mRNA transfection kit and detected by Western blot. (12D) HSV mRNA MTE - His was transfected into 293T cells using an mRNA transfection kit. After 48 hours, the MTE - His protein was concentrated from cell lysates and detected by Western blot. (12E - 12F) HSV mRNA gH - HA (12E) and UL - 19 (12F) were transfected into 293T cells using an mRNA transfection kit and detected by flow cytometry.

[0029] Figures 13A - 13B : Physicochemical characterization of lipid nanoparticle formulations. (13A) Size and polydispersity index (PDI) of LNP formulations of ARV - L002 (ARV - T1) with the indicated mRNA. (13B) mRNA vaccine encapsulation efficiency of ARV - L002 LNP formulations with the indicated mRNA.

[0030] Figure 14A -14G: HSV mRNA vaccines elicit potent T - cell responses and strong antibody responses in BALB / c mice. (14A) BALB / c mice were immunized intramuscularly with 5 μg of LNP - formulated mRNA vaccines (gD - wt, gD, gB / gD, gB / gD + gH / gL, and gH / gL + MTE / UL19) on days 0, 21, and 42. Mouse sera were collected on days 14, 35, and 56, respectively. Mouse spleens were collected on day 56. (14B) gD - specific IgG was detected from serum samples by ELISA. (14C) Neutralization assays were performed by counting plaques with the HSV - 2 MS strain in Vero cells. (14D - 14G) Spleen cells were isolated from mouse spleens and subjected to ELISPOT assays (14D - 14E) or flow cytometry intracellular staining (14F - 14G) in the presence of a pool of UL - 19 peptides (14E - 14F) or a pool of gD peptides (14D and 14G).

[0031] Figures 15A - 15D: HSV mRNA vaccine protects mice from HSV-2 challenge. (15A-15C) 50% lethal dose (LD50) of HSV2 MS in BALB / c. (15A) Female BALB / c mice were injected with 2 mg medroxyprogesterone on days -7 and -3, and challenged intravaginally with different PFU of HSV-2. Mouse body weight and survival rate were recorded after challenge. (15B) Survival curve of mice after challenge. (15C) Value of LD50 of HSV-2 MS strain in BALB / c mice. (15D) Female BALB / c mice were immunized intramuscularly with 5 μg LNP-formulated mRNA vaccines (gB / gD, gB / gD+MTE / UL19, gB / gD+gH / gL+MTE / UL19, and gB / gD+gH / gL+gC / gE+MTE / UL19) on days 0 and 21. Mouse serum samples were collected on days 14 and 28. On days 35 and 39, mice were injected with 2 mg medroxyprogesterone, and on day 42 with 10 4 PFU HSV2 MS strain or HSV-1 HF was used for intravaginal challenge. Vaginal cultures were collected on days 44 and 46 to determine the copy number of HSV2 or HSV-1. Mouse reproductive disease, body weight, and survival rate were recorded until day 56.

[0032] Figure 16 A: Synthesis and formulation of FIPV mRNA vaccine. Pre-in vitro transcription DNA plasmid linearized with BspQI. Respectively, 1, 2 represent before and after digestion.

[0033] Figures 17A - 17B : In vitro verification of FIPV mRNA vaccine. (17A) FIPV spike mRNA (Fcov-I-S-2P-HA, Fcov-I-S-4P-HA, and Fcov-I-S-2P2Cb-HA) was synthesized by in vitro transcription with T7 RNA polymerase, and the mRNA was run on a 0.8% MOPS agarose gel. (17B) FIPV spike mRNA was transfected into 293T cells using an mRNA transfection kit and detected by Western blot.

[0034] Figures 18A - 18C : mRNA with human-derived UTR can be highly expressed in feline cells. eGFP mRNA labeled with human-derived UTR was transfected into feline cell line FCWF-4cu and human cell line 293T cells using an mRNA transfection kit. The expression level of eGFP was detected by flow cytometry (18A-18B) and fluorescence microscopy (18C).

[0035] Figures 19A - 19B: Generation of Fcov-I S1 antigen and antibody. (19A) Recombinant Fcov-I S1 subunit His-tagged protein was generated in vitro and detected with His antibody. (19B) Rabbits were immunized with 4 doses of mRNA formulated with ARV-T1LNP expressing the full-length S protein of FCov-I. Serum samples were collected 3 weeks after immunization and subjected to ELISA for titration of S1 antibody against the recombinant Fcov-IS1-His protein.

[0036] Figures 20A - 20D : FIPV mRNA vaccines elicit potent T cell responses and strong antibody responses in BALB / c mice. (20A) BALB / c mice were immunized intramuscularly with 2 μg of mRNA vaccines (Fcov-I-S-wt, Fcov-I-S-2P, Fcov-I-S-2P2Cb, Fcov-I-S-4P, and Fcov-II-RBD-MN) formulated with LNP on days 0 and 21. Mouse serum samples were collected on days 14 and 35. Mouse spleens were collected on day 35. (20B) S1-specific IgG was detected by ELISA from the serum samples. (20C - 20D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assay in the presence of stimulation with S1 protein (20C) or N peptide pool (20D).

[0037] Figure 21 : FIPV mRNA vaccines protect cats from Fcov-I and Fcov-II challenge. Cats were immunized intramuscularly with 10 μg of mRNA vaccines (Fcov-I-S-2P, Fcov-II-RBD-MN, and Fcov-I-S-2P / Fcov-II-RBD-MN) formulated with LNP on days 0 and 21. Cat sera were collected on days 14 and 28. Cats were challenged with Fcov-I virus and Fcov-II virus on day 35. Cat body weight and survival rate were recorded until day 49. Detailed Description

[0038] The present disclosure provides compositions comprising a nucleic acid sequence encoding an infectious agent antigen polypeptide (e.g., mRNA) and a nucleic acid sequence encoding at least one universal T cell epitope (UTE), as well as compositions comprising a nucleic acid sequence encoding an infectious agent antigen polypeptide and at least one T cell epitope. In some embodiments, the compositions can be nanoparticles (e.g., lipid nanoparticles) or vaccines. The present disclosure also provides methods of using the compositions to deliver the nucleic acid sequences (e.g., mRNA) described herein to a subject.

[0039] Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings and examples. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions are provided to facilitate a full understanding of the terms used in this specification.

[0041] Definitions

[0042] General Definitions

[0043] As used in this specification and the following claims, the terms "comprise" (and its forms, derivatives, or variants such as "comprising" and "comprises") and "include" (and its forms, derivatives, or variants such as "including" and "includes") are inclusive (i.e., open-ended) and do not exclude additional elements or steps. For example, when used in this specification, the terms "comprise" and / or "comprising" indicate the presence of the stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Unless otherwise specified, all numbers representing quantities of ingredients, reaction conditions, geometries, dimensions, etc. used in the specification and claims should be understood to be interpreted at least in accordance with the number of significant figures and ordinary rounding methods, rather than attempting to limit the application of the doctrine of equivalents to the scope of the claims.

[0044] Accordingly, these terms are not only intended to cover the recited elements or steps, but may also include other elements or steps not expressly recited. In addition, as used herein, when used in conjunction with an element, the use of the terms "a", "an", and "the" may mean "one", but it is also consistent with the meanings of "one or more", "at least one", and "one or more than one". Thus, in the absence of further constraints, an element beginning with "a" or "an" does not exclude the presence of additional identical elements.

[0045] In this text, ranges may be expressed as from “about” a particular value and / or to “about” another particular value. “About” means within 5% of the stated value, such as within 4%, 3%, 2%, or 1% of the stated value. When expressing such a range, on the other hand, it includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by use of the antecedent “about”, it should be understood that the particular value forms on the other hand. It will be further understood that each endpoint of each range is significant both relative to and independent of the other endpoint. It should also be understood that many values are disclosed herein, and in addition to the value itself, each value is also disclosed herein as “about” the particular value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. A range can be interpreted to include the beginning and end of the range. For example, a range of 10% to 20% (i.e., a range of 10% - 20%) can include 10% and also include 20%, and include percentages between 10% and 20%, unless expressly stated otherwise herein.

[0046] As used herein, the terms “may”, “optionally”, and “may optionally” are used interchangeably and mean to include the case where the condition occurs and the case where the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is intended to include cases where the formulation includes an excipient and cases where the formulation does not include an excipient.

[0047] It should be understood that when combinations, subsets, groups, etc. of elements are disclosed (e.g., combinations of components in a composition, or combinations of steps in a method), although each specific reference to each individual and collective combination and permutation of these elements may not be expressly disclosed, each is specifically contemplated and described herein.

[0048] "Administering" to a subject includes any route of introducing or delivering an agent to the subject. Administration can be effected by any suitable route, including oral, topical, percutaneous, transdermal, intra-articular, intra-arterial, intradermal, intraventricular, intralesional, intranasal, rectal, vaginal, by inhalation, via an implantable reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), etc. As used herein, "co-administering", "combining", "simultaneously administering", or "administering simultaneously" means that the compounds are administered at the same point in time or substantially one right after the other. In the latter case, the administrations of the two compounds are close enough in time such that the observed results are indistinguishable from those obtained when the compounds are administered at the same point in time. "Systemic administration" means introducing or delivering an agent to a subject by a route that introduces or delivers the agent to a broad area of the subject's body (e.g., greater than 50% of the body), such as through an entry into the circulatory system or lymphatic system. In contrast, "local administration" means introducing or delivering an agent to a subject by a route that introduces or delivers the agent to the area at or immediately adjacent to the site of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, an agent administered locally can be readily detected in the vicinity of the site of administration, but is undetectable or present in negligible amounts in distal parts of the subject's body. Administration includes self-administration and administration by another person.

[0049] As used herein, the terms "controlled release" or "controlled release drug delivery" or "extended release" refer to the release or administration of a drug from a given dosage form in a controlled manner so as to obtain a desired in vivo pharmacokinetic profile. One aspect of "controlled" drug delivery is the ability to manipulate the formulation and / or dosage form so as to establish the desired drug release kinetics.

[0050] "Reducing" can mean any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance should also be understood to reduce the gene output of a gene when the gene output of the gene product of the substance is lower relative to the output of the gene product of the substance not being utilized. Also, for example, a reduction can be a change in the symptoms of a medical condition such that the symptoms are less than the symptoms previously observed. A reduction can be any individual, median, or average reduction in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction, so long as the reduction is statistically significant.

[0051] "Inhibit", "inhibiting", and "inhibition" mean to reduce an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to the native or control level. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between compared to the native or control level.

[0052] "Inactivate", "inactivating", and "inactivation" refer to the reduction or elimination of an activity, response, condition, disease, or other biological parameter due to a chemical (covalent bond formation) between a ligand and its biological target.

[0053] "Reduce" or other forms of the word such as "reducing" or "reduction" mean to lower an event or characteristic (e.g., tumor growth). It should be understood that this is generally relative to a certain standard or expected value, in other words it is relative, but is not always required by the cited standard or relative value. For example, "reduce tumor growth" means to lower the rate of tumor growth relative to a standard or control.

[0054] As used herein, the terms "treating" a subject or "treatment" of a subject include administering a medicament to a subject for the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, improving, enhancing, stabilizing, or affecting a disease or disorder or the symptoms of a disease or disorder. The terms "treating" and "treatment" can refer to reducing the severity and / or frequency of symptoms, eliminating the symptoms and / or underlying cause, preventing the occurrence of the symptoms and / or their underlying cause, and / or improving or repairing an injury.

[0055] "Prevent" or other forms of the word such as "preventing" or "prevention" mean to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chance of a particular event or characteristic occurring. Prevention does not require a comparison to a control as it is generally more absolute than, for example, reduction. As used herein, one can reduce but not prevent something, but one can prevent something that is reduced. Similarly, one can prevent but not reduce something, but one can also reduce something that is prevented. It should be understood that the use of other words is also explicitly disclosed in the context of using reduction or prevention, unless otherwise explicitly stated. For example, the terms "prevent" or "inhibit" can refer to a treatment that prevents or slows the onset of a disease or medical condition or reduces the severity of a disease or medical condition in advance. Thus, if a treatment can treat a disease in a subject having the symptoms of the disease, the treatment can also prevent or inhibit the disease in a subject who has not yet suffered some or all of the symptoms. As used herein, the term "prevent" a disease or an unwanted physiological event in a subject specifically means preventing the occurrence of symptoms and / or its underlying cause, where the subject may or may not exhibit a high susceptibility to the condition or event.

[0056] The term "effective amount" of a therapeutic agent means an amount of a beneficial agent that is non-toxic but sufficient to provide the desired effect. The "effective" amount of a beneficial agent will vary from subject to subject depending on the subject's age and general condition, one or more particular beneficial agents, etc. Thus, it is not always possible to specify an exact "effective amount". However, a person of ordinary skill in the art can use routine experimentation to determine the appropriate "effective amount" in any subject situation. Moreover, as used herein and unless otherwise explicitly stated, the beneficial "effective amount" can also refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount.

[0057] The "effective amount" of a drug required to achieve a therapeutic effect can vary depending on factors such as the age, sex, and weight of the subject. The dosage regimen can be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced, as indicated by the exigencies of the treatment situation.

[0058] As used herein, a "therapeutically effective amount" of a therapeutic agent refers to the amount that is effective to achieve a desired therapeutic outcome, and a "prophylactically effective amount" of a therapeutic agent refers to the amount that is effective to prevent an unwanted physiological condition. The therapeutically effective amount and prophylactically effective amount of a given therapeutic agent will generally vary depending on factors such as the type and severity of the condition or disease being treated and the age, sex, and weight of the subject. The term "therapeutically effective amount" can also refer to the amount of a therapeutic agent or the rate of delivery of a therapeutic agent (e.g., an amount that varies over time) that is effective to promote a desired therapeutic effect. The exact desired therapeutic effect will vary depending on the condition being treated, the tolerance of the subject, the drug and / or pharmaceutical formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of the drug in the formulation, etc.), and various other factors understood by one of ordinary skill in the art.

[0059] As used herein, the term "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component can be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a harmful manner with any other component of the formulation containing it. When the term "pharmaceutically acceptable" is used to refer to an excipient, the term generally means that the component has met the desired toxicological and manufacturing test criteria or that the component is included in the Inactive Ingredient Guide established by the U.S. Food and Drug Administration.

[0060] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that can be used to prepare a pharmaceutical composition or therapeutic composition that is generally safe and non-toxic and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline solutions, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other materials well known in the art for use in pharmaceutical formulations and further described herein.

[0061] As used herein, "pharmaceutically acceptable salts" are derivatives of the disclosed compounds in which the parent compound is modified by preparing its inorganic and organic, non-toxic, acid or base addition salts. The salts of the compounds of the present invention can be synthesized from the parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of an appropriate base (such as Na, Ca, Mg or K hydroxides, carbonates, bicarbonates, etc.) or by reacting the free base forms of these compounds with a stoichiometric amount of an appropriate acid. Such reactions are generally carried out in water or an organic solvent or a mixture of both. Generally, when feasible, non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol or acetonitrile are typical. The salts of the compounds of the present invention further include solvates of the compounds and the compound salts.

[0062] Examples of pharmaceutically acceptable salts include, but are not limited to: mineral salts or organic salts of basic residues such as amines; base salts or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts and quaternary ammonium salts of the parent compounds formed from non-toxic inorganic acids or organic acids. For example, conventional non-toxic acid salts include: those salts derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and salts prepared from the following organic acids or different acids that produce the same counterions: acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, HOOC-(CH2)n-COOH (where n is 0-4), etc. A list of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., p., 1418, (1985).

[0063] In addition, as used herein, the term "pharmacologically active" in "pharmacologically active" derivatives or analogs (or simply, "active") can refer to derivatives or analogs (such as salts, esters, amides, conjugates, metabolites, isomers, fragments, etc.) having a type of pharmacological activity that is the same as and approximately equal in degree to the type of pharmacological activity of the parent compound.

[0064] A "control" is an alternative subject or sample used for comparison purposes in an experiment. A control can be "positive" or "negative".

[0065] As used herein, "subject" means an individual. Thus, "subjects" can include domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subjects" can include mammals such as primates or humans. Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., a physician). Administration of a therapeutic agent can be carried out at a dosage and for a period of time effective to treat the subject. In some embodiments, the subject is a human.

[0066] As used herein, the term "nucleic acid" means a polymer composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides).

[0067] As used herein, the terms "ribonucleic acid" and "RNA" mean polymers composed of ribonucleotides.

[0068] As used herein, the terms "deoxyribonucleic acid" and "DNA" mean polymers composed of deoxyribonucleotides.

[0069] The term "oligonucleotide" refers to a single-stranded or double-stranded nucleotide polymer having a length of from about 2 to up to about 100 nucleotides. Suitable oligonucleotides can be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett. 22:1859-1862 (1981) or by the triester method according to Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981) (both incorporated herein by reference) or by other chemical methods using commercial automated oligonucleotide synthesizers or VLSIPS TM technology. When an oligonucleotide is referred to as "double-stranded", those skilled in the art will understand that typically there is a pair of oligonucleotides in a hydrogen-bonded helical array associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, as used herein, the term "double-stranded" is also intended to refer to forms that include structural features such as bulges and loops, which are more fully described in biochemical texts such as Stryer, Biochemistry, Third Edition, (1988), which is incorporated herein by reference for all purposes.

[0070] The term "polynucleotide" refers to a single-stranded or double-stranded polymer composed of nucleotide monomers. In some embodiments, the polynucleotide is composed of nucleotide monomers having a length generally greater than 100 nucleotides and a length of up to about 8,000 or more nucleotides.

[0071] The nucleic acid can be or can include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA (a diastereoisomer of LNA) having an α-L-ribose configuration, 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-α-LNA having a 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexyl nucleic acid (CeNA), or a chimera or combination thereof.

[0072] In some embodiments, the polynucleotides of the present disclosure act as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (a naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. Those skilled in the art will understand that, unless otherwise indicated, the polynucleotide sequences presented in this application will set forth "T" in the representative DNA sequences, but in cases where the sequence represents RNA (e.g., mRNA), the "T" will be replaced by "U". Thus, any RNA polynucleotide encoded by a DNA identified by a specific sequence identifier can also include the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each "T" in the DNA sequence is replaced by "U".

[0073] The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail. The polynucleotides of the present disclosure can act as mRNA but can be distinguished from wild-type mRNA in terms of their function and / or structural design features, which are used to overcome existing problems of efficient polypeptide expression using nucleic acid-based therapeutic agents.

[0074] In some embodiments, the RNA polynucleotide of the RNA (e.g., mRNA) vaccine encodes 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 antigenic polypeptides. In some embodiments, the nucleic acid sequence (e.g., mRNA) encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 antigenic polypeptides. In some embodiments, the nucleic acid sequence (e.g., mRNA) encodes at least 100 or at least 200 antigenic polypeptides. In some embodiments, the RNA polynucleotide encodes 1 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 1 to 50, 1 to 100, 2 to 50, or 2 to 100 antigenic polypeptides.

[0075] In some embodiments, the polynucleotides of the present disclosure are codon-optimized. Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to match codon frequencies in the target organism and the host organism to ensure proper folding; bias the GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional control regions and translational control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust the translation rate to allow proper folding of the individual domains of the protein; or reduce or eliminate problematic secondary structure within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park, Calif.) and / or proprietary methods. In some embodiments, an open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0076] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity, less than 90% sequence identity, less than 85% sequence identity, less than 80% sequence identity, or less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0077] In some embodiments, the codon-optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares between 65% and 75% or about 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0078] In some embodiments, the codon-optimized RNA (e.g., mRNA) can be, for example, an RNA in which the level of G / C is enhanced. The G / C content of a nucleic acid molecule can affect the stability of the RNA. An RNA having an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than a nucleic acid containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses pharmaceutical compositions containing mRNAs stabilized by sequence modifications in the translation region. Due to the degeneracy of the genetic code, the modifications act by replacing existing codons with codons that promote greater RNA stability without changing the codons of the resulting amino acids. The method is limited to the coding region of the RNA.

[0079] The term "polypeptide" refers to a compound composed of a single chain of D-amino acids or L-amino acids or a mixture of D-amino acids and L-amino acids linked by peptide bonds.

[0080] In some embodiments, the polypeptide is longer than 25 amino acids and shorter than 50 amino acids. The term "antigenic polypeptide" includes full-length polypeptides / proteins and immunogenic fragments thereof (immunogenic fragments capable of inducing an immune response against an infective agent). Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. The polypeptide can be a single polypeptide or can be a multimolecular complex, such as a dimer, trimer, or tetramer. The polypeptide can also comprise a single-chain polypeptide or a multi-chain polypeptide, such as an antibody or insulin, and can associate or be linked. Most commonly, disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also apply to an amino acid polymer in which at least one amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid.

[0081] A "polypeptide variant" is a molecule whose amino acid sequence is different from the native sequence or a reference sequence. Compared to the native sequence or reference sequence, the amino acid sequence variant can have substitutions, deletions, insertions, or combinations of any two or three of the foregoing at certain positions within the amino acid sequence. Generally, the variant has at least 50% identity with the native sequence or reference sequence. In some embodiments, the variant shares at least 80% identity or at least 90% identity with the native sequence or reference sequence.

[0082] In some embodiments, "variant mimics" are provided. A "variant mimic" contains at least one amino acid that will mimic an activated sequence. For example, glutamic acid can act as a mimic of phospho-threonine and / or phospho-serine. Alternatively, the variant mimic can inactivate a product containing the mimic or produce an inactivated product containing the mimic. For example, phenylalanine can serve as an inactivating substitution for tyrosine, or alanine can serve as an inactivating substitution for serine.

[0083] An "ortholog" refers to a gene in different species that has evolved from a common ancestral gene through speciation. Generally, orthologs retain the same function during evolution. Identification of orthologs is important for reliable prediction of gene function in newly sequenced genomes.

[0084] An "analog" is intended to include polypeptide variants that differ due to one or more amino acid alterations, such as substitutions, additions, or deletions of amino acid residues that still retain one or more of the properties of the parent polypeptide or starting polypeptide.

[0085] The present disclosure provides several types of compositions based on polynucleotides or polypeptides (including variants and derivatives). These compositions include, for example, substitutional, insertional, deletion, and covalent variants and derivatives. The term "derivative" is synonymous with the term "variant" and generally refers to a molecule that is modified and / or altered in any way relative to a reference molecule or starting molecule.

[0086] Accordingly, polynucleotides encoding peptides or polypeptides that contain substitutions, insertions, and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly the polypeptide sequences disclosed herein, are included within the scope of the present disclosure. For example, a sequence tag or an amino acid such as one or more lysines can be added to a peptide sequence (e.g., at the N-terminus or C-terminus). The sequence tag can be used for peptide detection, purification, or localization. Lysine can be used to increase peptide solubility or to permit biotinylation. Alternatively, amino acid residues located at the carboxyl-terminal region and the amino-terminal region of the amino acid sequence of a peptide or protein can optionally be deleted, thereby providing a truncated sequence. Certain amino acids (e.g., C-terminal residues or N-terminal residues) can alternatively be deleted depending on the use of the sequence, such as for expressing a sequence as part of a larger sequence that is soluble or linked to a solid support.

[0087] When referring to a polypeptide, a "substitutional variant" is a variant in which at least one amino acid residue in the native or starting sequence is removed and a different amino acid is inserted in its place at the same position. The substitution can be single, in which case only one amino acid in the molecule is replaced, or the substitution can be multiple, in which case two or more (e.g., 3, 4, or 5) amino acids in the same polypeptide molecule are replaced.

[0088] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid normally present in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitutions include the replacement of one nonpolar (hydrophobic) residue with another nonpolar residue such as isoleucine, valine, or leucine. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophilic) residue with another polar residue, such as a substitution between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the replacement of one basic residue with another basic residue such as lysine, arginine, or histidine or the replacement of one acidic residue with another acidic residue such as aspartic acid or glutamic acid are additional examples of conservative substitutions. Examples of non-conservative substitutions include the replacement of a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine with a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine and / or the replacement of a nonpolar residue with a polar residue.

[0089] When referring to a polypeptide or polynucleotide, a "feature" is defined, respectively, as a distinct amino acid sequence-based or nucleotide-based component of the molecule. Features of a polypeptide encoded by a polynucleotide include surface presentation, local conformational shape, fold, loop, half-loop, domain, half-domain, site, terminus, and any combination thereof.

[0090] As used herein, when referring to a polypeptide, the term "domain" refers to a motif of a polypeptide that has one or more identifiable structural or functional features or characteristics (e.g., binding ability, serving as a site for protein-protein interaction).

[0091] As used herein, when referring to a polypeptide, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" because it pertains to amino acid-based embodiments. As used herein, when referring to a polynucleotide, the term "site" is used synonymously with "nucleotide" because it pertains to nucleotide-based embodiments. A site represents a position within a peptide or polypeptide or polynucleotide that can be modified, manipulated, altered, derivatized, or varied within a polypeptide- or polynucleotide-based molecule.

[0092] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the polypeptides of interest. For example, provided herein are any protein fragments (meaning polypeptide sequences that are at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein that are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or longer than 100 amino acids in length. In another example, any protein can be utilized in accordance with the present disclosure that includes stretches of 20, 30, 40, 50, or 100 (consecutive) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein. In some embodiments, the polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided herein or cited herein. In another example, any protein can be utilized in accordance with the present disclosure that includes stretches of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has stretches of 5, 10, 15, 20, 25, or 30 amino acids that are less than 80%, 75%, 70%, 65% to 60% identical to any of the sequences described herein.

[0093] The polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or a reference polynucleotide), e.g., with molecules described in the art (e.g., engineered or designed molecules or wild-type molecules). The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between two sequences, as determined by the number of matches between strings composed of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gap alignments (if any) are resolved by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of related polypeptides can be readily calculated by known methods. "Percent identity" when applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or nucleic acid sequence of the second sequence after aligning the sequences and introducing gaps as needed to achieve the maximum percentage of identity. Methods and computer programs for alignment are well known in the art. Identity depends on the calculation of percent identity, but its value may vary due to gaps and penalties introduced in the calculation. Generally, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity with the particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include the tools of the BLAST suite (Stephen F. Altschul et al. (1997). "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25: 3389-3402).Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F. and Waterman, M.S. (1981) "Identification of common molecular subsequences." Journal of Molecular Biology (J. Mol. Biol.) 147: 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. and Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins" Journal of Molecular Biology 48: 443-453). Recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to produce global alignments of nucleotide and protein sequences faster than other optimal global alignment algorithms, including the Needleman-Wunsch algorithm. Other tools are described in this article, specifically in the definition of "identity" below.

[0094] As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymeric molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are said to be homologous. Homology is a qualitative term describing the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term defining the degree of sequence match between two compared sequences. In some embodiments, polymeric molecules are considered to be "homologous" to each other where the sequences of the polymeric molecules are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (e.g., polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered to be homologous where the polypeptides encoded by the two polynucleotide sequences are at least 50%, 60%, 70%, 80%, 90%, 95% or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous where the proteins are at least 50%, 60%, 70%, 80% or 90% identical for at least one stretch of at least 20 amino acids.

[0095] Homology implies that the compared sequences from a common source diverged during evolution. The term "homolog" refers to a first amino acid sequence or nucleic acid sequence (e.g., a gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by inheritance from a common ancestral sequence. The term "homolog" can apply to the relationship between genes and / or proteins separated by a speciation event, or to the relationship between genes and / or proteins separated by a gene duplication event. "Orthologs" are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function during evolution. "Paralogs" are genes (or proteins) that are related due to duplication within a genome. Orthologs retain the same function during evolution, while paralogs evolve new functions, even if these functions are related to the original function.

[0096] The term "identity" refers to the overall relatedness between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the percent identity of two polynucleotide molecules can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second nucleic acid sequences, and for comparison purposes, non-identical sequences can be ignored). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. When the position in the first sequence is occupied by a nucleotide that is the same as the nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, which takes into account the number of gaps and the length of each gap, which are introduced to achieve an optimal alignment of the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using mathematical algorithms.For example, the percent identity between two nucleic acid sequences can be determined using the following methods: Computational Molecular Biology, Lesk, A.M. Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. Ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G. Eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. Eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, the method disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math. 48:1073 (1988); which is incorporated herein by reference. Methods for determining identity are incorporated into publicly available computer programs.Exemplary computer programs for determining homology between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., Journal of Molecular Biology 215, 403 (1990)).

[0097] The term "complementary" refers to the topological compatibility or matching of the interacting surfaces of a probe molecule and its target. Thus, a target and its probe can be described as complementary, and furthermore, the contact surface features are complementary to each other.

[0098] The term "hybridization" refers to the process of establishing non-covalent sequence-specific interactions between two or more complementary nucleic acid strands to form a single hybrid, which in the case of two strands is called a duplex.

[0099] The term "annealing" refers to the process by which a single-stranded nucleic acid sequence pairs with a complementary sequence through hydrogen bonds to form a double-stranded nucleic acid sequence, which includes the process of reformation (renaturation) of complementary strands separated by heat (thermal denaturation).

[0100] The term "melting" refers to the denaturation of a double-stranded nucleic acid sequence due to high temperature, resulting in the separation of the double-stranded into two single strands due to the breakage of the hydrogen bonds between the double strands.

[0101] The term "target" refers to a molecule that has an affinity for a given probe. The target can be a naturally occurring molecule or an artificial molecule. In addition, the target can be taken in its unaltered state or as an aggregate with other species.

[0102] The term "promoter" or "regulatory element" refers to a region or sequence determinant that is located upstream or downstream of the start of transcription and that participates in the recognition of RNA polymerase and its binding to other proteins to initiate transcription. A promoter need not be of bacterial origin; for example, promoters derived from viruses or other organisms can be used in the compositions, systems, or methods described herein. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRESs), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., (1990). Regulatory elements include elements that direct constitutive expression of a nucleotide sequence in many types of host cells and elements that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest such as muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner (such as a cell cycle-dependent or developmental stage-dependent manner), which expression can be or can not be tissue-specific or cell type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter.The term "regulatory element" also encompasses enhancer elements such as the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA, Vol. 78(3), pp. 1527-31, 1981). Those skilled in the art will appreciate that the design of an expression vector can depend on, for example, the choice of host cell to be transformed, the desired level of expression, etc.

[0103] The term "recombinant" refers to a human-manipulated nucleic acid (e.g., polynucleotide) or a copy or complement of a human-manipulated nucleic acid (e.g., polynucleotide), or, in the case of a protein (i.e., "recombinant protein"), a protein encoded by a recombinant nucleic acid (e.g., polynucleotide). In an embodiment, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) can include a promoter heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) or Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette can contain nucleic acids (e.g., polynucleotides) combined in such a way that the nucleic acids (e.g., polynucleotides) are highly unlikely to exist in nature. For example, human-manipulated restriction sites or plasmid vector sequences can flank the promoter or separate the promoter from the second nucleic acid (e.g., polynucleotide). Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be manipulated in a variety of ways and are not limited to the above examples.

[0104] The term "expression cassette" refers to a nucleic acid construct that, when introduced into a host cell, causes transcription and / or translation of RNA or a polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) can include a promoter heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) or Current Protocols in Molecular Biology, Volumes 1 - 3, John Wiley & Sons (1994 - 1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g., a polynucleotide) can include a terminator heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In some embodiments, an expression cassette comprises a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) and a terminator operably linked to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In some embodiments, an expression cassette comprises an endogenous promoter. In some embodiments, an expression cassette comprises an endogenous terminator. In some embodiments, an expression cassette comprises a synthetic (or non - native) promoter. In some embodiments, an expression cassette comprises a synthetic (or non - native) terminator.

[0105] A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, if DNA for a pre - sequence or secretory leader sequence is expressed as a pre - protein that participates in the secretion of a polypeptide, it is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to the sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to the coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous to each other and, in the case of a secretory leader sequence, are continuous and in reading frame. However, operably linked nucleic acids (e.g., an enhancer and a coding sequence) need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or adaptors are used according to conventional practice. In embodiments, a promoter is operably linked to a coding sequence when the promoter is capable of affecting (regulating relative to the absence of the promoter) the expression of a protein from the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

[0106] The term "nucleobase" refers to the portion of a nucleotide that has Watson / Crick base-pairing functionality. The most common naturally occurring nucleobases, namely adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), have hydrogen bonding functionality that enables one nucleic acid strand to bind to another nucleic acid strand in a sequence-specific manner.

[0107] A nucleic acid sequence is "heterologous" to a second nucleic acid sequence if the nucleic acid sequence and the second nucleic acid sequence are from foreign species or, in the case where they are from the same species, are modified by human action relative to their original forms. For example, a heterologous promoter (or heterologous 5' untranslated region (5'UTR)) operably linked to a coding sequence refers to a coding sequence from a species different from the species from which the promoter is derived or, in the case where they are from the same species, refers to a coding sequence different from the naturally occurring allelic variant (e.g., a 5'UTR or 3'UTR from a different gene is operably linked to a nucleic acid encoding a costimulatory molecule).

[0108] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass and fragments of such antibodies, provided that they exhibit the desired antagonistic activity.

[0109] The disclosed monoclonal antibodies can be prepared using any procedure for producing monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method as described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0110] Monoclonal antibodies can also be prepared by recombinant DNA methods. The DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of murine antibodies). Antibody or active antibody fragment libraries can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.).

[0111] In vitro methods are also suitable for preparing monovalent antibodies. Conventional techniques known in the art can be used to accomplish digestion of the antibody to produce its fragments, particularly Fab fragments. For example, digestion can be carried out using papain. Examples of papain digestion are described in WO 94 / 29348 published on December 22, 1994 and U.S. Patent No. 4,342,566. Papain digestion of an antibody typically produces two identical antigen-binding fragments (referred to as Fab fragments) and a residual Fc fragment, each of the two identical antigen-binding fragments having a single antigen-binding site. Pepsin treatment yields fragments that have two antigen-combining sites and are still capable of cross-linking antigens.

[0112] As used herein, the term “antibody or its antigen-binding fragment” or “antibody or its fragment” encompasses chimeric and hybrid antibodies and fragments having dual or multiple antigen or epitope specificities, such as F(ab')2, Fab', Fab, Fv, sFv, scFv, etc., including hybrid fragments. Thus, fragments of an antibody that retain the ability to bind to a particular antigen are provided. For example, fragments of an antibody that retain binding activity are included within the meaning of the term “antibody or its antigen-binding fragment”. Such antibodies and fragments can be prepared by techniques known in the art and can be screened for specificity and activity according to the methods shown in the examples and the general methods for generating antibodies and screening antibodies for specificity and activity (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York, (1988)).

[0113] Also included within the meaning of “antibody or its antigen-binding fragment” are conjugates of antibody fragments with antigen-binding proteins (single-chain antibodies). Also included within the meaning of “antibody or its antigen-binding fragment” are immunoglobulin single variable domains, e.g., nanobodies.

[0114] Whether attached to other sequences or not, the fragment may also include insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide some additional properties, such as removing / adding amino acids capable of disulfide bonding, increasing its biological lifespan, altering its secretion characteristics, etc. In any case, the antibody or antibody fragment must have biological activity characteristics, such as specific binding to its cognate antigen. The functional or active regions of an antibody or antibody fragment can be identified by mutagenizing specific regions of the protein followed by expressing the polypeptide and testing the expressed polypeptide. Such methods are obvious to those skilled in the art and may include site-directed mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0115] As used herein, the term "antibody" or "antibodies" may also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., antibodies derived from mice, rats or rabbits) are immunogenic in humans and thus, when administered to humans, can elicit an unwanted immune response. Accordingly, human antibodies or humanized antibodies are used in the methods to reduce the chance that the antibody administered to a human will elicit an unwanted immune response.

[0116] As used herein, "immune effector cell" refers to a cell capable of binding to an antigen or peptide and mediating an immune response. These cells include, but are not limited to, T cells (including CD4+ and CD8+ T cells), B cells, monocytes, macrophages, NK cells and cytotoxic T lymphocytes (CTL).

[0117] Chemical definition

[0118] As used herein, it is contemplated that the term "substituted" includes all permissible substituents of an organic compound. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of an organic compound. Illustrative substituents include, for example, those described below. For a suitable organic compound, the permissible substituents can be one or more and can be the same or different. For the purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of the organic compounds described herein that satisfy the valence of the heteroatom. The present disclosure is not intended to be limited in any way by the permissible substituents of organic compounds. Also, the terms "substituted" or "substituted with" include the implicit proviso that such substitution is consistent with the permissible valences of the substituted atoms and substituents and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0119] "Z 1 ", "Z 2 ", "Z 3 ", and "Z 4 " are used herein as general symbols to represent various specific substituents. These symbols can be any substituent, not limited to the substituents disclosed herein, and when these symbols are defined as certain substituents in one instance, they can be defined as some other substituents in another instance.

[0120] As used herein, the term "aliphatic" refers to non-aromatic hydrocarbon groups and includes branched and unbranched groups, alkyl, alkenyl, or alkynyl.

[0121] As used herein, the term "alkyl" is a branched or unbranched saturated hydrocarbon group having from 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. The alkyl can also be substituted or unsubstituted. The aryl can be substituted with one or more groups including but not limited to the following: alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0122] Throughout this specification, the term "alkyl" is generally used to refer to both unsubstituted alkyl and substituted alkyl; however, substituted alkyl is also specifically referred to herein by identifying specific substituents on the alkyl. For example, the term "haloalkyl" specifically refers to alkyl substituted with one or more halides, such as fluorine, chlorine, bromine, or iodine. The term "alkoxyalkyl" specifically refers to alkyl substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to alkyl substituted with one or more amino groups, etc., as described below. When the term "alkyl" is used in one context and a specific term such as "alkyl alcohol" is used in another context, this is not intended to imply that the term "alkyl" does not also refer to specific terms such as "alkyl alcohol".

[0123] This practice is also used for other groups described herein. That is, although terms such as "cycloalkyl" refer to both unsubstituted cycloalkyl moieties and substituted cycloalkyl moieties, the additionally described substituted moieties can be specifically identified herein; for example, a specific substituted cycloalkyl can be referred to as, for example, "alkylcycloalkyl". Similarly, a substituted alkoxy can be specifically referred to as, for example, "haloalkoxy", and a specific substituted alkenyl can be, for example, "alkenyl alcohol", etc. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" does not mean to imply that the general term does not also include the specific term.

[0124] As used herein, the term "alkoxy" is alkyl bonded through a single terminal ether bond; that is, "alkoxy" can be defined as -OZ 1 , where Z 1 is alkyl as defined above.

[0125] As used herein, the term "alkenyl" is a hydrocarbon group having from 2 to 24 carbon atoms and having at least one carbon-carbon double bond in its structure. Asymmetric structures such as (Z 1 Z 2 )C=C(Z 3 Z 4 ) are intended to include both E isomers and Z isomers. This can be inferred in the structural formulas herein where there are asymmetric alkenes, or it can be explicitly indicated by the bond symbol C=C. Alkenyl can be substituted with one or more groups including but not limited to the following: alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0126] As used herein, the term "alkynyl" is a hydrocarbyl group having from 2 to 24 carbon atoms and having at least one carbon-carbon triple bond in its structure. The alkynyl group may be substituted with one or more groups including but not limited to the following: alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol.

[0127] As used herein, the term "aryl" is a group containing any carbon-based aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, phenoxyphenyl, etc. The term "heteroaryl" is defined as a group containing an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include but are not limited to nitrogen, oxygen, sulfur and phosphorus. The term "non-heteroaryl", which is also included within the term "aryl", is defined as a group containing an aromatic group that does not contain a heteroatom. The aryl or heteroaryl group may be substituted or unsubstituted. The aryl or heteroaryl group may be substituted with one or more groups including but not limited to the following: alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol. The term "biaryl" is a specific type of aryl and is included within the definition of "aryl". A biaryl refers to two aryl groups joined together by a fused ring structure as in naphthalene or attached by one or more carbon-carbon bonds as in biphenyl.

[0128] As used herein, the term "cycloalkyl" is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "heterocycloalkyl" is a cycloalkyl group as defined above in which at least one of the carbon atoms of the ring is replaced by a heteroatom such as but not limited to nitrogen, oxygen, sulfur or phosphorus. The cycloalkyl and heterocycloalkyl groups may be substituted or unsubstituted. The cycloalkyl and heterocycloalkyl groups may be substituted with one or more groups including but not limited to the following: alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol.

[0129] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bond, i.e., C═C. Examples of cycloalkenyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl as defined above and is included within the meaning of the term "cycloalkenyl", where in the case of cycloalkenyl, at least one of the carbon atoms in the ring is replaced by a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl may be substituted or unsubstituted. Cycloalkenyl and heterocycloalkenyl may be substituted by one or more groups as described herein including, but not limited to, the following: alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0130] The term "cyclic group" is used herein to refer to an aryl, non-aryl (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl), or both. A cyclic group has one or more ring systems that may be substituted or unsubstituted. A cyclic group may contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0131] As used herein, the term "aldehyde" is represented by the formula -C(O)H. Throughout this specification, "C(O)" or "CO" is a shorthand notation for C═O.

[0132] As used herein, the term "amine" or "amino" is represented by the formula -NZ 1 Z 2 where Z 1 and Z 2 may each be a substituent group as described herein, such as hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0133] As used herein, the term "carboxylic acid" is represented by the formula -C(O)OH. As used herein, the term "carboxylic ester" or "carboxy" is represented by the formula -C(O)O - as shown.

[0134] As used herein, the term "ester" is represented by the formula -OC(O)Z 1 or -C(O)OZ 1 where Z 1 may be an alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0135] As used herein, the term "ether" is represented by the formula Z 1 OZ2 is represented, where Z 1 and Z 2 can each independently be an alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl as described above.

[0136] As used herein, the term "ketone" is represented by the formula Z 1 C(O)Z 2 is represented, where Z 1 and Z 2 can each independently be an alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl as described above.

[0137] As used herein, the term "halide" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0138] As used herein, the term "hydroxyl" is represented by the formula -OH.

[0139] As used herein, the term "nitro" is represented by the formula -NO2.

[0140] As used herein, the term "silyl" is represented by the formula -SiZ 1 Z 2 Z 3 is represented, where Z 1 , Z 2 and Z 3 can each independently be hydrogen, an alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl as described above.

[0141] The term "sulfonyl" is used herein to refer to a sulfo-oxo group represented by the formula -S(O)2Z 1 is represented, where Z 1 can be hydrogen, an alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl as described above.

[0142] As used herein, the term "sulfonylamino" or "sulfonamide" is represented by the formula -S(O)2NH-.

[0143] The term "phosphonyl" is used herein to refer to a phospho-oxo group represented by the formula -P(O)(OZ 1 )2, where Z 1 can be hydrogen, an alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl as described above.

[0144] As used herein, the term "thiol" is represented by the formula -SH.

[0145] As used herein, the term "thio" is represented by the formula -S-.

[0146] As used herein, "R 1 ", "R 2 ", "R 3 ", "R n " (where n is an integer) can independently have one or more of the groups listed above. For example, if R 1 is a straight-chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an amine group, an alkyl group, a halide, etc. Depending on the groups selected, the first group can be incorporated within the second group, or alternatively, the first group can be a side group of the second group (i.e., attached to the second group). For example, in the case of the phrase "alkyl group containing an amino group", the amino group can be incorporated within the main chain of the alkyl group. Alternatively, the amino group can be attached to the main chain of the alkyl group. The nature of the groups selected will determine whether the first group is embedded within the second group or attached to the second group.

[0147] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines rather than wedges or dashes contemplates each possible isomer (e.g., each enantiomer, diastereomer, and meso compound) and mixtures of isomers, such as a racemic mixture or a scalemic mixture.

[0148] Now, specific aspects of the disclosed materials, compounds, compositions, articles, and methods will be referred to in detail. Examples of the above-disclosed content are illustrated in the accompanying examples and figures.

[0149] Compositions

[0150] Compositions are described herein that include nucleic acid sequences (e.g., mRNA) encoding an infectious agent antigen polypeptide and nucleic acid sequences (e.g., mRNA) encoding at least one universal T cell epitope, as well as compositions that include nucleic acid sequences (e.g., mRNA) encoding an infectious agent antigen polypeptide and at least one universal T cell epitope.

[0151] The compositions described herein can include multiple nucleic acid sequences (e.g., mRNA) each encoding a single infectious agent antigen polypeptide and multiple nucleic acid sequences (e.g., mRNA) each encoding a single universal T cell epitope, as well as compositions that include a single nucleic acid sequence (e.g., mRNA) encoding one or more infectious agent antigen polypeptides and a single nucleic acid sequence (e.g., mRNA) encoding one or more universal T cell epitopes.

[0152] The compositions described herein can include multiple nucleic acid sequences (e.g., mRNA) each encoding a single pathogen antigen polypeptide and a single universal T cell epitope, as well as compositions including a single nucleic acid sequence (e.g., mRNA) encoding one or more pathogen antigen polypeptides and one or more universal T cell epitopes.

[0153] Accordingly, a composition comprising a nucleic acid sequence encoding a pathogen antigen polypeptide and at least one universal T cell epitope encompasses a composition comprising nucleic acid sequences encoding a first pathogen antigen polypeptide, a second pathogen antigen polypeptide, a first universal T cell epitope, and a second universal T cell epitope. In some embodiments, a composition comprising a nucleic acid sequence encoding a pathogen antigen polypeptide and a nucleic acid sequence encoding at least one universal T cell epitope encompasses a composition comprising a first nucleic acid sequence encoding a first pathogen antigen polypeptide, a second nucleic acid sequence encoding a second pathogen antigen polypeptide, a third nucleic acid sequence encoding a first universal T cell epitope, and a fourth nucleic acid sequence encoding a second universal T cell epitope.

[0154] In some embodiments, the compositions described herein include 2 - 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleic acid sequences (e.g., mRNA) each having an open reading frame and encoding different pathogen antigen polypeptides (or a single nucleic acid sequence (e.g., mRNA) encoding 2 - 10 or more different pathogen antigen polypeptides). In some embodiments, the compositions described herein include 2 - 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleic acid sequences (e.g., mRNA) each having an open reading frame and encoding different universal T cell epitopes (or a single nucleic acid sequence (e.g., mRNA) encoding 2 - 10 or more different universal T cell epitopes).

[0155] In some embodiments, a nucleic acid sequence (e.g., mRNA) encoding a pathogen antigen polypeptide can elicit an antibody response in a subject. In some embodiments, a nucleic acid sequence (e.g., mRNA) encoding a pathogen antigen polypeptide can elicit a broad T cell response in a subject. In some embodiments, a nucleic acid sequence (e.g., mRNA) encoding a pathogen antigen polypeptide can elicit both an antibody response and a T cell response in a subject.

[0156] In some embodiments, a nucleic acid sequence encoding a pathogen antigen polypeptide (e.g., mRNA) can elicit a cellular immune response, a humoral immune response, or a combination thereof. In some embodiments, a nucleic acid sequence encoding a pathogen antigen polypeptide (e.g., mRNA) can elicit a cellular immune response, a humoral immune response, or a combination thereof without the risk of possible insertional mutagenesis. In some embodiments, a universal T cell epitope can provide broader protection against pathogen variants.

[0157] In some embodiments, the composition can further comprise a pharmaceutically acceptable carrier. In some aspects, a pharmaceutical composition is disclosed herein that can comprise a nucleic acid sequence encoding a pathogen antigen polypeptide (e.g., mRNA), a nucleic acid sequence encoding at least one universal T cell epitope (e.g., mRNA), and a pharmaceutically acceptable carrier. In some aspects, a pharmaceutical composition is disclosed herein that comprises a nucleic acid sequence encoding a pathogen antigen polypeptide and at least one T cell epitope (e.g., mRNA) and a pharmaceutically acceptable carrier. In some embodiments, the composition can be a nanoparticle, a lipid nanoparticle dispersion, a liposome formulation, a lipid emulsion, a vaccine, a vector, or any combination thereof.

[0158] In some embodiments, the nucleic acid sequence can include DNA, RNA, any combination thereof (e.g., plasmid DNA, minicircle DNA, minimally immunogenic defined gene expression (MIDGE) and Doggybone, messenger RNA (mRNA), circular (cirRNA), self-amplifying RNA (saRNA, also referred to as SAM), or DNA-initiated SAM (DLSAM)). In some embodiments, the composition can include plasmid DNA encoding a pathogen antigen polypeptide and a universal T cell epitope.

[0159] Signal peptide

[0160] In some embodiments, the antigen polypeptide encoded by a nucleic acid sequence (e.g., mRNA) comprises a signal peptide. In some embodiments, the T cell epitope encoded by a nucleic acid sequence (e.g., mRNA) comprises a signal peptide. Signal peptides, which typically comprise the N-terminal 15 - 60 amino acids of a protein, are generally required for transmembrane translocation in the secretory pathway and thus generally govern entry of most proteins in eukaryotes and prokaryotes into the secretory pathway. Signal peptides generally include three regions: an N-terminal region of variable length that typically contains positively charged amino acids; a hydrophobic region; and a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates translocation of the growing peptide chain across the membrane for processing. ER processing yields a mature protein, where the signal peptide is typically cleaved from the preprotein by an ER-resident signal peptidase of the host cell, or it remains uncleaved and serves as a membrane anchor. Signal peptides can also facilitate protein targeting to the cell membrane. However, signal peptides are not responsible for the ultimate destination of the mature protein. Secretory proteins lacking additional address tags in their sequence are secreted by default into the external environment. During recent years, a more sophisticated view of signal peptides has evolved, which suggests that the functions and immunodominance of certain signal peptides are more diverse than previously anticipated.

[0161] The compositions described herein can include, for example, a nucleic acid sequence (e.g., mRNA) encoding an artificial signal peptide, in which case the signal peptide coding sequence is operably linked to the coding sequence of an antigen polypeptide, at least one universal T cell epitope, or any combination thereof, and is in-frame with the coding sequence. Thus, in some embodiments, the compositions of the present disclosure produce an antigen polypeptide, at least one universal T cell epitope, or any combination thereof, comprising an antigen polypeptide fused to a signal peptide. In some embodiments, the signal peptide is fused to the N-terminus of an antigen polypeptide, at least one universal T cell epitope, or any combination thereof. In some embodiments, the signal peptide is fused to the C-terminus of an antigen polypeptide, at least one universal T cell epitope, or any combination thereof.

[0162] In some embodiments, the signal peptide fused to the antigen polypeptide, at least one universal T cell epitope, or any combination thereof, is an artificial signal peptide. In some embodiments, the artificial signal peptide fused to the antigen polypeptide encoded by the composition is obtained from an immunoglobulin (e.g., IgE signal peptide or IgG signal peptide). In some embodiments, the signal peptide fused to the antigen polypeptide encoded by the composition, at least one universal T cell epitope, or any combination thereof, is the Ig heavy chain epsilon-1 signal peptide (IgE HC SP) having the sequence: MDWTWILFLVAAATRVHS (SEQ ID NO:16). In some embodiments, the signal peptide fused to the antigen polypeptide encoded by the composition, at least one universal T cell epitope, or any combination thereof, is the IgGk chain V-III region HAH signal peptide (IgGk SP) having the sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO:15). In some embodiments, the signal peptide is selected from: Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO:17), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO:18), and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ ID NO:19).

[0163] In some embodiments, the antigen polypeptide encoded by the composition comprises an amino acid sequence identified by any one of SEQ ID NOs: 5-8, 12-13, 24-34, 47-50, or 54-56 fused to a signal peptide identified by any one of SEQ ID NOs: 15-19. The examples disclosed herein are not intended to be limiting, and any signal peptide known in the art for promoting targeting of a protein to the ER for processing and / or targeting of a protein to the cell membrane can be used in accordance with the present disclosure.

[0164] The length of the signal peptide can be 15 - 60 amino acids. For example, the length of the signal peptide can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids. In some embodiments, the length of the signal peptide is 20 - 60, 25 - 60, 30 - 60, 35 - 60, 40 - 60, 45 - 60, 50 - 60, 55 - 60, 15 - 55, 20 - 55, 25 - 55, 30 - 55, 35 - 55, 40 - 55, 45 - 55, 50 - 55, 15 - 50, 20 - 50, 25 - 50, 30 - 50, 35 - 50, 40 - 50, 45 - 50, 15 - 45, 20 - 45, 25 - 45, 30 - 45, 35 - 45, 40 - 45, 15 - 40, 20 - 40, 25 - 40, 30 - 40, 35 - 40, 15 - 35, 20 - 35, 25 - 35, 30 - 35, 15 - 30, 20 - 30, 25 - 30, 15 - 25, 20 - 25 or 15 - 20 amino acids.

[0165] The signal peptide is typically cleaved from the nascent polypeptide at the cleavage junction during ER processing. The mature antigen polypeptide produced by the compositions of the present disclosure typically does not contain a signal peptide.

[0166] Chemical modification

[0167] In some embodiments, the antigen polypeptide encoded by a nucleic acid sequence (e.g., mRNA) comprises at least one chemical modification. In some embodiments, the T cell epitope encoded by a nucleic acid sequence (e.g., mRNA) comprises at least one chemical modification.

[0168] The terms “chemical modification” and “chemically modified” refer to a modification made to a ribonucleoside or deoxyribonucleoside of adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) in at least one of its position, pattern, percentage or population. Generally, these terms do not refer to ribonucleotide modifications in the naturally occurring 5′ - terminal mRNA cap moiety. In terms of polypeptides, the term “modification” refers to a modification relative to the canonical set of 20 amino acids. As provided herein, a polypeptide is also considered to be “modified” because it contains amino acid substitutions, insertions or a combination of substitutions and insertions.

[0169] In some embodiments, a nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises various (more than one) different modifications. In some embodiments, a particular region of the polynucleotide contains one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or an organism exhibits reduced degradation in the cell or the organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or an organism can exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or the organism, respectively.

[0170] Modifications of the polynucleotide include, but are not limited to, those described herein. A nucleic acid sequence (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) can contain naturally occurring modifications, non-naturally occurring modifications, or the polynucleotide can contain a combination of naturally occurring and non-naturally occurring modifications. The polynucleotide can include, for example, any useful modification to the sugar, nucleobase, or internucleoside bond (e.g., to a linking phosphate, to a phosphodiester bond, or to a phosphodiester backbone).

[0171] In some embodiments, a nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) contains non-natural modified nucleotides introduced during or after the synthesis of the polynucleotide to achieve desired functions or properties. The modifications can be present on the internucleoside bond, purine or pyrimidine base, or sugar. The modifications can be introduced by chemical synthesis or with a polymerase at the end of the chain or at any other position in the chain. Any region of the polynucleotide can be chemically modified.

[0172] The present disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA polynucleotides, such as mRNA polynucleotides). A "nucleoside" refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method, such as chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A polynucleotide can contain one or more regions composed of linked nucleosides. Such regions can have variable backbone bonds. The bonds can be standard phosphodiester bonds, in which case the polynucleotide will contain regions composed of nucleotides.

[0173] Modified nucleobase pairings include not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides that contain non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between non-standard and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of bases / sugars or linkers can be incorporated into the polynucleotides of the present disclosure.

[0174] Modifications of nucleic acids (e.g., RNA polynucleotides such as mRNA polynucleotides) that can be used in the compositions of the present disclosure include, but are not limited to, the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxy-norvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1-methyl-adenosine; N6,N6(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2(aminopropyl)adenine; 2(methylthio)N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy-ATP; 2'-azido-2'-deoxy-ATP; 2'-deoxy-2'-α-aminoadenosine TP; 2'-deoxy-2'-α-azidoadenosine TP; 6(alkyl)adenine; 6(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7(deaza)adenine; 8(alkenyl)adenine; 8(alkynyl)adenine; 8(amino)adenine; 8(thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; azidoadenine; deazaadenine; N6(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-azido-adenosine; 7-methyladenine; 1-deazaadenosine TP; 2'fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-α-ethynyladenosine TP; 2-aminoadenine; 2-aminoadenosine TP; 2-amino-ATP; 2'-α-trifluoromethyladenosine TP; 2-azidoadenosine TP;2'-β-ethynyladenosine TP; 2-bromoadenosine TP; 2'-β-trifluoromethyladenosine TP; 2-chloroadenosine TP; 2'-deoxy-2',2'-difluoroadenosine TP; 2'-deoxy-2'-α-mercaptoadenosine TP; 2'-deoxy-2'-α-thiomethoxyadenosine TP; 2'-deoxy-2'-β-aminoadenosine TP; 2'-deoxy-2'-β-azidoadenosine TP; 2'-deoxy-2'-β-bromoadenosine TP; 2'-deoxy-2'-β-chloroadenosine TP; 2'-deoxy-2'-β-fluoroadenosine TP; 2'-deoxy-2'-β-iodoadenosine TP; 2'-deoxy-2'-β-mercaptoadenosine TP; 2'-deoxy-2'-β-thiomethoxyadenosine TP; 2-fluoroadenosine TP; 2-iodoadenosine TP; 2-mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-trifluoromethyladenosine TP; 3-deaza-3-bromoadenosine TP; 3-deaza-3-chloroadenosine TP; 3-deaza-3-fluoroadenosine TP; 3-deaza-3-iodoadenosine TP; 3-deazaadenosine TP; 4'-azidoadenosine TP; 4'-carbocyclic adenosine TP; 4'-ethynyladenosine TP; 5'-homoadenosine TP; 8-aza-ATP; 8-bromo-adenosine TP; 8-trifluoromethyladenosine TP; 9-deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine; 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-azacytidine; pseudoisocytidine; pyrrolo-cytidine; α-thio-cytidine; 2-(thio)cytosine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'-deoxy-2'-α-aminocytidine TP; 2'-deoxy-2'-α-azidocytidine TP; 3(deaza)5(azido)cytosine; 3(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza)5(azido)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5(halo)cytosine; 5(methyl)cytosine; 5(propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine;5-Propynylcytosine; 6-(Azido)cytosine; 6-Aza-cytidine; Azacytosine; Deazacytosine; N4(Acetyl)cytosine; 1-Methyl-1-deaza-pseudoisocytidine; 1-Methyl-pseudoisocytidine; 2-Methoxy-5-methyl-cytidine; 2-Methoxy-cytidine; 2-Thio-5-methyl-cytidine; 4-Methoxy-1-methyl-pseudoisocytidine; 4-Methoxy-pseudoisocytidine; 4-Thio-1-methyl-1-deaza-pseudoisocytidine; 4-Thio-1-methyl-pseudoisocytidine; 4-Thio-pseudoisocytidine; 5-Aza-zebularine; 5-Methyl-zebularine; Pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-Anhydro-cytidine TP Hydrochloride; 2'-Fluoro-N4-Bz-cytidine TP; 2'-Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP; 2'O-Methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine TP; 2'-Deoxy-2'-a-mercaptocytidine TP; 2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP; 2'-Deoxy-2'-b-thiomethoxycytidine TP; 2'-O-Methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5'-Homocytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-cytidine TP; N4-Aminocytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-Methylguanosine; N2,2'-O-Dimethylguanosine; N2-Methylguanosine; Wyosine; 1,2'-O-Dimethylguanosine; 1-Methylguanosine; 2'-O-Methylguanosine; 2'-O-Ribosylguanosine (Phosphate); 2'-O-Methylguanosine; 2'-O-Ribosylguanosine (Phosphate); 7-Aminomethyl-7-deazaguanosine; 7-Cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-Dimethylguanosine; N2,N2,2'-O-Trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deazaguanosine; 8-oxoguanosine; N1-methylguanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2'-amino-2'-deoxy-GTP; 2'-azido-2'-deoxy-GTP; 2'-deoxy-2'-a-aminoguanosine TP; 2'-deoxy-2'-a-azidoguanosine TP; 6(methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methylguanosine; 7(alkyl)guanine; 7(deaza)guanine; 7(methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8(alkyl)guanine; 8(alkynyl)guanine; 8(halo)guanine; 8(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deazaguanine; N(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxyguanosine; 6-thio-7-deaza-8-azaguanosine; 6-thio-7-deazaguanosine; 6-thio-7-methylguanosine; 7-deaza-8-azaguanosine; 7-methyl-8-oxoguanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2'fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine TP; 2'-a-ethynylguanosine TP; 2'-a-trifluoromethylguanosine TP; 2'-b-ethynylguanosine TP; 2'-b-trifluoromethylguanosine TP; 2'-deoxy-2',2'-difluoroguanosine TP; 2'-deoxy-2'-a-mercapto-guanosine TP; 2'-deoxy-2'-a-thiomethoxy-guanosine TP; 2'-deoxy-2'-b-aminoguanosine TP; 2'-deoxy-2'-b-azidoguanosine TP; 2'-deoxy-2'-b-bromoguanosine TP; 2'-deoxy-2'-b-chloroguanosine TP; 2'-deoxy-2'-b-fluoroguanosine TP; 2'-deoxy-2'-b-iodoguanosine TP; 2'-deoxy-2'-b-mercapto-guanosine TP; 2'-deoxy-2'-b-thiomethoxy-guanosine TP; 4'-azidoguanosine TP; 4'-carbocyclicguanosine TP; 4'-ethynylguanosine TP; 5'-homo-guanosine TP; 8-bromo-guanosine TP; 9-deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; inosine; 1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O-methylinosine; epoxyqueuosine; galactosyl-queuosine; mannosylqueuosine;D-arabinofuranosyladenine; allylamino-thymidine; azidothymidine; deazathymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-tauromethyl-2-thiouridine; 5-tauromethyluridine; dihydrouridine; pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseudouridine; 1-methyl-pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-O-dimethyluridine; 3-methyl-pseudouridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methyldihydrouridine; 5-hydroxyacetate-uridine TP; 5-hydroxyacetate-methyl-uridine TP; N1-methyl-pseudouridine; uridine 5-hydroxyacetate; uridine 5-hydroxyacetate methyl ester; 3-(3-amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2'-O-methyluridine TP; 5-(isopentenylaminomethyl)uridine TP; 5-propynyluracil; α-thio-uridine; 1(aminoalkylaminocarbonylethynyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethynyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethynyl)-4(thio)pseudouracil; 1(aminoalkylaminocarbonylethynyl)-pseudouracil; 1(aminocarbonylethynyl)-2(thio)-pseudouracil; 1(aminocarbonylethynyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethynyl)-4(thio)pseudouracil; 1(aminocarbonylethynyl)-pseudouracil; 1-substituted 2(thio)-pseudouracil; 1-substituted 2,4-(dithio)pseudouracil; 1-substituted 4(thio)pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylaminocarbonylethynyl)-2-(thio)-pseudouracil;1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine 5'-triphosphate; 1-methyl-pseudouridine 5'-triphosphate; 2(thio)pseudouracil; 2'-deoxyuridine; 2'-fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-guanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azido-deoxyuridine 5'-triphosphate; 2'-O-methylpseudouridine; 2'-deoxyuridine; 2'-fluorouridine; 2'-deoxy-2'-α-aminouridine 5'-triphosphate; 2'-deoxy-2'-α-azidouridine 5'-triphosphate; 2-methylpseudouridine; 3(3-amino-3-carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouridine; 5(1,3-diazol-1-yl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidylalkyl)uracil; 5(methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5(methyl)2(thio)uracil; 5(methyl)2,4(dithio)uracil; 5(methyl)4(thio)uracil; 5(methylaminomethyl)-2(thio)uracil; 5(methylaminomethyl)-2,4(dithio)uracil; 5(methylaminomethyl)-4(thio)uracil; 5(propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4(dithio)pseudouracil; 5-(alkyl)-4(thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidylalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazol-1-yl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)2(thio)uracil; 5-(methyl)2,4(dithio)uracil; 5-(methyl)4(thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4(dithio)pseudouracil; 5-(methyl)-4(thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2(thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil;6(Azido)uracil; 6-(Azido)uracil; 6-Aza-uridine; Allylamino-uracil; Azauracil; Deazauracil; N3(Methyl)uracil; Pseudouridine-1-2-acetic acid; Pseudouridine; 4-Thio-pseudouridine triphosphate; 1-Carboxymethyl-pseudouridine; 1-Methyl-1-deaza-pseudouridine; 1-Propynyl-uridine; 1-Taurinomethyl-1-methyl-uridine; 1-Taurinomethyl-4-thio-uridine; 1-Taurinomethyl-pseudouridine; 2-Methoxy-4-thio-pseudouridine; 2-Thio-1-methyl-1-deaza-pseudouridine; 2-Thio-1-methyl-pseudouridine; 2-Thio-5-aza-uracil; 2-Thio-dihydropseudouridine; 2-Thio-dihydrouridine; 2-Thio-pseudouridine; 4-Methoxy-2-thio-pseudouridine; 4-Methoxy-pseudouridine; 4-Thio-1-methyl-pseudouridine; 4-Thio-pseudouridine; 5-Aza-uracil; Dihydropseudouridine; (±)1-(2-Hydroxypropyl)pseudouridine triphosphate; (2R)-1-(2-Hydroxypropyl)pseudouridine triphosphate; (2S)-1-(2-Hydroxypropyl)pseudouridine triphosphate; (E)-5-(2-Bromo-vinyl)arabinouridine triphosphate; (E)-5-(2-Bromo-vinyl)uridine triphosphate; (Z)-5-(2-Bromo-vinyl)arabinouridine triphosphate; (Z)-5-(2-Bromo-vinyl)uridine triphosphate; 1-(2,2,2-Trifluoroethyl)-pseudouridine triphosphate; 1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine triphosphate; 1-(2,2-Diethoxyethyl)pseudouridine triphosphate; 1-(2,4,6-Trimethylbenzyl)pseudouridine triphosphate; 1-(2,4,6-Trimethyl-benzyl)pseudouridine triphosphate; 1-(2,4,6-Trimethyl-phenyl)pseudouridine triphosphate; 1-(2-Amino-2-carboxyethyl)pseudouridine triphosphate; 1-(2-Amino-ethyl)pseudouridine triphosphate; 1-(2-Hydroxyethyl)pseudouridine triphosphate; 1-(2-Methoxyethyl)pseudouridine triphosphate; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine triphosphate; 1-(3,4-Dimethoxybenzyl)pseudouridine triphosphate; 1-(3-Amino-3-carboxypropyl)pseudouridine triphosphate; 1-(3-Amino-propyl)pseudouridine triphosphate; 1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine triphosphate; 1-(4-Amino-4-carboxybutyl)pseudouridine triphosphate; 1-(4-Amino-benzyl)pseudouridine triphosphate; 1-(4-Amino-butyl)pseudouridine triphosphate; 1-(4-Amino-phenyl)pseudouridine triphosphate; 1-(4-Azidobenzyl)pseudouridine triphosphate; 1-(4-Bromobenzyl)pseudouridine triphosphate; 1-(4-Chlorobenzyl)pseudouridine triphosphate; 1-(4-Fluorobenzyl)pseudouridine triphosphate; 1-(4-Iodobenzyl)pseudouridine triphosphate; 1-(4-Methanesulfonylbenzyl)pseudouridine triphosphate; 1-(4-Methoxybenzyl)pseudouridine triphosphate; 1-(4-Methoxy-benzyl)pseudouridine triphosphate; 1-(4-Methoxy-phenyl)pseudouridine triphosphate; 1-(4-Methylbenzyl)pseudouridine triphosphate; 1-(4-Methyl-benzyl)pseudouridine triphosphate;1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudouridine 5'-triphosphate; 1-(4-Nitro-phenyl)pseudouridine 5'-triphosphate; 1-(4-Methoxy-thiobenzyl)pseudouridine TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino-pentyl)pseudouridine 5'-triphosphate; 1-(6-Amino-hexyl)pseudouridine 5'-triphosphate; 1,6-Dimethyl-pseudouridine 5'-triphosphate; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propanoyl]pseudouridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propanoyl}pseudouridine TP; 1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudouridine 5'-triphosphate; 1-Alkyl-6-(2-propynyl)-pseudouridine 5'-triphosphate; 1-Alkyl-6-allyl-pseudouridine 5'-triphosphate; 1-Alkyl-6-ethynyl-pseudouridine 5'-triphosphate; 1-Alkyl-6-homoallyl-pseudouridine 5'-triphosphate; 1-Alkyl-6-vinyl-pseudouridine 5'-triphosphate; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudouridine 5'-triphosphate; 1-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudouridine 5'-triphosphate; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudouridine 5'-triphosphate; 1-Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudouridine 5'-triphosphate; 1-Cyclobutyl-pseudouridine 5'-triphosphate; 1-Cycloheptylmethyl-pseudouridine 5'-triphosphate; 1-Cycloheptyl-pseudouridine 5'-triphosphate; 1-Cyclohexylmethyl-pseudouridine 5'-triphosphate; 1-Cyclohexyl-pseudouridine 5'-triphosphate; 1-Cyclooctylmethyl-pseudouridine 5'-triphosphate; 1-Cyclooctyl-pseudouridine 5'-triphosphate; 1-Cyclopentylmethyl-pseudouridine 5'-triphosphate; 1-Cyclopentyl-pseudouridine 5'-triphosphate; 1-Cyclopropylmethyl-pseudouridine 5'-triphosphate; 1-Cyclopropyl-pseudouridine 5'-triphosphate; 1-Ethyl-pseudouridine 5'-triphosphate; 1-Hexyl-pseudouridine 5'-triphosphate; 1-Homoallylpseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-Isopropyl-pseudouridine 5'-triphosphate; 1-Me-2-thio-pseudouridine 5'-triphosphate; 1-Me-4-thio-pseudouridine 5'-triphosphate; 1-Me-α-thio-pseudouridine 5'-triphosphate; 1-Methanesulfonylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2-trifluoroethyl)pseudouridine 5'-triphosphate; 1-Methyl-6-(4-morpholino)-pseudouridine 5'-triphosphate; 1-Methyl-6-(4-thiomorpholino)-pseudouridine 5'-triphosphate; 1-Methyl-6-(substituted phenyl)pseudouridine 5'-triphosphate; 1-Methyl-6-amino-pseudouridine 5'-triphosphate; 1-Methyl-6-azido-pseudouridine 5'-triphosphate; 1-Methyl-6-bromo-pseudouridine 5'-triphosphate; 1-Methyl-6-butyl-pseudouridine 5'-triphosphate; 1-Methyl-6-chloro-pseudouridine 5'-triphosphate; 1-Methyl-6-cyano-pseudouridine 5'-triphosphate; 1-Methyl-6-dimethylamino-pseudouridine 5'-triphosphate; 1-Methyl-6-ethoxy-pseudouridine 5'-triphosphate; 1-Methyl-6-ethylcarboxy-pseudouridine 5'-triphosphate; 1-Methyl-6-ethyl-pseudouridine 5'-triphosphate; 1-Methyl-6-fluoro-pseudouridine 5'-triphosphate; 1-Methyl-6-formyl-pseudouridine 5'-triphosphate;1-methyl-6-hydroxyamino-pseudo-UTP; 1-methyl-6-hydroxy-pseudo-UTP; 1-methyl-6-iodo-pseudo-UTP; 1-methyl-6-isopropyl-pseudo-UTP; 1-methyl-6-methoxy-pseudo-UTP; 1-methyl-6-methylamino-pseudo-UTP; 1-methyl-6-phenyl-pseudo-UTP; 1-methyl-6-propyl-pseudo-UTP; 1-methyl-6-tert-butyl-pseudo-UTP; 1-methyl-6-trifluoromethoxy-pseudo-UTP; 1-methyl-6-trifluoromethyl-pseudo-UTP; 1-morpholinomethylpseudouridine TP; 1-pentyl-pseudo-UTP; 1-phenyl-pseudo-UTP; 1-pivaloylpseudouridine TP; 1-propynylpseudouridine TP; 1-propyl-pseudo-UTP; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-butyl-pseudo-UTP; 1-thiomethoxymethylpseudouridine TP; 1-thiomorpholinomethylpseudouridine TP; 1-trifluoroacetylpseudouridine TP; 1-trifluoromethyl-pseudo-UTP; 1-vinylpseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-α-ethynyluridine TP; 2'-α-trifluoromethyluridine TP; 2'-β-ethynyluridine TP; 2'-β-trifluoromethyluridine TP; 2'-deoxy-2',2'-difluorouridine TP; 2'-deoxy-2'-α-mercaptouridine TP; 2'-deoxy-2'-α-thiomethoxyuridine TP; 2'-deoxy-2'-β-aminouridine TP; 2'-deoxy-2'-β-azidouridine TP; 2'-deoxy-2'-β-bromouridine TP; 2'-deoxy-2'-β-chlorouridine TP; 2'-deoxy-2'-β-fluorouridine TP; 2'-deoxy-2'-β-iodouridine TP; 2'-deoxy-2'-β-mercaptouridine TP; 2'-deoxy-2'-β-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-O-methyl-5-(1-propynyl)uridine TP; 3-alkyl-pseudo-UTP; 4'-azidouridine TP; 4'-carbocyclic uridine TP; 4'-ethynyluridine TP; 5-(1-propynyl)ara-uridine TP; 5-(2-furyl)uridine TP; 5-cyano-uridine TP; 5-dimethylamino-uridine TP; 5'-homouridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5-phenylethynyluridine TP; 5-trideuteriomethyl-6-deuteriouridine TP; 5-trifluoromethyl-uridine TP; 5-vinylarabinouridine TP; 6-(2,2,2-trifluoroethyl)-pseudo-UTP; 6-(4-morpholino)-pseudo-UTP; 6-(4-thiomorpholino)-pseudo-UTP; 6-(substituted phenyl)-pseudo-UTP; 6-amino-pseudo-UTP; 6-azido-pseudo-UTP; 6-bromo-pseudo-UTP; 6-butyl-pseudo-UTP;6-chloro-pseudo-UTP; 6-cyano-pseudo-UTP; 6-dimethylamino-pseudo-UTP; 6-ethoxy-pseudo-UTP; 6-ethylcarboxylic acid-pseudo-UTP; 6-ethyl-pseudo-UTP; 6-fluoro-pseudo-UTP; 6-formyl-pseudo-UTP; 6-hydroxyamino-pseudo-UTP; 6-hydroxy-pseudo-UTP; 6-iodo-pseudo-UTP; 6-isopropyl-pseudo-UTP; 6-methoxy-pseudo-UTP; 6-methylamino-pseudo-UTP; 6-methyl-pseudo-UTP; 6-phenyl-pseudo-UTP; 6-phenyl-pseudo-UTP; 6-propyl-pseudo-UTP; 6-tert-butyl-pseudo-UTP; 6-trifluoromethoxy-pseudo-UTP; 6-trifluoromethyl-pseudo-UTP; α-thio-pseudo-UTP; pseudouridine 1-(4-methylbenzenesulfonic acid)TP; pseudouridine 1-(4-methylbenzoic acid)TP; pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-methylphosphate; pseudouridine TP 1-diethyl methylphosphate; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butyric acid; pseudo-UTP-N1-5-glycyl-valeric acid; pseudo-UTP-N1-6-hexanoic acid; pseudo-UTP-N1-7-heptanoic acid; pseudo-UTP-N1-methyl-p-benzoic acid; pseudo-UTP-N1-p-benzoic acid; wybutosine; hydroxywybutosine; isowybutoside; peroxwybutosine; under-modified hydroxywybutosine; 4-demethylisowybutoside; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diazabicyclo)-2-(oxo)-phenothiazin-1-yl; 1,3-(diazabicyclo)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxo)-naphthalene; 2(amino)purine; 2,4,5-(trimethyl)phenyl; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-cytidine; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-adenine; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-chloro-purine; 2-aza-inulinosyl; 2'-azido-2'-deoxyribose; 2'-fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propargyl)isoquinolinyl; 3-(methyl)isoquinolinyl;4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isoquinolyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz a)-2-(oxo)phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz a)-2-(oxo)phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz a)-2-(oxo)phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenoxazin1-yl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenoxazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz a)-2-(oxo)phenoxazin-1-yl; 7-(guanidylalkyl-hydroxy)-1,3-(diaz a)-2-(oxo)phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz a)-2-(oxo)phenoxazin-1-yl; 7-(propargyl)isoquinolyl; 7-(propargyl)isoquinolyl, propargyl-7-(aza)indolyl; 7-deaza-inulinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)phenoxazin-1-yl; 7-substituted 1,3-(diaz a)-2-(oxo)phenoxazin-1-yl; 9-(methyl)imidazolylpyridine; aminoindolyl; anthryl; bis-in situ(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-in situ-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazolylpyridine; inulinyl; isoquinolyl; isoguanosine; N2-substituted purine; N6-methyl-2-aminopurine; N6-substituted purine; N-alkylated derivative; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; Nubularine; 06-substituted purine; O-alkylated derivative; in situ-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; in situ-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;Para-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; condensed pentaphenyl; phenanthryl; phenyl; propargyl-7-(aza)indolyl; pyrenyl; pyrido[2,3-d]pyrimidin-3-yl; pyrido[2,3-d]pyrimidin-3-yl, 2-oxo-7-amino-pyrido[2,3-d]pyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyrazinyl; stilbenyl; substituted 1,2,4-triazole; quaterphenyl; tuberactinomycin; xanthine; xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-amino-ribonucleoside-TP; formycin A TP; formycin BTP; pyrrolysine TP; 2'-OH-arabinofuranosyladenosine TP; 2'-OH-arabinofuranosylcytidine TP; 2'-OH-arabinofuranosyluridine TP; 2'-OH-arabinofuranosylguanosine TP; 5-(2-methoxycarbonylvinyl)uridine TP; and N6-(19-amino-pentaoxanonadecyl)adenosine TP.;

[0175] In some embodiments, a nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises a combination of at least two (e.g., 2, 3, 4, or more) of the aforementioned modified nucleobases.

[0176] In some embodiments, the modified nucleobases in a nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) are selected from the group consisting of: pseudouridine (ψ), N1-methylpseudouridine (m 1 ψ), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, a nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises a combination of at least two (e.g., 2, 3, 4, or more) of the aforementioned modified nucleobases.

[0177] In some embodiments, the modified nucleobases in a nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) are selected from the group consisting of: 1-methyl-pseudouridine (m 1 ψ), 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5C), pseudouridine (ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, the nucleic acid comprises a combination of at least two (e.g., 2, 3, 4, or more) of the aforementioned modified nucleobases.

[0178] In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (m 5 C). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m 1 ψ). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m 1 ψ) and 5-methyl-cytidine (m 5 C). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2-thio-uridine (s 2 U). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2-thio-uridine and 5-methyl-cytidine (m 5 C). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises methoxy-uridine (mo 5 U). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 5-methoxy-uridine (mo 5 U) and 5-methyl-cytidine (m 5 C). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2'-O-methyl uridine. In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2'-O-methyl uridine and 5-methyl-cytidine (m 5 C). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m 6 A). In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m 6 A) and 5-methyl-cytidine (m 5 C).

[0179] In some embodiments, the nucleic acid (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) is uniformly modified (e.g., fully modified, modified throughout the sequence) for a particular modification. For example, the nucleic acid can be uniformly modified with 5-methyl-cytidine (m 5 C), which means that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5 C) Substitution. Similarly, nucleic acids can be uniformly modified for any type of nucleoside residue present in the sequence by substituting with modified residues such as those listed above.

[0180] Exemplary nucleobases and nucleosides having modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0181] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and in some embodiments modified nucleobases are modified cytosine. Nucleosides having modified uridine include 5-cyano-uridine and 4'-thio-uridine.

[0182] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0183] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0184] The nucleic acids described herein can be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) can be uniformly modified in the polynucleotides of the present disclosure or in its given predetermined sequence region (e.g., in an mRNA including or not including a polyA tail). In some embodiments, all nucleotides X in the polynucleotides of the present disclosure (or in its given sequence region) are modified nucleotides, where X can be any one of the nucleotides A, G, U, C or a combination of A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0185] The nucleic acid may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%) of modified nucleotides. Any remaining percentage accounts for the presence of unmodified A, G, U, or C.

[0186] The nucleic acid may contain at least 1% and at most 100% modified nucleotides, or any intermediate percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acid may contain modified pyrimidines, such as modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in the polynucleotide are replaced with modified uracil (e.g., 5-substituted uracil). The modified uracil may be replaced with a compound having a single unique structure, or may be replaced with multiple compounds having different structures (e.g., 2, 3, 4, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the polynucleotide are replaced with modified cytosine (e.g., 5-substituted cytosine). The modified cytosine may be replaced with a compound having a single unique structure, or may be replaced with multiple compounds having different structures (e.g., 2, 3, 4, or more unique structures).

[0187] Thus, in some embodiments, the composition comprises a 5'UTR element, an optionally codon-optimized open reading frame, and a 3'UTR element, a poly(A) sequence and / or a polyadenylation signal, wherein the RNA is not chemically modified.

[0188] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), methyl uridine 5-oxyacetate (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), methyl 5-carboxyhydroxymethyl-uridine (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyl-uridine (τm 5 U), 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine (τm 5 s2 U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, that is, having the nucleobase deoxythymine), 1-methyl-pseudouridine (m 1 ψ), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Urn), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-arabinouridine, 2'-F-uridine, 2'-0H-arabinouridine, 5-(2-methoxycarbonylviny)uridine and 5-[3-(1-E-propenylamino)]uridine.

[0189] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formylcytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethylcytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethylcytidine (m 4 Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-arabinocytidine, 2'-F-cytidine, and 2'-0H-arabinocytidine.

[0190] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m2 A), N6-methyl-adenosine (m 6 A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycylcarbamoyl-adenosine (g 6 A), N6-threonylcarbamoyl-adenosine (t 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A), N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxy-norvalylcarbamoyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxy-norvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabinofuranosyl-adenosine, 2'-F-adenosine, 2'-OH-arabinofuranosyl-adenosine, and N6-(19-amino-pentoxanonadecyl)-adenosine.

[0191] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m 1I), wogonoside (imG), methyl wogonoside (mimG), 4-demethyl-wogonoside (imG-14), isowogonoside (imG2), wodynoside (yW), peroxy-wodynoside (o2yW), hydroxy-wodynoside (OhyW), under-modified hydroxy-wodynoside (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxy-queuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G±), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2,N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-arabinoguanosine and 2'-F-guanosine.

[0192] N-linked glycosylation site mutants

[0193] The N-linked glycans of viral proteins play an important role in regulating immune responses. Glycans may be important for maintaining an appropriate antigen conformation, shielding potential neutralizing epitopes, and may alter the proteolytic sensitivity of the protein. Some viruses have putative N-linked glycosylation sites. Deletion or modification of N-linked glycosylation sites can enhance immune responses. Thus, in some embodiments, the present disclosure provides compositions comprising a nucleic acid (e.g., mRNA) encoding an antigen polypeptide, wherein the antigen polypeptide comprises a deletion or modification at one or more N-linked glycosylation sites.

[0194] In vitro transcription of RNA (e.g., mRNA)

[0195] The compositions of the present disclosure comprise at least one RNA polynucleotide, such as mRNA (e.g., modified mRNA). For example, mRNA is transcribed in vitro from a template DNA referred to as an "in vitro transcription template". In some embodiments, the in vitro transcription template encodes a 5' untranslated region (UTR), contains an open reading frame, and encodes a 3' UTR and a polyA tail. The specific nucleic acid sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0196] The "5' untranslated region" (5'UTR) refers to the region of an mRNA that is located immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript that is translated by a ribosome) and does not encode a polypeptide.

[0197] The "3' untranslated region" (3'UTR) refers to the region of an mRNA that is located immediately downstream (i.e., 3') of the stop codon (i.e., the codon in the mRNA transcript that signals the termination of translation) and does not encode a polypeptide.

[0198] An "open reading frame" is a continuous stretch of DNA that starts with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide.

[0199] The "polyA tail" is an mRNA region containing multiple consecutive adenosine monophosphates located downstream of the 3'UTR, for example, directly downstream (i.e., 3'). The polyA tail can contain 10 to 300 adenosine monophosphates. For example, the polyA tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological environment (e.g., in cells, in vivo), the poly(A) tail is used to protect mRNA from enzymatic degradation, for example, in the cytoplasm, and contributes to transcriptional termination, export of mRNA from the nucleus, and translation.

[0200] In some embodiments, the polynucleotide comprises 200 to 3,000 nucleotides. For example, the polynucleotide can comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0201] Flagellin adjuvant

[0202] Flagellin is a monomeric protein of approximately 500 amino acids that polymerizes to form flagella associated with bacterial motility. Flagellin is expressed by a variety of flagellated bacteria (e.g., Salmonella typhimurium) as well as non-flagellated bacteria (such as Escherichia coli). Sensing of flagellin by cells of the innate immune system (dendritic cells, macrophages, etc.) is mediated by Toll-like receptor 5 (TLR5) and Nod-like receptors (NLRs) Ipaf and Naip5. TLRs and NLRs have been identified as playing a role in the activation of innate and adaptive immune responses. Thus, flagellin provides an adjuvant effect in vaccines.

[0203] The nucleotide and amino acid sequences encoding known flagellin polypeptides are publicly available in the NCBI GenBank database. Flagellin sequences from Salmonella typhimurium, Helicobacter pylori (H. Pylori), Vibrio cholerae (V. Cholera), Serratia marcesens (S. marcesens), Shigella flexneri (S. flexneri), Treponema pallidum (T. Pallidum), Legionella pneumophila (L. pneumophila), Borrelia burgdorferi (B. burgdorferei), Clostridium difficile (C. difficile), Sinorhizobium meliloti (R. meliloti), Agrobacterium tumefaciens (A. tumefaciens), Rhizobium lupini (R. lupini), Bartonella clarridgeiae (B. clarridgeiae), Proteus mirabilis (P. Mirabilis), Bacillus subtilis (B. subtilus), Listeria monocytogenes (L. monocytogenes), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli are known.

[0204] As used herein, a flagellin polypeptide refers to a full-length flagellin, an immunogenic fragment thereof, and a peptide having at least 50% sequence identity to flagellin or an immunogenic fragment thereof. Exemplary flagellins include flagellins from Salmonella typhi (UniPro entry number: Q56086), Salmonella typhimurium (A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonella choleraesuis (Q6V2X8) as well as SEQ ID NO: 54 - 56. In some embodiments, the flagellin polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to flagellin or an immunogenic fragment thereof.

[0205] In some embodiments, the flagellin polypeptide is an immunogenic fragment. An immunogenic fragment is the portion of flagellin that elicits an immune response. In some embodiments, the immune response is a TLR5 immune response. An example of an immunogenic fragment is flagellin in which all or a portion of the hinge region has been deleted or replaced with other amino acids. For example, an antigen polypeptide can be inserted into the hinge region. The hinge region is a hypervariable region of flagellin. The hinge region of flagellin is also referred to as the "D3 domain or region", "propeller domain or region", "hypervariable domain or region", and "variable domain or region". As used herein, "at least a portion of the hinge region" also refers to any part or the entire hinge region of the hinge region of flagellin. In other embodiments, the immunogenic fragment of flagellin is a 20-, 25-, 30-, 35-, or 40-amino acid C-terminal fragment of flagellin.

[0206] Flagellin monomers are formed by domains D0 to D3. D0 and D1, which form the stalk, consist of tandem long α helices and are highly conserved among different bacteria. The D1 domain includes several amino acid stretches that are available for TLR5 activation. The entire D1 domain or one or more active regions within the domain are immunogenic fragments of flagellin. Examples of immunogenic regions within the D1 domain include residues 88 - 114 and residues 411 - 431 (in Salmonella typhimurium FliC flagellin). Within the 13 amino acids of the 88 - 100 region, at least 6 substitutions are allowed between Salmonella flagellin and other flagellins that still retain TLR5 activation. Thus, an immunogenic fragment of flagellin includes a flagellin-like sequence that activates TLR5 and contains a 13-amino acid motif that is 53% or more identical to the Salmonella sequence in 88 - 100 of FliC (LQRVRELAVQSAN; SEQ ID NO:84).

[0207] In some embodiments, the composition includes a nucleic acid sequence encoding a fusion protein of flagellin and one or more antigen polypeptides. As used herein, a "fusion protein" refers to the joining of two components of a construct. In some embodiments, the carboxyl terminus of the antigen polypeptide is fused or linked to the amino terminus of the flagellin polypeptide. In other embodiments, the amino terminus of the antigen polypeptide is fused or linked to the carboxyl terminus of the flagellin polypeptide. The fusion protein can include, for example, one, two, three, four, five, six, or more flagellin polypeptides linked to one, two, three, four, five, six, or more antigen polypeptides. When two or more flagellin polypeptides and / or two or more antigen polypeptides are linked, this construct can be referred to as a "multimer".

[0208] Each component of the fusion protein can be directly linked to each other, or it can be linked via a linker. For example, the linker can be an amino acid linker. The amino acid linker encoded by the composition to be linked to the components of the fusion protein can include, for example, at least one member selected from the group consisting of: lysine residue, glutamate residue, serine residue, and arginine residue. In some embodiments, the length of the linker is 1-30, 1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids.

[0209] In other embodiments, the composition includes at least three separate nucleic acid sequences, one nucleic acid sequence encoding one or more antigen polypeptides, one nucleic acid sequence encoding one or more universal T cell epitopes, and another nucleic acid sequence encoding a flagellin polypeptide. The at least three nucleic acid sequences can be co-formulated in a carrier such as a lipid nanoparticle.

[0210] Stabilizing element

[0211] It has been found that, in addition to other structural features such as a 5' cap structure or a 3'-poly(A) tail, naturally occurring eukaryotic mRNA molecules also contain stabilizing elements, including but not limited to untranslated regions (UTRs) located at their 5' end (5'UTR) and / or at their 3' end (3'UTR). Both the 5'UTR and the 3'UTR are typically transcribed from genomic DNA and are elements of the premature mRNA. During mRNA processing, characteristic structural features of the mature mRNA, such as a 5'-cap and a 3'-poly(A) tail, are typically added to the transcribed (premature) mRNA. The 3'-poly(A) tail is typically an extension of adenine nucleotides added to the 3' end of the transcribed mRNA. It can contain up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail can be an essential element related to the stability of the individual mRNA.

[0212] In some embodiments, a nucleic acid sequence (e.g., mRNA) can include one or more stabilizing elements. The stabilizing elements can include, for example, histone stem-loops. A stem-loop binding protein (SLBP) has been identified, which is a 32 kDa protein. The stem-loop binding protein associates with the histone stem-loop at the 3' end of the histone message in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S phase when histone mRNA levels are also elevated. The protein has been shown to be required for the efficient 3' end processing of histone precursor mRNA by U7 snRNP. SLBP continues to associate with the stem-loop after processing and then stimulates the translation of mature histone mRNA into histone in the cytoplasm. The RNA binding domain of SLBP is conserved in metazoans and protozoans; its binding to the histone stem-loop depends on the structure of the loop. The minimal binding site includes at least three nucleotides located 5' relative to the stem-loop and two nucleotides located 3'.

[0213] In some embodiments, a nucleic acid sequence (e.g., mRNA) includes a coding region, at least one histone stem-loop, and optionally a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the encoded protein is not a histone, a reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selectable protein (e.g., α-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0214] In some embodiments, the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, act synergistically to increase protein expression to levels exceeding those observed with either element alone. It has been found that the synergistic effect of the combination of poly(A) and at least one histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence.

[0215] In some embodiments, a nucleic acid sequence (e.g., mRNA) does not contain a histone downstream element (HDE). A "histone downstream element" (HDE) includes a purine-rich polynucleotide extension of about 15 to 20 nucleotides located 3' of the naturally occurring stem-loop, which represents the binding site of U7 snRNA and is involved in processing histone precursor mRNA into mature histone mRNA. Desirably, the nucleic acids of the present invention do not include introns.

[0216] In some embodiments, a nucleic acid sequence (e.g., mRNA) may or may not contain enhancer and / or promoter sequences, which may be modified or unmodified, or which may be activated or inactivated. In some embodiments, histone stem-loops typically originate from histone genes and include intramolecular base pairing of two adjacent partial or fully reverse complementary sequences separated by a spacer that forms a structured loop, the spacer comprising (consisting of) a short sequence. The unpaired loop region is generally unable to base pair with either of the stem-loop elements. This occurs more frequently in RNA, as is the case for many key components of RNA secondary structure, but may also be present in single-stranded DNA. The stability of the stem-loop structure generally depends on the length of the paired regions, the number of mismatches or bulges, and the composition of the bases. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may be generated. In some embodiments, at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

[0217] In other embodiments, a nucleic acid sequence (e.g., mRNA) may have one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES, are destabilizing sequences present in the 3'UTR. AURES can be removed from the nucleic acid sequence (e.g., mRNA). Alternatively, AURES can be retained in the nucleic acid sequence (e.g., mRNA).

[0218] Delivery methods

[0219] Suitable nucleic acid delivery agents are well known in the art and may include, but are not limited to, lipid-based (e.g., liposome formulations, lipid complexes, or lipid nanoparticles (LNPs)), virus-based, or physical methods such as injection, microinjection, electroporation, sonication, gene gun, hydrodynamic application, or any combination thereof.

[0220] Nanoparticles

[0221] Described herein are nanoparticles comprising a nucleic acid sequence (e.g., mRNA) encoding a pathogen antigen polypeptide and a nucleic acid sequence (e.g., mRNA) encoding at least one universal T cell epitope, and nanoparticles comprising a nucleic acid sequence (e.g., mRNA) encoding a pathogen antigen polypeptide and at least one universal T cell epitope.

[0222] The nanoparticles described herein may include multiple nucleic acid sequences (e.g., mRNA) each encoding a single pathogen antigen polypeptide and multiple nucleic acid sequences (e.g., mRNA) each encoding a single universal T cell epitope, as well as nanoparticles comprising a polynucleotide having a single nucleic acid sequence (e.g., mRNA) encoding one or more pathogen antigen polypeptides and a single nucleic acid sequence (e.g., mRNA) encoding one or more universal T cell epitopes.

[0223] The nanoparticles described herein may include multiple nucleic acid sequences (e.g., mRNA) each encoding a single pathogen antigen polypeptide and a single universal T cell epitope, as well as nanoparticles comprising a polynucleotide having a single nucleic acid sequence (e.g., mRNA) encoding one or more pathogen antigen polypeptides and one or more universal T cell epitopes.

[0224] In some embodiments, the nanoparticles described herein include 2 - 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleic acid sequences each having an open reading frame and encoding different pathogen antigen polypeptides (or a single nucleic acid sequence encoding 2 - 10 or more different pathogen antigen polypeptides). In some embodiments, the nanoparticles described herein include 2 - 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleic acid sequences each having an open reading frame and encoding different universal T cell epitopes (or a single nucleic acid sequence encoding 2 - 10 or more different universal T cell epitopes).

[0225] In some embodiments, the nanoparticles may be lipid nanoparticles. In some embodiments, the nanoparticles may be lipid - polycation complexes, referred to as cationic lipid nanoparticles. As a non - limiting example, the polycation may include a cationic peptide or polypeptide, such as but not limited to polylysine, polyornithine, and / or polyarginine. In some embodiments, the lipid nanoparticles may include non - cationic lipids, such as but not limited to cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0226] Lipid nanoparticle formulations can be affected by, but are not limited to, the choice of cationic lipid component, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all components, and biophysical parameters such as size. In one example of Semple et al. (Nature Biotech. 2010 28:172-176), the lipid nanoparticles can further include 57% cationic lipid, 7% dipalmitoyl phosphatidylcholine, 34% cholesterol, and 1.5% PEG-c-DMA. As another example, changing the composition of the cationic lipid can more effectively deliver siRNA to various antigen-presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200).

[0227] In some embodiments, the lipid nanoparticle formulation can comprise 35% to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid, and / or 55% to 65% cationic lipid. In some embodiments, the ratio of lipid to nucleic acid (e.g., mRNA) in the lipid nanoparticles can be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1, and / or at least 30:1.

[0228] Lipid nanoparticle formulations typically comprise lipids, specifically ionizable cationic lipids, and further comprise neutral lipids, sterols, and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids.

[0229] In some embodiments, the lipid nanoparticle formulation comprises at least one ionizable lipid, cationic lipid, vitamin-based lipid, or any combination thereof; a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, such as cholesterol; and (iv) a PEG lipid, such as PEG-DMG or PEG-cDMA, with a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol: 0.5-15% PEG lipid.

[0230] In some embodiments, the lipid nanoparticle formulation comprises 25% to 75% by mole of a cationic lipid selected from, for example, 35% to 65%, 45% to 65%, 60%, 57.5%, 50% or 40% by mole of a cationic lipid: 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and bis((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0231] In some embodiments, the lipid nanoparticle formulation comprises 25 - 75% by mole of a cationic lipid, 0.5 - 15% by mole of a neutral lipid, 5 - 50% by mole of a sterol, and 0.5 - 20% by mole of a PEG or PEGylated lipid.

[0232] In some embodiments, the lipid nanoparticle formulation comprises 35 - 65% by mole of a cationic lipid, 3 - 12% by mole of a neutral lipid, 15 - 45% by mole of a sterol, and 0.5 - 10% by mole of a PEG or PEGylated lipid.

[0233] In some embodiments, the lipid nanoparticle formulation comprises 45 - 65% by mole of a cationic lipid, 5 - 10% by mole of a neutral lipid, 25 - 40% by mole of a sterol, and 0.5 - 10% by mole of a PEG or PEGylated lipid.

[0234] In some embodiments, the lipid nanoparticle formulation comprises 60% by mole of a cationic lipid, 7.5% by mole of a neutral lipid, 31% by mole of a sterol, and 1.5% by mole of a PEG or PEGylated lipid.

[0235] In some embodiments, the lipid nanoparticle formulation comprises 50% by mole of a cationic lipid, 10% by mole of a neutral lipid, 38.5% by mole of a sterol, and 1.5% by mole of a PEG or PEGylated lipid.

[0236] In some embodiments, the lipid nanoparticle formulation comprises 50% by mole of a cationic lipid, 10% by mole of a neutral lipid, 35% by mole of a sterol, 4.5% or 5% by mole of a PEG or PEGylated lipid, and 0.5% by mole of a targeting lipid.

[0237] In some embodiments, the lipid nanoparticle formulation comprises 40% by mole of a cationic lipid, 15% by mole of a neutral lipid, 40% by mole of a sterol, and 5% by mole of a PEG or PEGylated lipid.

[0238] In some embodiments, the lipid nanoparticle formulation comprises 57.2% cationic lipid, 7.1% neutral lipid, 34.3% sterol, and 1.4% PEG or PEGylated lipid on a molar basis.

[0239] In some embodiments, the lipid nanoparticle formulation comprises 57.5% cationic lipid on a molar basis, a PEG lipid selected from PEG-cDMA (PEG-cDMA is further discussed in Reyes et al., J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety), 7.5% neutral lipid, 31.5% sterol, and 3.5% PEG or PEGylated lipid.

[0240] In some embodiments, the lipid nanoparticle formulation comprises a lipid mixture having a molar ratio of 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEGylated lipid. In some embodiments, the lipid nanoparticle formulation comprises a lipid mixture having a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEGylated lipid.

[0241] In some embodiments, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG, or PEG-DPG), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid, e.g., DPPC / Chol / PEG-modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DSG), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

[0242] Non-limiting examples of lipid nanoparticle compositions and methods for their preparation are described, for example, in Semple et al. (2010) Nature Biotechnology 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the content of each of the aforementioned references is incorporated herein by reference in its entirety).

[0243] In some embodiments, the lipid nanoparticle formulation can comprise a cationic lipid, a PEG lipid, and a structural lipid, and optionally a non-cationic lipid. As a non-limiting example, the lipid nanoparticle can comprise 40 - 60% cationic lipid, 5 - 15% non-cationic lipid, 1 - 2% PEG lipid, and 30 - 50% structural lipid. As another non-limiting example, the lipid nanoparticle can comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid, and 38.5% structural lipid. As yet another non-limiting example, the lipid nanoparticle can comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid, and 32.5% structural lipid.

[0244] In some embodiments, the lipid nanoparticle formulation described herein can be a four-component lipid nanoparticle. The lipid nanoparticle can comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structural lipid. As a non-limiting example, the lipid nanoparticle can comprise 40 - 60% cationic lipid, 5 - 15% non-cationic lipid, 1 - 2% PEG lipid, and 30 - 50% structural lipid. As another non-limiting example, the lipid nanoparticle can comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid, and 38.5% structural lipid. As yet another non-limiting example, the lipid nanoparticle can comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid, and 32.5% structural lipid.

[0245] In some embodiments, the lipid nanoparticle formulation described herein can comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structural lipid. As a non-limiting example, the lipid nanoparticle comprises 50% cationic lipid DLin-KC2-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprises 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprises 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DMG, and 38.5% structural lipid cholesterol. As yet another non-limiting example, the lipid nanoparticle comprises 55% cationic lipid L319, 10% non-cationic lipid DSPC, 2.5% PEG lipid PEG-DMG, and 32.5% structural lipid cholesterol.

[0246] In some embodiments, the nanoparticles can be the nanoparticles described in U.S. Patent No. 10,933,127, the content of which is incorporated herein by reference in its entirety. In some embodiments, the nanoparticles can be the nanoparticles described in U.S. Patent No. 10,933,127, for example, nanoparticles comprising a compound according to Formula (I), (Ia), (II), (IIa), (IIb), (IIc), (IId) or (IIe) described in columns 101 to 187.

[0247] In some embodiments, the lipid nanoparticles can comprise 20% to 80% ionizable lipid, cationic lipid, vitamin-based derivative lipid, or any combination thereof; greater than 0% to 5% polyethylene glycol lipid; greater than 0% to 20% helper lipid; 20% to 80% sterol; a nucleic acid sequence encoding an infectious agent antigen polypeptide; and a nucleic acid sequence encoding at least one T cell epitope encapsulated in the nanoparticles.

[0248] In some embodiments, the lipid nanoparticles can comprise 20% to 80% ionizable lipid, cationic lipid, vitamin-based derivative lipid, or any combination thereof; greater than 0% to 5% polyethylene glycol lipid; greater than 0% to 20% helper lipid; 20% to 80% sterol; a nucleic acid sequence encoding an infectious agent antigen polypeptide and at least one T cell epitope encapsulated in the nanoparticles.

[0249] In one embodiment, the nanoparticles can comprise 40% to 60% ionizable lipid, cationic lipid, vitamin-based derivative lipid, or any combination thereof; 1% to 2% polyethylene glycol lipid; 8% to 12% helper lipid; 35% to 40% sterol; a nucleic acid sequence encoding an infectious agent antigen polypeptide; and a nucleic acid sequence encoding at least one T cell epitope encapsulated in the nanoparticles.

[0250] In some embodiments, the nanoparticles can comprise 40% to 60% ionizable lipid, cationic lipid, vitamin-based derivative lipid, or any combination thereof; 1% to 2% polyethylene glycol lipid; 8% to 12% helper lipid; 35% to 40% sterol; and a nucleic acid sequence encoding an infectious agent antigen polypeptide and at least one T cell epitope encapsulated in the nanoparticles.

[0251] In some embodiments, the average diameter of the nanoparticles (e.g., lipid nanoparticles) is 10 - 500 nm, 20 - 400 nm, 30 - 300 nm, 40 - 200 nm. In some embodiments, the average diameter of the nanoparticles (e.g., lipid nanoparticles) is 50 - 150 nm, 50 - 200 nm, 80 - 100 nm, or 80 - 200 nm.

[0252] The lipid nanoparticles described herein can be prepared in a sterile environment.

[0253] In some embodiments, the LNP formulation can be formulated in nanoparticles such as nucleic acid-lipid particles. As a non-limiting example, the lipid particles can comprise one or more active agents or therapeutic agents; one or more cationic lipids, the cationic lipids comprising about 50 mol% to about 85 mol% of the total lipids present in the particles; one or more non-cationic lipids, the one or more non-cationic lipids comprising about 13 mol% to about 49.5 mol% of the total lipids present in the particles; and one or more conjugated lipids, the one or more conjugated lipids inhibiting aggregation of the particles, the one or more conjugated lipids comprising about 0.5 mol% to about 2 mol% of the total lipids present in the particles.

[0254] The nanoparticle formulation can comprise a phosphate conjugate. The phosphate conjugate can increase the in vivo circulation time and / or increase the targeted delivery of the nanoparticles. As a non-limiting example, the phosphate conjugate can include a compound having any one of the chemical formulas described in International Application No. WO2013033438, the content of which is incorporated herein by reference in its entirety.

[0255] The nanoparticle formulation can comprise a polymer conjugate. The polymer conjugate can be a water-soluble conjugate. The polymer conjugate can have the structure described in U.S. Patent Application No. 20130059360, the content of which is incorporated herein by reference in its entirety. In some embodiments, the polymer conjugate with the polynucleotide of the present disclosure can be prepared using the methods and / or segmented polymer reagents described in U.S. Patent Application No. 20130072709, the content of which is incorporated herein by reference in its entirety. In some embodiments, the polymer conjugate can have a side group comprising a ring moiety, such as, but not limited to, the polymer conjugate described in U.S. Patent Publication No. US20130196948, the content of which is incorporated herein by reference in its entirety.

[0256] The nanoparticle formulations can include conjugates for enhancing the delivery of the nanoparticles of the present disclosure in a subject. Further, the conjugates can inhibit macrophage clearance of the nanoparticles in the subject. In one aspect, the conjugate can be an "autologous" peptide engineered from the human membrane protein CD47 (e.g., the "autologous" particles described by Rodriguez et al. (Science 2013 339, 971-975), which is incorporated herein by reference in its entirety). As shown by Rodriguez et al., the autologous peptide delays macrophage-mediated clearance of the nanoparticles, and this delay enhances nanoparticle delivery. In another aspect, the conjugate can be the membrane protein CD47 (e.g., see Rodriguez et al. Science 2013 339, 971-975, which is incorporated herein by reference in its entirety). Rodriguez et al. showed that, similar to the "autologous" peptide, CD47 can increase the ratio of circulating particles in a subject compared to scrambled peptides and PEG-coated nanoparticles.

[0257] In some embodiments, the nanoparticles can include conjugates for enhancing the delivery of the nanoparticles of the present disclosure in a subject. The conjugate can be a CD47 membrane, or the conjugate can be derived from the CD47 membrane protein, such as the "autologous" peptide described previously. In some embodiments, the nanoparticles can include a conjugate or derivative thereof of PEG and CD47. In some embodiments, the nanoparticles can include both the "autologous" peptide and the membrane protein CD47 described above.

[0258] In some embodiments, the "autologous" peptide and / or the CD47 protein can be conjugated to virus-like particles or pseudovirions as described herein to deliver the nucleic acid sequences (e.g., mRNA) of the present disclosure.

[0259] In some embodiments, the composition includes a polynucleotide of the present disclosure and a conjugate that can have a degradable bond. Non-limiting examples of the conjugate include an aromatic moiety containing an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer. As a non-limiting example, a pharmaceutical composition containing a conjugate having a degradable bond and a method for delivering such a pharmaceutical composition are described in U.S. Patent Publication No. US20130184443, the content of which is incorporated herein by reference in its entirety.

[0260] Neutral lipid

[0261] In some embodiments, the lipid nanoparticle formulation comprises from 0.5% to 15% by mole of neutral lipids, e.g., from 3% to 12%, 5% to 10%, or 15%, 10%, or 7.5% by mole of neutral lipids. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises from 5% to 50% by mole of sterol (e.g., from 15% to 45%, 20% to 40%, 40%, 38.5%, 35%, or 31% by mole). Non-limiting examples of sterols are cholesterol. In some embodiments, the lipid nanoparticle formulation comprises from 0.5% to 20% by mole of PEG or PEG-modified lipid (e.g., from 0.5% to 10%, 0.5% to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% by mole). In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules having an average molecular weight of 2,000 Da. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules having an average molecular weight less than 2,000, e.g., about 1,500 Da, about 1,000 Da, or about 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA (further described in Reyes et al., Journal of Controlled Release, 107, 276-287 (2005), the content of which is incorporated herein by reference in its entirety).

[0262] zwitterionic lipid

[0263] In some embodiments, the composition can be encapsulated in, linked to, and / or associated with zwitterionic lipids. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No. US20130216607, the content of which is incorporated herein by reference in its entirety. In some aspects, zwitterionic lipids can be used in the liposomes and lipid nanoparticles described herein.

[0264] cationic lipid

[0265] Suitable cationic lipids can include, but are not limited to, 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in US20130150625); or a pharmaceutically acceptable salt or stereoisomer thereof. As a non-limiting example, the cationic lipid can be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-9-amine, (1Z,19Z)-N,N-dimethylpentacos-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocos-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylheneicos-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptadec-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, N,N-dimethylheptacos-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicos-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptac-20-en-10-amine, (15Z)-N,N-dimethylheptac-15-en-10-amine, (14Z)-N,N-dimethylnonac-14-en-10-amine, (17Z)-N,N-dimethylnonac-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonac-20-en-10-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentac-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylheneicos-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocos-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]triacont-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadec-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadec-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneicos-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadec-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadec-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradec-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodec-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadec-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadec-6-amine, N,N-dimethyl-1-(R1S,2R)-2-octylcyclopropylpentadec-8-amine, R-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-(R9Z,12Z)-octadeca-9,12-dien-1-yloxypropan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-(R9Z,12Z)-octadeca-9,12-dien-1-yloxypropan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-(R9Z,12Z)-octadeca-9,12-dien-1-yloxypropan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docos-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docos-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxypropan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-R1S,25)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-1[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,23-triene-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the lipid can be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and amino alcohol lipids. The amino alcohol cationic lipid can be the lipid described in U.S. Patent Publication No. US20130150625 and / or can be prepared by the method described in the said U.S. Patent Publication, the content of which is incorporated herein by reference in its entirety. In some embodiments, the nanoparticles described herein can include amine cationic lipids, such as the amine cationic lipids described in International Patent Application No. WO2013059496, the content of which is incorporated herein by reference in its entirety. In some embodiments, the cationic lipid can have an amino-amine or amino-amide moiety.,

[0266] In some embodiments, the cationic lipid can be a low molecular weight cationic lipid, such as the low molecular weight cationic lipid described in U.S. Patent Application No. 20130090372, the content of which is incorporated herein by reference in its entirety.

[0267] Ionizable lipid

[0268] Exemplary ionizable lipids are described in U.S. Patent Publication Nos. U.S. 2016 / 0311759, U.S. 2015 / 0376115, U.S. 2016 / 0151284, U.S. 2017 / 0210697, U.S. 2015 / 0140070, U.S. 2013 / 0178541, U.S. 2013 / 0303587, U.S. 2015 / 0141678, U.S. 2015 / 0239926, U.S. 2016 / 0376224, U.S. 2017 / 0119904, U.S. 2012 / 0149894, U.S. 2015 / 0057373, U.S. 2013 / 0090372, U.S. 2013 / 0274523, U.S. 2013 / 0274504, U.S. 2013 / 0274504, U.S. 2009 / 0023673, U.S. 2012 / 0128760, U.S. 2010 / 03241240, U.S. 2014 / 0200257, U.S. 2015 / 0203446, U.S. 2018 / 0005363, U.S. 2014 / 0308304, U.S. 2013 / 0338210, U.S. 2012 / 0101148, U.S. 2012 / 0027796, U.S. 2012 / 0058144, U.S. 2013 / 0323269, U.S. 2011 / 0117125, U.S. 2011 / 0256175, U.S. 2012 / 0202871, U.S. 2011 / 0076335, U.S. 2006 / 0083780, U.S. 2013 / 0123338, U.S. 2015 / 0064242, U.S. 2006 / 0051405, U.S. 2013 / 0065939, U.S. 2006 / 0008910, U.S. 2003 / 0022649, U.S. 2010 / 0130588, U.S. 2013 / 0116307, U.S. 2010 / 0062967, U.S. 2013 / 0202684, U.S. 2014 / 0141070, U.S. 2014 / 0255472, U.S. 2014 / 0039032, U.S. 2018 / 0028664, U.S. 2016 / 0317458, U.S. 2013 / 0195920, U.S. 2022 / 0062175, U.S.No. 2021 / 0121411, No. U.S.2022 / 0009878, No. U.S.2022 / 0040325, No. U.S.2012 / 61657480, No. U.S.2016 / 0074514, No. U.S.2013 / 0330401, No. U.S.2019 / 0185410, No. U.S.2012 / 61617468, No. U.S.2019 / 0032087, No. U.S.2015 / 62184188, No. U.S.2019 / 0127318, No. U.S.2021 / 0002813, No. U.S.2020 / 0345641, No. U.S.2014 / 61944336, No. U.S.2012 / 61657480, No. U.S.2021 / 0059953, No. U.S.2022 / 0162521, No. U.S.2022 / 0235377, No. U.S.2018 / 0085474, No. U.S.2018 / 0000953, No. U.S.2020 / 0129445, No. U.S.2021 / 0145982, No. U.S.2021 / 0378980, No. U.S.2020 / 0254086, No. U.S.2021 / 0346306 and U.S.2018 / 0000953, the entire content of the literature is incorporated herein by reference in its entirety.

[0269] In some embodiments, the nanoparticles comprise an ionizable lipid in a molar ratio of 0% to 80%. In some embodiments, the ionizable lipid may be present in a molar ratio of at least 0% (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80%). In some embodiments, the ionizable lipid may be present in a molar ratio of 80% or less (e.g., 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, 0.5% or less).

[0270] The ionizable lipid can be present in a molar ratio ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the ionizable lipid can be present in a molar ratio of 0% to 80% (e.g., greater than 0% to 80%, greater than 0% to 70%, greater than 0% to 60%, greater than 0% to 50%, greater than 0% to 40%, greater than 0% to 30%, greater than 0% to 20%, greater than 0% to 10%, greater than 0% to 5%, greater than 0% to 1%, greater than 0% to 0.5%, 1% to 30%, 1% to 20%, 1% to 10%, 1% to 5%, 5% to 30%, 5% to 20%, 5% to 10%, 10% to 30%, 10% to 20%, 20% to 30%, 20% to 40%, or 30% to 40%).

[0271] Helper lipid

[0272] In some embodiments, the nanoparticles comprise a helper lipid. In some embodiments, the helper lipid can be a non-cationic lipid. In some embodiments, the non-cationic lipid can include, but is not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho-(1'-rac-glycerol) (DOPG), or a combination thereof. In one embodiment, the non-cationic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the non-cationic lipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). In one embodiment, the non-cationic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the non-cationic lipid is 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). Although several non-cationic lipids are described herein, additional non-cationic lipids can be used in combination with the compounds disclosed herein.

[0273] In some embodiments, the nanoparticles comprise co-lipids in a molar ratio of 0% to 20%. In some embodiments, the nanoparticles comprise polyethylene glycol lipids in a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 15% or 20%.

[0274] Polyethylene glycol lipid

[0275] In some embodiments, the nanoparticles include polyethylene glycol lipids (PEG lipids). The PEG lipids are incorporated to form a hydrophilic outer layer and stabilize the particles. Non-limiting examples of polyethylene glycol lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol lipids include DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG. In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG). In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-XXXX, where XXXX represents the molecular weight of the polyethylene glycol moiety (e.g., DMG-PEG2000 or DMG-PEG5000).

[0276] In some embodiments, the nanoparticles comprise polyethylene glycol lipids in a molar ratio of 0% to 5%. In some embodiments, the nanoparticles comprise polyethylene glycol lipids in a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4% or 5%. In one embodiment, the nanoparticles comprise polyethylene glycol lipids in a molar ratio of 0.75%.

[0277] In some embodiments, the ratio of PEG in the lipid nanoparticle formulation can be increased or decreased and / or the carbon chain length of the PEG lipid can be changed from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, compared to the cationic lipid, DSPC, and cholesterol, the lipid nanoparticle formulation can contain PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol) 2000) carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) at a lipid molar ratio of 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0%, and / or 3.0% to 5.0%. In some embodiments, PEG-c-DOMG can be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol), and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).

[0278] In some embodiments, the LNP formulation can contain PEG-c-DOMG at a lipid molar ratio of 3%. In some embodiments, the LNP formulation can contain PEG-c-DOMG at a lipid molar ratio of 1.5%.

[0279] In some embodiments, the pharmaceutical composition can include at least one of the PEGylated lipids described in International Publication No. WO2012099755, the content of which is incorporated herein by reference in its entirety.

[0280] In some embodiments, the LNP formulation can contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). In some embodiments, the LNP formulation can contain PEG-DMG 2000, a cationic lipid known in the art, and at least one other component. In some embodiments, the LNP formulation can contain PEG-DMG 2000, a cationic lipid known in the art, DSPC, and cholesterol. As a non-limiting example, the LNP formulation can contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, the LNP formulation can contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol in a molar ratio of 2:40:10:48 (see, for example, Geall et al., Nonviral delivery of self-amplifying RNA (e.g., mRNA) vaccines, Proceedings of the National Academy of Sciences of the United States of America (PNAS) 2012; PMID: 22908294, the contents of each of which are incorporated herein by reference in their entirety).

[0281] Sterol

[0282] In some embodiments, the nanoparticle includes a sterol. Sterols are well known to those skilled in the art and generally refer to compounds having a cyclopentanoperhydrophenanthrene ring system and having one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, and the like.

[0283] In some embodiments, the sterol is selected from cholesterol-based lipids. In some embodiments, one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol, and DC-Choi (N,N-dimethyl-N-ethylformamido cholesterol) and 1,4-bis(3-N-oleylaminopropyl)piperazine or combinations thereof.

[0284] Sterols can tune particle permeability and fluidity based on their function in cell membranes. In some embodiments, the sterol is cholesterol.

[0285] In some embodiments, the nanoparticle contains a sterol in a molar ratio of 20% to 80%. In some embodiments, the nanoparticle contains a sterol in a molar ratio of 25%, 30%, 35%, 40%, 45%, or 50%. In one embodiment, the nanoparticle contains a sterol in a molar ratio of 40%.

[0286] The nanoparticle formulation can be a carbohydrate nanoparticle comprising a carbohydrate carrier and a nucleic acid sequence (e.g., mRNA) as described herein. As a non-limiting example, the carbohydrate carrier can include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-like material, octenyl succinic acid phytoglycogen, phytoglycogen β-cyclodextrin, anhydride-modified phytoglycogen β-cyclodextrin. (See, e.g., International Publication No. WO2012109121; the content of the international publication is incorporated herein by reference in its entirety).

[0287] The nanoparticle formulations of the present disclosure can be coated with a surfactant or a polymer to improve particle delivery. In some embodiments, the nanoparticles can be coated with a hydrophilic coating such as, but not limited to, a PEG coating and / or a coating having a neutral surface charge. The hydrophilic coating may facilitate the delivery of nanoparticles with a large payload within the central nervous system, such as, but not limited to, RNA (e.g., mRNA). As a non-limiting example, nanoparticles comprising a hydrophilic coating and methods of preparing such nanoparticles are described in U.S. Patent Publication No. US20130183244, the content of which is incorporated herein by reference in its entirety.

[0288] In some embodiments, the lipid nanoparticles of the present disclosure can be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods of preparing hydrophilic polymer particles are described in U.S. Patent Publication No. US20130210991, the content of which is incorporated herein by reference in its entirety.

[0289] In some embodiments, the lipid nanoparticles of the present disclosure can be hydrophobic polymer particles.

[0290] In some embodiments, an immune response can be elicited by delivering lipid nanoparticles that can include nanospecies, polymers, and immunogens. (U.S. Publication No. 20120189700 and International Publication No. WO2012099805; each of the international publications is incorporated herein by reference in its entirety). The polymer can encapsulate the nanospecies or partially encapsulate the nanospecies. The immunogen can be a recombinant protein, a modified RNA, and / or a polynucleotide as described herein. In some embodiments, the lipid nanoparticles can be formulated for use in vaccines against, for example, but not limited to, pathogens.

[0291] Lipid nanoparticles can be engineered to alter the surface properties of the particles such that the lipid nanoparticles can penetrate mucosal barriers. Mucus is located on mucosal tissues such as, but not limited to, the oral cavity (e.g., buccal and esophageal membranes and tonsil tissue), eye, gastrointestinal tract (e.g., stomach, small intestine, large intestine, colon, rectum), nose, respiratory tract (e.g., nasal, pharyngeal, tracheal, and bronchial membranes), genitalia (e.g., vaginal, cervical, and urethral membranes). Nanoparticles larger than 10 - 200 nm, which are preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of multiple drugs, are considered too large to rapidly diffuse through mucosal barriers. Mucus is constantly secreted, shed, discarded, or digested and recycled, and thus most trapped particles can be removed from mucosal tissues within seconds or hours. Large polymeric nanoparticles (200 nm - 500 nm in diameter) densely coated with low molecular weight polyethylene glycol (PEG) have a diffusion ability through mucus that is 1 / 6 to 1 / 4 of the ability of the same particles to diffuse in water (Lai et al., Proceedings of the National Academy of Sciences of the United States of America 2007 104(5):1482 - 487; Lai et al., Advanced Drug Delivery Reviews 2009 61(2):158 - 171, each of which is incorporated herein by reference in its entirety). The delivery of nanoparticles can be determined using permeation rate and / or fluorescence microscopy techniques, including but not limited to fluorescence recovery after photobleaching (FRAP) and high - resolution multi - particle tracking (MPT). As a non - limiting example, a composition that can penetrate mucosal barriers can be prepared as described in U.S. Patent No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of which are incorporated herein by reference in their entirety.

[0292] Engineered to penetrate mucus, the lipid nanoparticles can comprise a polymeric material (i.e., polymeric core) and / or a polymer-vitamin conjugate and / or a triblock copolymer. The polymeric material can include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, poly(styrene), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material can be biodegradable and / or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No. WO2013116804, the content of which is incorporated herein by reference in its entirety. The polymeric material can additionally be irradiated. As a non-limiting example, the polymeric material can be gamma irradiated (see, e.g., International Application No. WO201282165, which is incorporated herein by reference in its entirety).Non-limiting examples of specific polymers include poly(ε-caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-ε-caprolactone), poly(D,L-lactide-co-ε-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), alkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(ester amide), polyamide, poly(ether ester), polycarbonate, polyalkylene such as polyethylene and polypropylene, polyalkylene glycol such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalate such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl ester such as poly(vinyl acetate), polyvinyl halide such as poly(vinyl chloride) (PVC), polyvinyl pyrrolidone, polysiloxane, polystyrene (PS), polyurethane, derived cellulose (such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose), polymers of acrylic acid (such as poly((meth)acrylate methyl) (PMMA), poly((meth)acrylate ethyl), poly((meth)acrylate butyl), poly((meth)acrylate isobutyl), poly((meth)acrylate hexyl), poly((meth)acrylate isodecyl), poly((meth)acrylate lauryl), poly((meth)acrylate phenyl), poly(acrylate methyl), poly(acrylate isopropyl), poly(acrylate isobutyl), poly(acrylate octadecyl) and their copolymers and mixtures, polydioxanone and its copolymers, polyhydroxyalkanoate, polypropylene fumarate, polyformaldehyde, poloxamer, poly(ortho)ester, poly(butyric acid), poly(valeric acid), poly(lactide-co-ε-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinyl pyrrolidone.Lipid nanoparticles can be coated with or associated with a copolymer, such as but not limited to a block copolymer (e.g., a branched polyether-polyamide block copolymer as described in International Publication No. WO2013012476, which is incorporated herein by reference in its entirety) and a (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see, e.g., U.S. Publication No. 20120121718, U.S. Publication No. 20100003337, and U.S. Patent No. 8,263,665, the contents of each of which are incorporated herein by reference in their entirety). The copolymer can be a generally recognized as safe (GRAS) polymer, and the lipid nanoparticles can be formed in a manner that does not create a new chemical entity. For example, the lipid nanoparticles can contain poloxamer, which coats PLGA nanoparticles without forming a new chemical entity that is still capable of rapidly penetrating human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; the content of which is incorporated herein by reference in its entirety). Xu et al. described a non-limiting scalable method for generating nanoparticles that can penetrate human mucus (see Journal of Controlled Release 2013, 170(2):279-86; the content of which is incorporated herein by reference in its entirety).

[0293] The vitamin of the polymer-vitamin conjugate can be vitamin E. The vitamin moiety of the conjugate can be replaced by other suitable components such as but not limited to vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or the hydrophobic component of other surfactants (e.g., a sterol chain, a fatty acid, a hydrocarbon chain, and an oxyalkylene chain).

[0294] Engineered to penetrate mucus, lipid nanoparticles can include surface modifiers such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., N-acetylcysteine, artemisia, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, alfa-dornase, neltenexine, erdosteine), and various DNA enzymes including rhDNase. The surface modifiers can be entrapped or incorporated into the surface of the particles or disposed (e.g., by coating, adsorption, covalent bonding, or other processes) on the surface of the lipid nanoparticles. (See, e.g., U.S. Publication 20100215580 and U.S. Publication 20080166414 and US20130164343; the contents of each of which are hereby incorporated by reference in their entirety).

[0295] In some embodiments, the mucus-penetrating lipid nanoparticles can comprise at least one polynucleotide as described herein. The polynucleotide can be encapsulated within the lipid nanoparticles and / or disposed on the surface of the particles. The polynucleotide can be covalently coupled to the lipid nanoparticles. Formulations of the mucus-penetrating lipid nanoparticles can comprise a plurality of nanoparticles.

[0296] In some embodiments, the composition can be formulated as solid lipid nanoparticles. The solid lipid nanoparticles (SLN) can be spherical and have an average diameter between 10 nm and 1000 nm. The SLN has a solid lipid core matrix that can dissolve lipophilic molecules and can be stabilized by surfactants and / or emulsifiers. In some embodiments, the lipid nanoparticles can be self-assembled lipid polymer nanoparticles (see Zhang et al., ACS Nano, 2008, 2(8), pp. 1696-1702; the content of which is incorporated herein by reference in its entirety). As a non-limiting example, the SLN can be the SLN described in International Patent Publication No. WO2013105101, the content of which is incorporated herein by reference in its entirety. As another non-limiting example, the SLN can be prepared by the method or process described in International Patent Publication No. WO2013105101, the content of which is incorporated herein by reference in its entirety.

[0297] In some embodiments, the compositions of the present disclosure can be encapsulated in nanoparticles. The nanoparticles can be formulated by methods known in the art and described herein, such as, but not limited to, International Publications Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, U.S. Publications Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286, US20120288541, US20130123351, and US20130230567, and U.S. Patents Nos. 8,206,747, 8,293,276, 8,318,208, and 8,318,211, the content of each of which is incorporated herein by reference in its entirety. In some embodiments, the polymer nanoparticles can be identified by the method described in U.S. Publication No. US20120140790, the content of which is incorporated herein by reference in its entirety.

[0298] In some embodiments, the nanoparticles can be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that conforms to a certain release rate over a specific period of time. The period of time can include, but is not limited to, hours, days, weeks, months, and years. As a non-limiting example, the sustained release nanoparticles can comprise the polymers and nucleic acid sequences of the present disclosure (see International Publication No. 2010075072 and U.S. Publications Nos. US20100216804, US20110217377, and US20120201859, the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, the sustained release formulation can comprise agents that allow for sustained bioavailability, such as, but not limited to, crystals, macromolecular gels, and / or particulate suspensions (see U.S. Patent Publication No. US20130150295, the contents of which are incorporated herein by reference in their entirety).

[0299] In some embodiments, the nanoparticles of the present disclosure can comprise a polymeric matrix. As a non-limiting example, the nanoparticles can comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactic acid-co-L-lysine), poly(4-hydroxy-L-proline ester), or combinations thereof.

[0300] In some embodiments, the nanoparticles comprise a diblock copolymer. In some embodiments, the diblock copolymer can comprise a combination of PEG and a polymer, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactic acid-co-L-lysine), poly(4-hydroxy-L-proline ester), or combinations thereof. In yet another embodiment, the diblock copolymer can be a high-X diblock copolymer, such as the diblock copolymer described in International Patent Publication No. WO2013120052, the contents of which are incorporated herein by reference in their entirety.

[0301] As a non-limiting example, the nanoparticles comprise a PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, the contents of each of which are hereby incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticles are stealth nanoparticles comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968 and International Publication No. WO2012166923, the contents of each of which are hereby incorporated by reference in their entirety). In yet another non-limiting example, the nanoparticles are stealth nanoparticles or target-specific stealth nanoparticles as described in U.S. Patent Publication No. US20130172406, the content of which is hereby incorporated by reference in its entirety.

[0302] In some embodiments, the nanoparticles may comprise a multiblock copolymer (see, for example, U.S. Patent Nos. 8,263,665 and 8,287,910 and U.S. Patent Publication No. US20130195987, the contents of each of which are hereby incorporated by reference in their entirety).

[0303] In yet another non-limiting example, the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see, e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a Tgf-β1 gene delivery vehicle in Lee et al., Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle Enhances Diabetic Wound Healing, Pharmaceutical Research, 2003, 20(12):1995-2000; was used as a controlled gene delivery system in Li et al., Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel, Pharmaceutical Research, 2003, 20(6):884-888; and Chang et al., Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle, Journal of Controlled Release, 2007, 118:245-253, the contents of each of which are hereby incorporated by reference in their entirety). The nucleic acid (e.g., mRNA) vaccines of the present disclosure can be formulated in lipid nanoparticles comprising the PEG-PLGA-PEG block copolymer.

[0304] In some embodiments, the nanoparticles can comprise a multi-block copolymer (see, e.g., U.S. Patent Nos. 8,263,665 and 8,287,910 and U.S. Patent Publication No. US20130195987, the contents of each of which are hereby incorporated by reference in their entirety).

[0305] In some embodiments, the block copolymers described herein can be included in polyion complexes comprising non-polymeric micelles and block copolymers. (See U.S. Publication No. 20120076836; the contents of which are hereby incorporated by reference in their entirety).

[0306] In some embodiments, the nanoparticles can comprise at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, amine alkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, and combinations thereof.

[0307] In some embodiments, the nanoparticles can comprise at least one poly(vinyl ester) polymer. The poly(vinyl ester) polymer can be a copolymer such as a random copolymer. As a non-limiting example, the random copolymer can have a structure as described in International Application No. WO2013032829 or U.S. Patent Application No. US20130121954, the content of each of which is incorporated herein by reference in its entirety. In some embodiments, the poly(vinyl ester) polymer can be conjugated to the polynucleotides described herein.

[0308] In some embodiments, the nanoparticles can comprise at least one diblock copolymer. The diblock copolymer can be, but is not limited to, a poly(lactic acid)-poly(vinyl alcohol) copolymer (see, for example, International Patent Publication No. WO2013044219, the content of which is incorporated herein by reference in its entirety). As a non-limiting example, the nanoparticles can be used for treating cancer (see International Publication No. WO2013044219, the content of which is incorporated herein by reference in its entirety).

[0309] In some embodiments, the nanoparticles can comprise at least one cationic polymer described herein and / or known in the art.

[0310] In some embodiments, the nanoparticles can comprise at least one amine-containing polymer such as, but not limited to, polylysine, polyethyleneimine, poly(aminoamine) dendrimers, poly(β-amino esters) (see, for example, U.S. Patent No. 8,287,849, the content of which is incorporated herein by reference in its entirety), and combinations thereof.

[0311] In some embodiments, the nanoparticles can comprise at least one degradable polyester, and the at least one degradable polyester can contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyester can include PEG conjugation to form a PEGylated polymer.

[0312] In some embodiments, the composition can be formulated in colloidal nanocarriers as described in U.S. Patent Publication No. US20130197100, the contents of which are incorporated herein by reference in their entirety.

[0313] In some embodiments, the nanoparticles can be optimized for oral administration. The nanoparticles can comprise at least one cationic biopolymer, such as but not limited to chitosan or its derivatives. As a non-limiting example, the nanoparticles can be formulated by the method described in U.S. Publication No. 20120282343, the contents of which are incorporated herein by reference in their entirety.

[0314] In some embodiments, the LNP comprises lipid KL52 (an amino lipid disclosed in U.S. Application Publication No. 2012 / 0295832, the contents of which are incorporated herein by reference in their entirety). The activity and / or safety of LNP administration (as measured by examining, for example, one or more of ALT / AST, white blood cell count, and cytokine induction) can be enhanced by incorporating such lipids. The LNP comprising KL52 can be administered intravenously and / or in one or more doses. In some embodiments, the administration of the LNP comprising KL52 results in equal or enhanced mRNA and / or protein expression compared to the administration of the LNP comprising MC3.

[0315] In some embodiments, the lipid nanoparticles can be the lipid nanoparticles of limiting size as described in International Patent Publication No. WO2013059922, the contents of which are incorporated herein by reference in their entirety. The lipid nanoparticles of limiting size can comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; wherein the lipid bilayer can comprise phospholipids, such as but not limited to diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, C8-C20 fatty acid diacyl phosphatidylcholine, and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In some embodiments, the lipid nanoparticles of limiting size can comprise polyethylene glycol lipids, such as but not limited to DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0316] In some embodiments, the composition can be formulated in nanoparticles that comprise a core including acellular material and an outer surface including a cell membrane. The cell membrane can be derived from a cell or a virus-derived membrane. As a non-limiting example, the nanoparticles can be prepared by the method described in International Patent Publication No. WO2013052167, the content of which is incorporated herein by reference in its entirety. As another non-limiting example, the nanoparticles described in International Patent Publication No. WO2013052167 can be used to deliver the composition described herein, the content of which is incorporated herein by reference in its entirety.

[0317] In some embodiments, the composition can be formulated in a porous nanoparticle-supported lipid bilayer (protocell). Protocells are described in International Patent Publication No. WO2013056132, the content of which is incorporated herein by reference in its entirety.

[0318] In some embodiments, the composition described herein can be formulated in polymer nanoparticles as described in U.S. Pat. Nos. 8,420,123 and 8,518,963 and European Patent No. EP2073848B1, or can be prepared by the methods described in those references, the content of each of which is incorporated herein by reference. As a non-limiting example, the glass transition temperature of the polymer nanoparticles can be high, such as the nanoparticles described in U.S. Pat. No. 8,518,963 or prepared by the methods described in that reference, the content of which is incorporated herein by reference. As another non-limiting example, polymer nanoparticles for oral and parenteral formulations can be prepared by the method described in European Patent No. EP2073848B1, the content of which is incorporated herein by reference.

[0319] In some embodiments, the composition described herein can be formulated in nanoparticles for imaging. The nanoparticles can be liposome nanoparticles, such as the liposome nanoparticles described in U.S. Patent Publication No. US20130129636, which is incorporated herein by reference. As a non-limiting example, the liposome can comprise gadolinium(III) 2-{4,7-bis-carboxymethyl-10-[(N,N-distearoyl-aminomethyl-N'-amino-methyl]-1,4,7,10-tetra-azacyclododecane-1-yl}-acetate and a neutral fully saturated phospholipid component (see, e.g., U.S. Patent Publication No. US20130129636, the content of which is incorporated herein by reference).

[0320] In some embodiments, the nanoparticles that can be used in the present disclosure are formed by the method described in U.S. Patent Application No. US20130130348, the content of which is incorporated herein by reference in its entirety.

[0321] In some embodiments, the compositions of the present disclosure can be formulated in the form of swellable nanoparticles. The swellable nanoparticles can be, but are not limited to, the swellable nanoparticles described in U.S. Patent No. 8,440,231, the content of which is incorporated herein by reference in its entirety. As a non-limiting example, the swellable nanoparticles can be used to deliver the RNA (e.g., mRNA) vaccines of the present disclosure to the pulmonary system (see, e.g., U.S. Patent No. 8,440,231, the content of which is incorporated herein by reference in its entirety).

[0322] The compositions of the present disclosure can be formulated in polyanhydride nanoparticles, such as, but not limited to, the nanoparticles described in U.S. Patent No. 8,449,916, the content of which is incorporated herein by reference in its entirety.

[0323] The nanoparticles and microparticles of the present disclosure can be geometrically engineered to modulate macrophages and / or immune responses. In some embodiments, the geometrically engineered particles can have different shapes, sizes, and / or surface charges in order to incorporate the polynucleotides of the present disclosure for targeted delivery, such as, but not limited to, pulmonary delivery (see, e.g., International Publication No. WO2013082111, the content of which is incorporated herein by reference in its entirety). Other physical characteristics that the geometrically engineered particles can have include, but are not limited to, fenestrations, angled arms, asymmetry, and surface roughness, and charges that can alter the interaction with cells and tissues. As a non-limiting example, the nanoparticles of the present disclosure can be prepared by the method described in International Publication No. WO2013082111, the content of which is incorporated herein by reference in its entirety.

[0324] In some embodiments, the nanoparticles of the present disclosure can be water-soluble nanoparticles, such as, but not limited to, the water-soluble nanoparticles described in International Publication No. WO2013090601, the content of which is incorporated herein by reference in its entirety. The nanoparticles can be inorganic nanoparticles that have compact and zwitterionic ligands in order to exhibit good water solubility. The nanoparticles can also have a small hydrodynamic diameter (HD), stability in terms of time, pH, and salinity, and a low level of non-specific protein binding.

[0325] In some embodiments, the nanoparticles of the present disclosure can be developed by the method described in U.S. Patent Publication No. US20130172406, the content of which is incorporated herein by reference in its entirety.

[0326] In some embodiments, the nanoparticles of the present disclosure are stealth nanoparticles or target - specific stealth nanoparticles, such as but not limited to the stealth nanoparticles or target - specific stealth nanoparticles described in U.S. Patent Publication No. US20130172406, the content of which is incorporated herein by reference in its entirety. The nanoparticles of the present disclosure can be prepared by the method described in U.S. Patent Publication No. US20130172406, the content of which is incorporated herein by reference in its entirety.

[0327] In some embodiments, the stealth nanoparticles or target - specific stealth nanoparticles can comprise a polymer matrix. The polymer matrix can comprise two or more polymers, such as but not limited to polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(ortho ester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polyester, polyanhydride, polyether, polyurethane, polymethacrylate, polyacrylate, polycyanoacrylate, or combinations thereof.

[0328] In some embodiments, the nanoparticles can be nanoparticle - nucleic acid hybrid structures having a high - density nucleic acid layer. As a non - limiting example, the nanoparticle - nucleic acid hybrid structure can be prepared by the method described in U.S. Patent Publication No. US20130171646, the content of which is incorporated herein by reference in its entirety. The nanoparticles can comprise nucleic acids, such as but not limited to polynucleotides described herein and / or known in the art.

[0329] At least one of the nanoparticles of the present disclosure can be embedded in a core nanostructure or coated with a low - density porous 3D structure or coating that is capable of carrying at least one payload or associating with the at least one payload within or on the surface of the nanostructure. Non - limiting examples of nanostructures comprising at least one nanoparticle are described in International Patent Publication No. WO2013123523, the content of which is incorporated herein by reference in its entirety.

[0330] In some embodiments, the composition can be delivered using smaller LNPs. Such particles can have a diameter of less than 0.1 μm down to 100 nm, such as but not limited to less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, less than 250 μm, less than 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm, less than 450 μm, less than 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, less than 675 μm, less than 700 μm, less than 725 μm, less than 750 μm, less than 775 μm, less than 800 μm, less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm, less than 925 μm, less than 950 μm, less than 975 μm or less than 1000 μm.

[0331] In some embodiments, the composition can be delivered using smaller LNPs, which can have diameters of about 1 nm to about 100 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60 nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 10 nm to about 50 nm, about 20 nm to about 50 nm, about 30 nm to about 50 nm, about 40 nm to about 50 nm, about 20 nm to about 60 nm, about 30 nm to about 60 nm, about 40 nm to about 60 nm, about 20 nm to about 70 nm, about 30 nm to about 70 nm, about 40 nm to about 70 nm, about 50 nm to about 70 nm, about 60 nm to about 70 nm, about 20 nm to about 80 nm, about 30 nm to about 80 nm, about 40 nm to about 80 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 20 nm to about 90 nm, about 30 nm to about 90 nm, about 40 nm to about 90 nm, about 50 nm to about 90 nm, about 60 nm to about 90 nm, and / or about 70 nm to about 90 nm.

[0332] In some embodiments, such LNPs are synthesized using a method that includes a microfluidic mixer. Examples of microfluidic mixers can include, but are not limited to, a slotted interdigital micromixer and / or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al.), the slotted interdigital micromixer including, but not limited to, a slotted interdigital micromixer manufactured by Microinnova (Microinnova) (Allerheiligen bei Wildon, Austria), for which the bottom-up design and synthesis of a lipid nanoparticle system of ultimate size with aqueous and triglyceride cores using millisecond microfluidic mixing has been published (Langmuir. 2012. 28:3633-40; Belliveau, N.M. et al., Microfluidic synthesis of highly effective ultimate-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51, the content of each of which is incorporated herein by reference in its entirety). In some embodiments, the method of producing LNPs that include an SHM further includes mixing at least two input streams, wherein the mixing occurs by microstructured-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern, thereby causing a swirling flow and folding the fluids around each other. This method can also include a surface for fluid mixing, wherein the surface changes orientation during fluid circulation. The methods of producing LNPs using an SHM include the methods disclosed in U.S. Application Publication Nos. 2004 / 0262223 and 2012 / 0276209, the content of each of which is incorporated herein by reference in its entirety.

[0333] In some embodiments, the compositions of the present disclosure can be formulated in lipid nanoparticles produced using a micromixer, such as but not limited to a mixer with a slotted interdigital microstructure (SIMM-V2) or a standard slotted interdigital micromixer (SSIMM) or a Caterpillar (CPMM) or an impinging jet (IJMM) from the Institut für Mikrotechnik Mainz GmbH, Mainz, Germany.

[0334] In some embodiments, the compositions of the present disclosure can be formulated in lipid nanoparticles produced using microfluidic techniques (see, e.g., Whitesides, George M. The Origins and the Future of Microfluidics. Nature 2006 442:368 - 373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647 - 651; each of which is hereby incorporated by reference in its entirety). As a non - limiting example, controlled microfluidic formulations include passive methods for mixing streams of a steady pressure - driven flow in microchannels at low Reynolds number (see, e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647 - 651, the contents of which are hereby incorporated by reference in its entirety).

[0335] In some embodiments, the compositions of the present disclosure can be formulated in lipid nanoparticles produced using a micromixer chip, such as but not limited to a micromixer chip from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). The micromixer chip can be used for rapid mixing of two or more fluid streams with a splitting and recombining mechanism.

[0336] In some embodiments, the compositions of the present disclosure can be formulated in lipid nanoparticles having a diameter of from about 10 nm to about 100 nm, such as, but not limited to, from about 10 nm to about 20 nm, from about 10 nm to about 30 nm, from about 10 nm to about 40 nm, from about 10 nm to about 50 nm, from about 10 nm to about 60 nm, from about 10 nm to about 70 nm, from about 10 nm to about 80 nm, from about 10 nm to about 90 nm, from about 20 nm to about 30 nm, from about 20 nm to about 40 nm, from about 20 nm to about 50 nm, from about 20 nm to about 60 nm, from about 20 nm to about 70 nm, from about 20 nm to about 80 nm, from about 20 nm to about 90 nm, from about 20 nm to about 100 nm, from about 30 nm to about 40 nm, from about 30 nm to about 50 nm, from about 30 nm to about 60 nm, from about 30 nm to about 70 nm, from about 30 nm to about 80 nm, from about 30 nm to about 90 nm, from about 30 nm to about 100 nm, from about 40 nm to about 50 nm, from about 40 nm to about 60 nm, from about 40 nm to about 70 nm, from about 40 nm to about 80 nm, from about 40 nm to about 90 nm, from about 40 nm to about 100 nm, from about 50 nm to about 60 nm, from about 50 nm to about 70 nm, from about 50 nm to about 80 nm, from about 50 nm to about 90 nm, from about 50 nm to about 100 nm, from about 60 nm to about 70 nm, from about 60 nm to about 80 nm, from about 60 nm to about 90 nm, from about 60 nm to about 100 nm, from about 70 nm to about 80 nm, from about 70 nm to about 90 nm, from about 70 nm to about 100 nm, from about 80 nm to about 90 nm, from about 80 nm to about 100 nm, and / or from about 90 nm to about 100 nm.

[0337] In some embodiments, the diameter of the lipid nanoparticles can be from about 10 nm to 500 nm.

[0338] In some embodiments, the diameter of the lipid nanoparticles can be greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm.

[0339] The present disclosure also describes a composition comprising an effective amount of the nanoparticles described herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition can comprise a pharmaceutically acceptable carrier and nanoparticles comprising a nucleic acid sequence encoding a pathogen antigen polypeptide (e.g., mRNA) and a nucleic acid sequence encoding at least one universal T cell epitope (e.g., mRNA), and a composition comprising a nucleic acid sequence encoding a pathogen antigen polypeptide and at least one universal T cell epitope (e.g., mRNA). In some aspects, the pharmaceutical composition can comprise a pharmaceutically acceptable carrier and lipid nanoparticles comprising a nucleic acid sequence encoding a pathogen antigen polypeptide (e.g., mRNA) and a nucleic acid sequence encoding at least one universal T cell epitope (e.g., mRNA), and a composition comprising a nucleic acid sequence encoding a pathogen antigen polypeptide and at least one universal T cell epitope (e.g., mRNA).

[0340] Liposomes, lipid complexes, or lipid nanoparticles can be used to enhance the efficacy of polynucleotide-directed protein production, as these formulations may be able to increase cellular transfection by RNA (e.g., mRNA) polynucleotides; and / or increase the translation of the encoded protein. One such example involves the use of lipid encapsulation to enable efficient systemic delivery of polyplexed plasmid DNA (Heyes et al., Molecular Therapy 2007 15:713-720; the contents of which are incorporated herein by reference in their entirety). Liposomes, lipid complexes, or lipid nanoparticles can also be used to enhance the stability of polynucleotides.

[0341] Liposomes are artificially prepared vesicles that can consist primarily of a lipid bilayer and can be used as delivery vehicles for administering nutrient and drug formulations. Liposomes can have different sizes, such as but not limited to multilamellar vesicles (MLV), which can have a diameter of several hundred nanometers and can contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUV), which can have a diameter of less than 50 nm; and large unilamellar vesicles (LUV), which can have a diameter between 50 nm and 500 nm. Liposome design can include but not limited to opsonins or ligands to improve the attachment of liposomes to unhealthy tissues or to activate events such as but not limited to endocytosis. Liposomes can contain a low or high pH to improve the delivery of drug formulations.

[0342] The formation of liposomes can depend on physicochemical characteristics such as but not limited to, the entrapped drug formulation and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimized size, the polydispersity of the vesicles for the intended application, and the shelf life, as well as the batch-to-batch reproducibility and the possibility of large-scale production of a safe and efficient liposome product.

[0343] In some embodiments, the pharmaceutical compositions described herein may include, but are not limited to, liposomes such as 1,2-dioleoyl-oxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleoyl-oxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; which is hereby incorporated by reference in its entirety), and liposomes formed from small molecule drugs such as, but not limited to, those from Janssen Biotech, Inc. (Horsham, Pa.).

[0344] ​In some embodiments, the pharmaceutical compositions described herein can include, but are not limited to, liposomes such as those formed by the synthesis of stable plasmid-lipid particles (SPLP) or stable nucleic acid lipid particles (SNALP) which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al., Gene Therapy. 1999 6:271-281; Zhang et al., Gene Therapy. 1999 6:1438-1447; Jeffs et al., Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al., Journal of Controlled Release. 2005 107:276-287; Semple et al., Nat Biotechnol. 2010 28:172-176; Judge et al., J Clin Invest. 2009 119:661-673; de Fougerolles, Hum Gene Ther. 2008 19:125-132; US Patent Publication No. US20130122104; the entire contents of the aforementioned documents are incorporated herein by reference in their entirety). The initial manufacturing method of Wheeler et al. was the detergent dialysis method, which was later improved by Jeffs et al. and is known as the spontaneous vesicle formation method. In addition to polynucleotides, liposome formulations are composed of 3 to 4 lipid components. As an example, liposomes can contain, but are not limited to, 55% cholesterol, 20% distearoylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleoyl-oxy-N,N-dimethylaminopropane (DODMA) as described by Jeffs et al. As another example, certain liposome formulations can contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid as described by Heyes et al., where the cationic lipid can be 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinoleyloxy-3-dimethylaminopropane (DLenDMA).

[0345] In some embodiments, the liposome formulation can comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol, and / or from about 48.5% cholesterol to about 60% cholesterol. In some embodiments, the formulation can comprise a percentage of cholesterol selected from the group consisting of: 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0%, and 43.5%. In some embodiments, the formulation can comprise from about 5.0% to about 10.0% DSPC and / or from about 7.0% to about 15.0% DSPC.

[0346] In some embodiments, the composition can be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al., Cancer Biology & Therapy 2006 5(12)1708-1713); which is hereby incorporated by reference in its entirety), and hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel).

[0347] In some embodiments, the composition can be formulated in lipid vesicles that can have crosslinks between functionalized lipid bilayers.

[0348] In some embodiments, the composition can be formulated in lipid-polycation complexes. Formation of the lipid-polycation complexes can be accomplished by methods known in the art and / or as described in U.S. Publication No. 20120178702, which is hereby incorporated by reference in its entirety. As a non-limiting example, the polycation can include cationic peptides or polypeptides such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the composition can be formulated in lipid polycation complexes that can further include non-cationic lipids such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0349] In some embodiments, the composition can be formulated as a lipid complex such as, but not limited to, ATUPLEX from Silence Therapeutics (London, United Kingdom)TM systems, DACC systems, DBTC systems, and other siRNA-lipid complex technologies, from (STEMFECT, Cambridge, Mass.) TM and the targeted and non-targeted delivery of nucleic acids based on polyethylenimine (PEI) or protamine (Aleku et al., Cancer Res. 2008, 68:9788-9798; Strumberg et al., Int J Clin Pharmacol Ther. 2012, 50:76-78; Santel et al., Gene Ther. 2006, 13:1222-1234; Santel et al., Gene Ther. 2006, 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010, 23:334-344; Kaufmann et al., Microvasc Res. 2010, 80:286-293; Weide et al., J Immunother. 2009, 32:498-507; Weide et al., J Immunother. 2008, 31:180-188; Pascolo, Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., J Immunother. 2011, 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007, 104:4095-4100; de Fougerolles, Hum Gene Ther. 2008, 19:125-132; the content of each of the documents is incorporated herein by reference in its entirety).

[0350] In some embodiments, such formulations can also be engineered or the composition altered such that it is passively or actively directed in vivo to different cell types, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and white blood cells (Akinc et al., Molecular Therapy 2010 18:1357-1364; Song et al., Nature Biotechnology 2005 23:709-717; Judge et al., The Journal of Clinical Investigation 2009 119:661-673; Kaufmann et al., Microvascular Research 2010 80:286-293; Santel et al., Gene Therapy 2006 13:1222-1234; Santel et al., Gene Therapy 2006 13:1360-1370; Gutbier et al., Pulmonary Pharmacology & Therapeutics 2010 23:334-344; Basha et al., Molecular Therapy 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science 2008 319:627-630; Peer and Lieberman, Gene Therapy 2011 18:1127-1133, the contents of each of which are incorporated herein by reference in their entirety). An example of a formulation that passively targets hepatocytes includes lipid nanoparticle formulations based on DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA, which have been shown to bind to apolipoprotein E and facilitate the binding and uptake of these formulations into hepatocytes in vivo (Akinc et al., Molecular Therapy 2010 18:1357-1364, the contents of which are incorporated herein by reference in their entirety).The formulations can also be selectively targeted by the expression of different ligands on their surface, as exemplified by but not limited to folic acid, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeting methods (Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release 20:63-68; Peer et al., Proc Natl Acad Sci U S A. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133, the contents of each of which are incorporated herein by reference in their entirety).

[0351] In some embodiments, the compositions of the present disclosure can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a drug composition or compound release profile that conforms to a specific release pattern to achieve a therapeutic outcome. In some embodiments, the composition can be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulation" means enclosing, surrounding, or encapsulating. Since it relates to the formulation of the compounds of the present disclosure, the encapsulation can be substantial, complete, or partial. The term "substantially encapsulated" means that at least greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.9% or greater than 99.999% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded, or encapsulated within the delivery agent. "Partially encapsulated" means that less than 10%, 10%, 20%, 30%, 40%, 50% or less of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded, or encapsulated within the delivery agent. Advantageously, the encapsulation can be determined by measuring the escape or activity of the pharmaceutical composition or compound of the present disclosure using fluorescence and / or electron micrographs. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or greater than 99.99% of the pharmaceutical composition or compound of the present disclosure is encapsulated in the delivery agent.

[0352] In some embodiments, the controlled release formulation can include, but is not limited to, triblock copolymers. As a non-limiting example, the formulation can include two different types of triblock copolymers (International Publication Nos. WO2012131104 and

[0353] WO2012131106, the contents of each of which are incorporated herein by reference in their entirety).

[0354] In some embodiments, the composition can be encapsulated into lipid nanoparticles or rapidly eliminated lipid nanoparticles, and the lipid nanoparticles or rapidly eliminated lipid nanoparticles can then be encapsulated into a polymer, hydrogel, and / or surgical sealant described herein and / or known in the art. As a non-limiting example, the polymer, hydrogel, or surgical sealant can be PLGA, ethylene vinyl acetate (EVAc), poloxamer, (Nanotherapeutics, Inc. Alachua, Fla.) (Halozyme Therapeutics, San Diego, Calif.)、surgical sealants such as fibrinogen polymers (Ethicon Inc., Cornelia, Ga.), (Baxter International, Inc., Deerfield, Ill.), PEG-based sealants, and (Baxter International, Inc., Deerfield, Ill.).

[0355] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery may further include at least one degradable polyester, which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine esters), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline esters), and combinations thereof. In some embodiments, the degradable polyester may include PEG conjugated to form a PEGylated polymer.

[0356] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery may further include at least one PEG and / or PEG-related polymer derivative as described in U.S. Patent No. 8,404,222, the contents of which are incorporated herein by reference in their entirety.

[0357] In some embodiments, the compositions described herein formulated for controlled release delivery may be the controlled release polymer system described in US20130130348, the contents of which are incorporated herein by reference in their entirety.

[0358] In some embodiments, synthetic nanocarriers may be formulated for targeted release. In some embodiments, synthetic nanocarriers are formulated to release polynucleotides at a specified pH and / or after a desired time interval. As a non-limiting example, synthetic nanoparticles may be formulated to release an RNA (e.g., mRNA) vaccine after 24 hours and / or at a pH of 4.5 (see International Publications No. WO2010138193 and No. WO2010138194 and U.S. Publications No. US20110020388 and No. US20110027217, each of which is incorporated herein by reference in its entirety).

[0359] In some embodiments, the synthetic nanocarriers can be formulated for controlled and / or sustained release of the nucleic acids described herein. As a non-limiting example, synthetic nanocarriers for sustained release can be formulated by methods known in the art, described herein, and / or as described in International Publication No. WO2010138192 and U.S. Publication No. 20100303850, each of which is incorporated herein by reference in its entirety.

[0360] In some embodiments, the synthetic nanocarriers can be formulated to be used as vaccines. In some embodiments, the synthetic nanocarriers can encapsulate at least one polynucleotide encoding at least one antigen. As a non-limiting example, the synthetic nanocarriers can include at least one antigen and an excipient for a vaccine formulation (see International Publication No. WO2011150264 and U.S. Publication No. US20110293723, the content of each of which is incorporated herein by reference in its entirety). As another non-limiting example, the vaccine formulation can include at least two synthetic nanocarriers with the same or different antigens and excipients (see International Publication No. WO2011150249 and U.S. Publication No. US20110293701, the content of each of which is incorporated herein by reference in its entirety). The vaccine formulation can be selected by methods described herein, known in the art, and / or as described in International Publication No. WO2011150258 and U.S. Publication No. US20120027806, the content of each of which is incorporated herein by reference in its entirety).

[0361] In some embodiments, the synthetic nanocarriers can encapsulate at least one polynucleotide encoding a peptide, fragment, or region from a virus. As a non-limiting example, the synthetic nanocarriers can include, but are not limited to, any of the nanocarriers described in International Publication Nos. WO2012024621, WO201202629, WO2012024632 and U.S. Publication Nos. US20120064110, US20120058153 and US20120058154, the content of each of which is incorporated herein by reference in its entirety.

[0362] In some embodiments, the composition can be formulated in colloidal nanocarriers as described in U.S. Patent Publication No. US20130197100, the content of which is incorporated herein by reference in its entirety.

[0363] In some embodiments, the compositions of the present disclosure can be formulated for delivery using nanospheres encapsulating a drug as described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, the contents of each of which are incorporated herein by reference in their entirety. The microspheres can comprise compounds of formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are incorporated herein by reference in their entirety. In some embodiments, amino acids, peptides, polypeptides, lipids (APPL) can be used to deliver the RNA (e.g., mRNA) polynucleotides of the present disclosure to cells (see International Patent Publication No. WO2013063468, the contents of which are incorporated herein by reference in their entirety).

[0364] In some embodiments, the compositions can be delivered, positioned, and / or concentrated at a specified location using the delivery methods described in International Patent Publication No. WO2013063530, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, empty polymer particles can be administered to a subject before, simultaneously with, or after delivering a nucleic acid (e.g., mRNA) composition to the subject. The empty polymer particles undergo a volume change upon contact with the subject and become stuck, embedded, fixed, or trapped at a specific location within the subject's body.

[0365] In some embodiments, the compositions can be formulated in an active agent release system (see, e.g., U.S. Patent Publication No. US20130102545, the contents of which are incorporated herein by reference in their entirety). The active agent release system can comprise 1) at least one nanoparticle bonded to an oligonucleotide inhibitor strand that hybridizes to a catalytically active nucleic acid and 2) a compound bonded to a substrate molecule that is bonded to at least one therapeutic active agent (e.g., the polynucleotides described herein), wherein the therapeutic active agent is released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0366] In some embodiments, the compositions can be associated with a cationic compound or polycationic compound, the cationic compound or polycationic compound including protamine, nucleolin, spermine or spermidine or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell-penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP 22 derived peptides or mimetic peptides, Pestivirus Ems, HSV, VP 22(Herpes simplex virus), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptide, arginine-rich peptide, lysine-rich peptide, MPG peptide, Pep-1, L-oligomer, calcitonin peptide, Antennapedia-derived peptide from Drosophila melanogaster (especially from the antennapedia mutant of Drosophila), pAntp, plsl, FGF, lactoferrin, transferrin, magainin-2, Bac7 15-24, SynB, SynB(1), pVEC, hCT-derived peptide, SAP, histone, cationic polysaccharide (e.g., chitosan), polybrene, cationic polymer (e.g., polyethyleneimine (PEI)), cationic lipid (e.g., DOTMA: [1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleoyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycyl spermine, DIMRI: dimyristyloxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-tetracosanoyl-N-α-trimethylammonioacetyl)diethanolammonium chloride, CLIP 1: rac-[(2,3-di-octadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-di-hexadecyloxypropoxy methoxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-di-hexadecyloxypropoxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine or cationic or polycationic polymer, e.g., modified polyamino acid (such as β-amino acid polymer or reverse polyamide, etc.), modified polyethylene (such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc.), modified acrylate (such as pDMAEMA (poly(dimethylaminoethyl methacrylate)), etc.), modified amidoamine (such as pAMAM (poly(amidoamine)), etc.), modified poly-β-amino ester (PBAE) (such as diamine-terminally modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymer, etc.), dendrimers (such as polypropylamine dendrimers or pAMAM-based dendrimers, etc.), polyimines (such as PEI: poly(ethylene imine), poly(propylene imine), etc.), polyallylamine, sugar backbone-based polymers (such as cyclodextrin-based polymers, dextran-based polymers, chitosan, etc.), silane backbone-based polymers (such as PMOXA-PDMS copolymers, etc.), block polymers composed of a combination of one or more cationic blocks (e.g., selected from the cationic polymers mentioned above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol), etc.

[0367] In other embodiments, the composition does not associate with a cationic compound or a polycationic compound.

[0368] Vaccine

[0369] Vaccines comprising the compositions described herein are described herein. In some aspects, vaccines comprising a nucleic acid sequence (e.g., mRNA) encoding an infectious agent antigen polypeptide and a nucleic acid sequence (e.g., mRNA) encoding at least one universal T cell epitope, and vaccines comprising a nucleic acid sequence (e.g., mRNA) encoding an infectious agent antigen polypeptide and at least one universal T cell epitope are described herein.

[0370] The vaccines described herein can include multiple nucleic acid sequences (e.g., mRNA) each encoding a single infectious agent antigen polypeptide and multiple nucleic acid sequences (e.g., mRNA) each encoding a single universal T cell epitope, and vaccines comprising a single nucleic acid sequence (e.g., mRNA) encoding one or more infectious agent antigen polypeptides and a single nucleic acid sequence (e.g., mRNA) encoding one or more universal T cell epitopes.

[0371] The vaccines described herein can include multiple nucleic acid sequences (e.g., mRNA) each encoding a single infectious agent antigen polypeptide and a single universal T cell epitope, and vaccines comprising a single nucleic acid sequence (e.g., mRNA) encoding one or more infectious agent antigen polypeptides and one or more universal T cell epitopes.

[0372] In some embodiments, the vaccines of the present disclosure include two to ten (e.g., two, three, four, five, six, seven, eight, nine, or ten) or more nucleic acid sequences (or a single nucleic acid sequence encoding two to ten or more different infectious agent antigen polypeptides) each having an open reading frame and encoding a different infectious agent antigen polypeptide. In some embodiments, the vaccines of the present disclosure include two to ten (e.g., two, three, four, five, six, seven, eight, nine, or ten) or more nucleic acid sequences (or a single nucleic acid sequence encoding two to ten or more different universal T cell epitopes) each having an open reading frame and encoding a different universal T cell epitope.

[0373] In some embodiments, the vaccine can be formulated in the nanoparticles described herein. In some embodiments, the vaccine can be formulated in the lipid nanoparticles described herein. In some embodiments, the vaccine can be formulated in a lipid-polycation complex known as cationic lipid nanoparticles. The vaccines of the present disclosure can be formulated using one or more liposomes, lipid complexes, or lipid nanoparticles described herein. In some embodiments, the pharmaceutical composition of the vaccine can include the liposomes described herein. In some embodiments, the vaccine can be formulated in lipid vesicles that can have crosslinks between functionalized lipid bilayers. In some embodiments, the vaccine can be formulated in lipid-polycation complexes.

[0374] In some embodiments, the nucleic acid sequence encoding the infectious agent antigen polypeptide can elicit an antibody response in a subject. In some embodiments, the nucleic acid sequence encoding the infectious agent antigen polypeptide can elicit a broad T cell response in a subject. In some embodiments, the nucleic acid sequence encoding the infectious agent antigen polypeptide can elicit both an antibody response and a T cell response in a subject.

[0375] In some embodiments, the nucleic acid sequence encoding the infectious agent antigen polypeptide can elicit a cellular immune response, a humoral immune response, or a combination thereof. In some embodiments, the nucleic acid sequence encoding the infectious agent antigen polypeptide can elicit a cellular immune response, a humoral immune response, or a combination thereof without the risk of possible insertional mutagenesis.

[0376] In some embodiments, the universal T cell epitope can provide broader protection against infectious agent variants.

[0377] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding: human metapneumovirus (hMPV) antigen polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigen polypeptides, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, varicella-zoster antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus (e.g., smallpox, monkeypox) antigen polypeptide, cytomegalovirus antigen polypeptide, Epstein-Barr virus antigen polypeptide, rotavirus antigen polypeptide, rhinovirus antigen polypeptide, adenovirus antigen polypeptide, papillomavirus antigen polypeptide, poliovirus antigen polypeptide, mumps antigen polypeptide, rabies antigen polypeptide, rubella antigen polypeptide, coxsackievirus antigen polypeptide, equine encephalitis antigen polypeptide, Japanese encephalitis antigen polypeptide, yellow fever antigen polypeptide, Rift Valley fever antigen polypeptide, hepatitis A, B, C, D, and E virus antigen polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigen polypeptide (see, e.g., Esper F. et al., Emerging Infectious Diseases, 12(5), 2006; and Pyrc K. et al., Journal of Virology 81(7):3051-57, 2007, the contents of each of which are hereby incorporated by reference in their entirety), African swine fever (ASF) antigen polypeptide, foot-and-mouth disease virus (FMDV) antigen polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigen polypeptide, canine distemper antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or any combination thereof.

[0378] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding a monkeypox antigen polypeptide, a herpes simplex virus 1 (HSV1) antigen polypeptide, a herpes simplex virus 2 (HSV2) antigen polypeptide, a coronavirus (e.g., SARS-CoV2) antigen polypeptide, or any combination thereof.

[0379] In some embodiments, the vaccine can comprise a nucleic acid sequence (e.g., mRNA) encoding: human metapneumovirus (hMPV) antigenic polypeptides, human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively) antigenic polypeptides, respiratory syncytial virus (RSV) antigenic polypeptides, measles virus (MeV) antigenic polypeptides, varicella-zoster antigenic polypeptides, influenza virus antigenic polypeptides, herpes simplex virus 1 (HSV1) antigenic polypeptides, herpes simplex virus 2 (HSV2) antigenic polypeptides, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptides, cytomegalovirus antigenic polypeptides, Epstein-Barr virus antigenic polypeptides, rotavirus antigenic polypeptides, rhinovirus antigenic polypeptides, adenovirus antigenic polypeptides, papillomavirus antigenic polypeptides, poliovirus antigenic polypeptides, mumps antigenic polypeptides, rabies antigenic polypeptides, rubella antigenic polypeptides, coxsackievirus antigenic polypeptides, equine encephalitis antigenic polypeptides, Japanese encephalitis antigenic polypeptides, yellow fever antigenic polypeptides, Rift Valley fever antigenic polypeptides, hepatitis A, B, C, D, and E virus antigenic polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptides (see, e.g., Esper F. et al., Emerging Infectious Diseases, 12(5), 2006; and Pyrc K. et al., Journal of Virology 81(7):3051-57, 2007, the contents of each of which are hereby incorporated by reference in their entirety), African swine fever (ASF) antigenic polypeptides, foot-and-mouth disease virus (FMDV) antigenic polypeptides, feline herpesvirus-1 / feline viral rhinotracheitis antigenic polypeptides, canine distemper antigenic polypeptides, or any combination thereof.

[0380] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding: a human metapneumovirus (hMPV) antigen polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigen polypeptides, a respiratory syncytial virus (RSV) antigen polypeptide, a measles virus (MeV) antigen polypeptide, a varicella-zoster antigen polypeptide, an influenza virus antigen polypeptide, a herpes simplex virus 1 (HSV1) antigen polypeptide, a herpes simplex virus 2 (HSV2) antigen polypeptide, a poxvirus (e.g., smallpox, monkeypox) antigen polypeptide, a cytomegalovirus antigen polypeptide, an Epstein-Barr virus antigen polypeptide, a rotavirus antigen polypeptide, a rhinovirus antigen polypeptide, an adenovirus antigen polypeptide, a papillomavirus antigen polypeptide, a poliovirus antigen polypeptide, a mumps antigen polypeptide, a rabies antigen polypeptide, a rubella antigen polypeptide, a coxsackievirus antigen polypeptide, an equine encephalitis antigen polypeptide, a Japanese encephalitis antigen polypeptide, a yellow fever antigen polypeptide, a Rift Valley fever antigen polypeptide, hepatitis A, B, C, D, and E virus antigen polypeptides, an African swine fever (ASF) antigen polypeptide, a foot-and-mouth disease virus (FMDV) antigen polypeptide, a feline herpesvirus-1 / feline viral rhinotracheitis antigen polypeptide, a canine distemper antigen polypeptide, a feline coronavirus (FCoV) antigen polypeptide, or any combination thereof.

[0381] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding: human metapneumovirus (hMPV) antigen polypeptides, human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively) antigen polypeptides, respiratory syncytial virus (RSV) antigen polypeptides, measles virus (MeV) antigen polypeptides, varicella-zoster antigen polypeptides, influenza virus antigen polypeptides, poxvirus (e.g., smallpox, monkeypox) antigen polypeptides, cytomegalovirus antigen polypeptides, Epstein-Barr virus antigen polypeptides, rotavirus antigen polypeptides, rhinovirus antigen polypeptides, adenovirus antigen polypeptides, papillomavirus antigen polypeptides, poliovirus antigen polypeptides, mumps antigen polypeptides, rabies antigen polypeptides, rubella antigen polypeptides, coxsackievirus antigen polypeptides, equine encephalitis antigen polypeptides, Japanese encephalitis antigen polypeptides, yellow fever antigen polypeptides, Rift Valley fever antigen polypeptides, hepatitis A, B, C, D, and E virus antigen polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigen polypeptides (see, e.g., Esper F. et al., Emerging Infectious Diseases, 12(5), 2006; and Pyrc K. et al., Journal of Virology 81(7):3051-57, 2007, the contents of each of which are hereby incorporated by reference in their entirety), African swine fever (ASF) antigen polypeptides, foot-and-mouth disease virus (FMDV) antigen polypeptides, feline herpesvirus-1 / feline viral rhinotracheitis antigen polypeptides, canine distemper antigen polypeptides, feline coronavirus (FCoV) antigen polypeptides, or any combination thereof.

[0382] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding: human metapneumovirus (hMPV) antigen polypeptides, human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively) antigen polypeptides, respiratory syncytial virus (RSV) antigen polypeptides, measles virus (MeV) antigen polypeptides, varicella-zoster antigen polypeptides, influenza virus antigen polypeptides, poxvirus (e.g., smallpox, monkeypox) antigen polypeptides, cytomegalovirus antigen polypeptides, Epstein-Barr virus antigen polypeptides, rotavirus antigen polypeptides, rhinovirus antigen polypeptides, adenovirus antigen polypeptides, papillomavirus antigen polypeptides, poliovirus antigen polypeptides, mumps antigen polypeptides, rabies antigen polypeptides, rubella antigen polypeptides, coxsackievirus antigen polypeptides, equine encephalitis antigen polypeptides, Japanese encephalitis antigen polypeptides, yellow fever antigen polypeptides, Rift Valley fever antigen polypeptides, hepatitis A, B, C, D, and E virus antigen polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigen polypeptides (see, e.g., Esper F. et al., Emerging Infectious Diseases, 12(5), 2006; and Pyrc K. et al., Journal of Virology 81(7):3051-57, 2007, the contents of each of which are hereby incorporated by reference in their entirety), African swine fever (ASF) antigen polypeptides, foot-and-mouth disease virus (FMDV) antigen polypeptides, feline herpesvirus-1 / feline viral rhinotracheitis antigen polypeptides, canine distemper antigen polypeptides, or any combination thereof.

[0383] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding: human metapneumovirus (hMPV) antigen polypeptides, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigen polypeptides, respiratory syncytial virus (RSV) antigen polypeptides, measles virus (MeV) antigen polypeptides, varicella-zoster antigen polypeptides, influenza virus antigen polypeptides, poxvirus (e.g., smallpox, monkeypox) antigen polypeptides, cytomegalovirus antigen polypeptides, Epstein-Barr virus antigen polypeptides, rotavirus antigen polypeptides, rhinovirus antigen polypeptides, adenovirus antigen polypeptides, papillomavirus antigen polypeptides, poliovirus antigen polypeptides, mumps antigen polypeptides, rabies antigen polypeptides, rubella antigen polypeptides, coxsackievirus antigen polypeptides, equine encephalitis antigen polypeptides, Japanese encephalitis antigen polypeptides, yellow fever antigen polypeptides, Rift Valley fever antigen polypeptides, hepatitis A, B, C, D, and E virus antigen polypeptides, African swine fever (ASF) antigen polypeptides, foot-and-mouth disease virus (FMDV) antigen polypeptides, feline herpesvirus-1 / feline viral rhinotracheitis antigen polypeptides, canine distemper antigen polypeptides, feline coronavirus (FCoV) antigen polypeptides, or any combination thereof.

[0384] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding: human metapneumovirus (hMPV) antigen polypeptides, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigen polypeptides, respiratory syncytial virus (RSV) antigen polypeptides, measles virus (MeV) antigen polypeptides, varicella-zoster antigen polypeptides, influenza virus antigen polypeptides, herpes simplex virus 1 (HSV1) antigen polypeptides, herpes simplex virus 2 (HSV2) antigen polypeptides, poxvirus (e.g., smallpox, monkeypox) antigen polypeptides, cytomegalovirus antigen polypeptides, Epstein-Barr virus antigen polypeptides, rotavirus antigen polypeptides, rhinovirus antigen polypeptides, adenovirus antigen polypeptides, papillomavirus antigen polypeptides, poliovirus antigen polypeptides, mumps antigen polypeptides, rabies antigen polypeptides, rubella antigen polypeptides, coxsackievirus antigen polypeptides, equine encephalitis antigen polypeptides, Japanese encephalitis antigen polypeptides, yellow fever antigen polypeptides, Rift Valley fever antigen polypeptides, hepatitis A, B, C, D, and E virus antigen polypeptides, African swine fever (ASF) antigen polypeptides, foot-and-mouth disease virus (FMDV) antigen polypeptides, feline herpesvirus-1 / feline viral rhinotracheitis antigen polypeptides, canine distemper antigen polypeptides, or any combination thereof.

[0385] In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding a monkeypox antigen polypeptide. In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding a herpes simplex virus 1 (HSV1) antigen polypeptide. In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding a herpes simplex virus 2 (HSV2) antigen polypeptide. In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding a coronavirus (e.g., SARS-CoV2) antigen polypeptide. In some embodiments, the vaccine can include a nucleic acid sequence (e.g., mRNA) encoding a feline coronavirus (FCoV) antigen polypeptide.

[0386] In some embodiments, the present disclosure also provides a combination vaccine, in which the nucleic acid sequence (e.g., mRNA) encodes more than one antigen polypeptide selected from the following: human metapneumovirus (hMPV) antigen polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigen polypeptides, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, varicella-zoster antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus (e.g., smallpox, monkeypox) antigen polypeptide, cytomegalovirus antigen polypeptide, Epstein-Barr virus antigen polypeptide, rotavirus antigen polypeptide, rhinovirus antigen polypeptide, adenovirus antigen polypeptide, papillomavirus antigen polypeptide, poliovirus antigen polypeptide, mumps antigen polypeptide, rabies antigen polypeptide, rubella antigen polypeptide, coxsackievirus antigen polypeptide, equine encephalitis antigen polypeptide, Japanese encephalitis antigen polypeptide, yellow fever antigen polypeptide, Rift Valley fever antigen polypeptide, hepatitis A, B, C, D, and E virus antigen polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV2, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigen polypeptide, African swine fever (ASF) antigen polypeptide, foot-and-mouth disease virus (FMDV) antigen polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigen polypeptide, canine distemper antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or any combination thereof.

[0387] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one antigenic polypeptide selected from: human metapneumovirus (hMPV) antigenic polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigenic polypeptides, respiratory syncytial virus (RSV) antigenic polypeptide, measles virus (MeV) antigenic polypeptide, varicella-zoster antigenic polypeptide, influenza virus antigenic polypeptide, herpes simplex virus 1 (HSV1) antigenic polypeptide, herpes simplex virus 2 (HSV2) antigenic polypeptide, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptide, cytomegalovirus antigenic polypeptide, Epstein-Barr virus antigenic polypeptide, rotavirus antigenic polypeptide, rhinovirus antigenic polypeptide, adenovirus antigenic polypeptide, papillomavirus antigenic polypeptide, poliovirus antigenic polypeptide, mumps antigenic polypeptide, rabies antigenic polypeptide, rubella antigenic polypeptide, coxsackievirus antigenic polypeptide, equine encephalitis antigenic polypeptide, Japanese encephalitis antigenic polypeptide, yellow fever antigenic polypeptide, Rift Valley fever antigenic polypeptide, hepatitis A, B, C, D, and E virus antigenic polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptide, African swine fever (ASF) antigenic polypeptide, foot-and-mouth disease virus (FMDV) antigenic polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigenic polypeptide, canine distemper antigenic polypeptide, or any combination thereof.

[0388] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one antigenic polypeptide selected from: human metapneumovirus (hMPV) antigenic polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigenic polypeptides, respiratory syncytial virus (RSV) antigenic polypeptide, measles virus (MeV) antigenic polypeptide, varicella-zoster antigenic polypeptide, influenza virus antigenic polypeptide, herpes simplex virus 1 (HSV1) antigenic polypeptide, herpes simplex virus 2 (HSV2) antigenic polypeptide, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptide, cytomegalovirus antigenic polypeptide, Epstein-Barr virus antigenic polypeptide, rotavirus antigenic polypeptide, rhinovirus antigenic polypeptide, adenovirus antigenic polypeptide, papillomavirus antigenic polypeptide, poliovirus antigenic polypeptide, mumps antigenic polypeptide, rabies antigenic polypeptide, rubella antigenic polypeptide, coxsackievirus antigenic polypeptide, equine encephalitis antigenic polypeptide, Japanese encephalitis antigenic polypeptide, yellow fever antigenic polypeptide, Rift Valley fever antigenic polypeptide, hepatitis A, B, C, D, and E virus antigenic polypeptides, African swine fever (ASF) antigenic polypeptide, foot-and-mouth disease virus (FMDV) antigenic polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigenic polypeptide, canine distemper antigenic polypeptide, feline coronavirus (FCoV) antigenic polypeptide, or any combination thereof.

[0389] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one antigenic polypeptide selected from: human metapneumovirus (hMPV) antigenic polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigenic polypeptides, respiratory syncytial virus (RSV) antigenic polypeptide, measles virus (MeV) antigenic polypeptide, varicella-zoster antigenic polypeptide, influenza virus antigenic polypeptide, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptide, cytomegalovirus antigenic polypeptide, Epstein-Barr virus antigenic polypeptide, rotavirus antigenic polypeptide, rhinovirus antigenic polypeptide, adenovirus antigenic polypeptide, papillomavirus antigenic polypeptide, poliovirus antigenic polypeptide, mumps antigenic polypeptide, rabies antigenic polypeptide, rubella antigenic polypeptide, coxsackievirus antigenic polypeptide, equine encephalitis antigenic polypeptide, Japanese encephalitis antigenic polypeptide, yellow fever antigenic polypeptide, Rift Valley fever antigenic polypeptide, hepatitis A, B, C, D, and E virus antigenic polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptide, African swine fever (ASF) antigenic polypeptide, foot-and-mouth disease virus (FMDV) antigenic polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigenic polypeptide, canine distemper antigenic polypeptide, feline coronavirus (FCoV) antigenic polypeptide, or any combination thereof.

[0390] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one antigenic polypeptide selected from: human metapneumovirus (hMPV) antigenic polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigenic polypeptides, respiratory syncytial virus (RSV) antigenic polypeptide, measles virus (MeV) antigenic polypeptide, varicella-zoster antigenic polypeptide, influenza virus antigenic polypeptide, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptide, cytomegalovirus antigenic polypeptide, Epstein-Barr virus antigenic polypeptide, rotavirus antigenic polypeptide, rhinovirus antigenic polypeptide, adenovirus antigenic polypeptide, papillomavirus antigenic polypeptide, poliovirus antigenic polypeptide, mumps antigenic polypeptide, rabies antigenic polypeptide, rubella antigenic polypeptide, coxsackievirus antigenic polypeptide, equine encephalitis antigenic polypeptide, Japanese encephalitis antigenic polypeptide, yellow fever antigenic polypeptide, Rift Valley fever antigenic polypeptide, hepatitis A, B, C, D, and E virus antigenic polypeptides, coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptide, African swine fever (ASF) antigenic polypeptide, foot-and-mouth disease virus (FMDV) antigenic polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigenic polypeptide, canine distemper antigenic polypeptide, or any combination thereof.

[0391] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one antigenic polypeptide selected from: human metapneumovirus (hMPV) antigenic polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigenic polypeptides, respiratory syncytial virus (RSV) antigenic polypeptide, measles virus (MeV) antigenic polypeptide, varicella-zoster antigenic polypeptide, influenza virus antigenic polypeptide, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptide, cytomegalovirus antigenic polypeptide, Epstein-Barr virus antigenic polypeptide, rotavirus antigenic polypeptide, rhinovirus antigenic polypeptide, adenovirus antigenic polypeptide, papillomavirus antigenic polypeptide, poliovirus antigenic polypeptide, mumps antigenic polypeptide, rabies antigenic polypeptide, rubella antigenic polypeptide, coxsackievirus antigenic polypeptide, equine encephalitis antigenic polypeptide, Japanese encephalitis antigenic polypeptide, yellow fever antigenic polypeptide, Rift Valley fever antigenic polypeptide, hepatitis A, B, C, D, and E virus antigenic polypeptides, African swine fever (ASF) antigenic polypeptide, foot-and-mouth disease virus (FMDV) antigenic polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigenic polypeptide, canine distemper antigenic polypeptide, feline coronavirus (FCoV) antigenic polypeptide, or any combination thereof.

[0392] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one antigenic polypeptide selected from: human metapneumovirus (hMPV) antigenic polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3) antigenic polypeptides, respiratory syncytial virus (RSV) antigenic polypeptide, measles virus (MeV) antigenic polypeptide, varicella-zoster antigenic polypeptide, influenza virus antigenic polypeptide, herpes simplex virus 1 (HSV1) antigenic polypeptide, herpes simplex virus 2 (HSV2) antigenic polypeptide, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptide, cytomegalovirus antigenic polypeptide, Epstein-Barr virus antigenic polypeptide, rotavirus antigenic polypeptide, rhinovirus antigenic polypeptide, adenovirus antigenic polypeptide, papillomavirus antigenic polypeptide, poliovirus antigenic polypeptide, mumps antigenic polypeptide, rabies antigenic polypeptide, rubella antigenic polypeptide, coxsackievirus antigenic polypeptide, equine encephalitis antigenic polypeptide, Japanese encephalitis antigenic polypeptide, yellow fever antigenic polypeptide, Rift Valley fever antigenic polypeptide, hepatitis A, B, C, D, and E virus antigenic polypeptides, African swine fever (ASF) antigenic polypeptide, foot-and-mouth disease virus (FMDV) antigenic polypeptide, feline herpesvirus-1 / feline viral rhinotracheitis antigenic polypeptide, canine distemper antigenic polypeptide, or any combination thereof.

[0393] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one infectious agent antigenic polypeptide selected from: hMPV antigenic polypeptide, PIV3 antigenic polypeptide, RSV antigenic polypeptide, MeV antigenic polypeptide, and coronavirus antigenic polypeptide.

[0394] In some embodiments, a combination vaccine in which a nucleic acid sequence (e.g., mRNA) encodes more than one infectious agent antigenic polypeptide selected from: monkeypox antigenic polypeptide, herpes simplex virus 1 (HSV1) antigenic polypeptide, herpes simplex virus 2 (HSV2) antigenic polypeptide, coronavirus (e.g., SARS-CoV-2) antigenic polypeptide, or any combination thereof.

[0395] In some embodiments, the vaccine may further comprise a pharmaceutically acceptable carrier.

[0396] In some embodiments, the vaccine comprises an adjuvant as provided herein, such as a flagellin adjuvant.

[0397] Multiprotein and multicomponent vaccine

[0398] The compositions described herein can include multiple nucleic acid sequences (e.g., mRNA) each encoding a single pathogen antigen polypeptide and multiple nucleic acid sequences (e.g., mRNA) each encoding a single universal T cell epitope, as well as compositions comprising a single nucleic acid sequence (e.g., mRNA) encoding one or more pathogen antigen polypeptides and a single nucleic acid sequence (e.g., mRNA) encoding one or more universal T cell epitopes.

[0399] The compositions described herein can include multiple nucleic acid sequences (e.g., mRNA) each encoding a single pathogen antigen polypeptide and a single universal T cell epitope, as well as compositions comprising a single nucleic acid sequence (e.g., mRNA) encoding one or more pathogen antigen polypeptides and one or more universal T cell epitopes.

[0400] Thus, a composition comprising a nucleic acid sequence encoding a pathogen antigen polypeptide and at least one universal T cell epitope encompasses a composition comprising nucleic acid sequences encoding a first pathogen antigen polypeptide, a second pathogen antigen polypeptide, a first universal T cell epitope, and a second universal T cell epitope. In some embodiments, a composition comprising a nucleic acid sequence encoding a pathogen antigen polypeptide and a nucleic acid sequence encoding at least one universal T cell epitope encompasses a composition comprising a first nucleic acid sequence encoding a first pathogen antigen polypeptide, a second nucleic acid sequence encoding a second pathogen antigen polypeptide, a third nucleic acid sequence encoding a first universal T cell epitope, and a fourth nucleic acid sequence encoding a second universal T cell epitope.

[0401] In some embodiments, the compositions described herein include 2 - 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleic acid sequences (e.g., mRNA) each having an open reading frame and encoding different pathogen antigen polypeptides (or a single nucleic acid sequence (e.g., mRNA) encoding 2 - 10 or more different pathogen antigen polypeptides). In some embodiments, the compositions described herein include 2 - 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleic acid sequences (e.g., mRNA) each having an open reading frame and encoding different universal T cell epitopes (or a single nucleic acid sequence (e.g., mRNA) encoding 2 - 10 or more different universal T cell epitopes).

[0402] In some embodiments, the vaccine comprises nucleic acid sequences (e.g., mRNA) having open reading frames encoding viral capsid proteins, nucleic acid sequences (e.g., mRNA) having open reading frames encoding viral pre-membrane / membrane proteins, and nucleic acid sequences (e.g., mRNA) having open reading frames encoding viral envelope proteins. In some embodiments, the vaccine comprises a nucleic acid sequence (e.g., mRNA) having an open reading frame encoding a viral fusion (F) protein and a nucleic acid sequence having an open reading frame encoding a viral major surface glycoprotein (G protein). In some embodiments, the vaccine comprises a nucleic acid sequence (e.g., mRNA) having an open reading frame encoding a viral F protein. In some embodiments, the vaccine comprises a nucleic acid sequence (e.g., mRNA) having an open reading frame encoding a viral G protein. In some embodiments, the vaccine comprises a nucleic acid sequence (e.g., mRNA) having an open reading frame encoding an HN protein.

[0403] In some embodiments, the multi-component vaccine comprises a nucleic acid sequence (e.g., mRNA) encoding an infectious agent antigen polypeptide and at least one universal T cell epitope and a signal peptide (e.g., any one of SEQ ID NOs: 15-19). In some embodiments, the multi-component vaccine comprises a nucleic acid sequence (e.g., mRNA) encoding an infectious agent antigen polypeptide fused to a signal peptide (e.g., any one of SEQ ID NOs: 15-19). In some embodiments, the multi-component vaccine comprises a nucleic acid sequence (e.g., mRNA) encoding at least one universal T cell epitope fused to a signal peptide (e.g., any one of SEQ ID NOs: 15-19). The signal peptide can be fused at the N-terminus and / or C-terminus of the infectious agent antigen polypeptide and / or the universal T cell epitope.

[0404] Broad-spectrum vaccine

[0405] There are situations in which a subject is at risk of infection with more than one of the following strains: human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), respiratory syncytial virus (RSV), measles virus (MeV), varicella-zoster, influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus (e.g., smallpox, monkeypox), cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, coronaviruses (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), African swine fever (ASF), foot-and-mouth disease virus (FMDV), feline herpesvirus-1 / feline viral rhinotracheitis, canine distemper, feline coronavirus (FCoV), or any combination thereof.

[0406] Due to many factors, including but not limited to manufacturing speed, the ability to rapidly customize vaccines to address known perceived geographical threats, etc., the vaccines are particularly suitable for combination vaccination methods. Additionally, since the vaccines utilize the human body to produce infectious antigen polypeptides, the vaccines are suitable for generating larger and more complex antigen proteins, thereby allowing for proper folding, surface expression, antigen presentation, etc. in human subjects. To protect against more than one strain, a combination vaccine can be administered, the combination vaccine including a nucleic acid sequence (e.g., mRNA) encoding an infectious antigen polypeptide (or an antigenic portion thereof), a nucleic acid encoding at least one second infectious antigen polypeptide (or an antigenic portion thereof), and a nucleic acid encoding at least one universal T cell epitope. The nucleic acid sequences (e.g., mRNA) can be co-formulated, for example, in a single lipid nanoparticle (LNP), or can be formulated in separate LNPs for co-administration.

[0407] Combination vaccine

[0408] Embodiments of the present disclosure also provide a combination vaccine. The "combination vaccine" of the present disclosure refers to a vaccine comprising at least one (e.g., at least 2, 3, 4, or 5) nucleic acid sequence (e.g., mRNA) having an open reading frame encoding a combination of any two or more (or all) of the following infectious agent antigen polypeptides and a nucleic acid sequence (e.g., mRNA) having an open reading frame encoding at least one universal T cell epitope: human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3), respiratory syncytial virus (RSV), measles virus (MeV), varicella-zoster, influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus (e.g., smallpox, monkeypox), cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, coronaviruses (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), African swine fever (ASF), foot-and-mouth disease virus (FMDV), feline herpesvirus-1 / feline viral rhinotracheitis, canine distemper, feline coronavirus (FCoV), or any combination thereof.

[0409] In some embodiments, the combination vaccine comprises a nucleic acid sequence (e.g., mRNA) encoding an hMPV antigen polypeptide, a PIV3 antigen polypeptide, an RSV antigen polypeptide, an MeV antigen polypeptide, a monkeypox antigen polypeptide, a herpes simplex virus 1 (HSV1) antigen polypeptide, a herpes simplex virus 2 (HSV2) antigen polypeptide, a coronavirus antigen polypeptide (e.g., selected from MERS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, and HCoV-HKU1), and at least one universal T cell epitope. In some embodiments, the combination vaccine comprises a nucleic acid sequence (e.g., mRNA) encoding a monkeypox antigen polypeptide, a herpes simplex virus 1 (HSV1) antigen polypeptide, a herpes simplex virus 2 (HSV2) antigen polypeptide, a SARS-CoV-2 antigen polypeptide, or any combination thereof, and at least one universal T cell epitope.

[0410] In some embodiments, the combination vaccine comprises nucleic acid sequences (e.g., mRNA) encoding hMPV antigen polypeptides, PIV3 antigen polypeptides, RSV antigen polypeptides, MeV antigen polypeptides, monkeypox antigen polypeptides, herpes simplex virus 1 (HSV1) antigen polypeptides, herpes simplex virus 2 (HSV2) antigen polypeptides, coronavirus antigen polypeptides (e.g., selected from MERS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, and HCoV-HKU1), and nucleic acid sequences (e.g., mRNA) encoding at least one universal T cell epitope. In some embodiments, the combination vaccine comprises nucleic acid sequences (e.g., mRNA) encoding monkeypox antigen polypeptides, herpes simplex virus 1 (HSV1) antigen polypeptides, herpes simplex virus 2 (HSV2) antigen polypeptides, SARS-CoV-2 antigen polypeptides, or any combination thereof, and nucleic acid sequences (e.g., mRNA) encoding at least one universal T cell epitope.

[0411] In some embodiments, the combination vaccine comprises at least one (e.g., at least 2, 3, 4, or 5) nucleic acid sequences (e.g., mRNA) having an open reading frame encoding a combination of any two or more (or all) of the following infectious agent antigen polypeptides: human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3), respiratory syncytial virus (RSV), measles virus (MeV), varicella-zoster, influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus (e.g., smallpox, monkeypox), cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, coronavirus (e.g., MERS-CoV, SARS-CoV2, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), African swine fever (ASF), foot-and-mouth disease virus (FMDV), feline herpesvirus-1 / feline viral rhinotracheitis, canine distemper, feline coronavirus (FCoV), or any combination thereof.

[0412] In some embodiments, a combination vaccine comprising at least one (e.g., at least 2, 3, 4, or 5) nucleic acid sequences (e.g., mRNA) having an open reading frame encoding a combination of any two or more (or all) of the following infectious agent antigenic polypeptides: human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3), respiratory syncytial virus (RSV), measles virus (MeV), varicella-zoster, influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus (e.g., smallpox, monkeypox), cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, coronavirus (e.g., MERS-CoV, SARS-CoV2, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), African swine fever (ASF), foot-and-mouth disease virus (FMDV), feline herpesvirus-1 / feline viral rhinotracheitis, canine distemper, or any combination thereof.

[0413] In some embodiments, a combination vaccine comprising at least one (e.g., at least 2, 3, 4, or 5) nucleic acid sequences (e.g., mRNA) having an open reading frame encoding a combination of any two or more (or all) of the following infectious agent antigenic polypeptides: human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (respectively, hPIV1, hPIV2, and hPIV3), respiratory syncytial virus (RSV), measles virus (MeV), varicella-zoster, influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus (e.g., smallpox, monkeypox), cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, African swine fever (ASF), foot-and-mouth disease virus (FMDV), feline herpesvirus-1 / feline viral rhinotracheitis, canine distemper, feline coronavirus (FCoV), or...

Claims

1. A composition comprising: a nucleic acid sequence encoding an infectious agent antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

2. A composition comprising: a nucleic acid sequence encoding an infectious agent antigen polypeptide and at least one universal T cell epitope.

3. The composition according to any one of claims 1 to 2, wherein the at least one universal T cell epitope is as shown in Tables 1-5.

4. The composition according to any one of claims 1 to 3, wherein the infectious agent is a virus.

5. The composition according to claim 4, wherein the virus is human metapneumovirus (hMPV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), measles virus (MeV), influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus, feline coronavirus (FCoV), or coronavirus or any combination thereof.

6. The composition according to any one of claims 1 to 5, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide or any combination thereof.

7. The composition according to any one of claims 1 to 5, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide or any combination thereof.

8. The composition according to any one of claims 5 to 7, wherein the coronavirus is a human coronavirus.

9. The composition according to any one of claims 5 to 7, wherein the coronavirus is selected from HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL, HCoV-NH, HCoV-NL63, SARS-CoV, SARS-CoV-2, and MERS-CoV.

10. The composition according to any one of claims 5 to 8, wherein the coronavirus is SARS-CoV-2.

11. The composition according to any one of claims 5 to 7, wherein the poxvirus is monkeypox.

12. The composition according to any one of claims 1 to 5, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or any combination thereof.

13. A composition comprising: a nucleic acid sequence encoding a feline coronavirus (FCoV) antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

14. A composition comprising: a nucleic acid sequence encoding a feline coronavirus (FCoV) antigen polypeptide and at least one universal T cell epitope.

15. A composition comprising: a nucleic acid sequence encoding a coronavirus antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

16. A composition comprising: a nucleic acid sequence encoding a coronavirus antigen polypeptide and at least one universal T cell epitope.

17. A composition comprising: a nucleic acid sequence encoding a herpes simplex virus (HSV-1 and / or HSV-2) antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

18. A composition comprising: a nucleic acid sequence encoding a herpes simplex virus (HSV-1 and / or HSV-2) antigen polypeptide and at least one universal T cell epitope.

19. The composition according to any one of claims 13 to 18, wherein the at least one universal T cell epitope is as shown in Tables 1-5.

20. The composition according to any one of claims 1 to 19, wherein the nucleic acid sequence encoding the infectious agent antigen polypeptide elicits an antibody response in a subject.

21. The composition according to any one of claims 1 to 20, wherein the nucleic acid sequence encoding the infectious agent antigen polypeptide elicits a broad T cell response in a subject.

22. The composition according to any one of claims 1 to 21, wherein the nucleic acid sequence encoding the infectious agent antigen polypeptide elicits both an antibody response and a T cell response in a subject.

23. The composition according to any one of claims 1 to 22, wherein the nucleic acid sequence is DNA, RNA, any combination thereof (e.g., plasmid DNA, minicircle DNA, minimally immunogenic defined gene expression (MIDGE) and Doggybone, messenger RNA (mRNA), circular (cirRNA), self-amplifying RNA (saRNA, also known as SAM) or DNA-initiated SAM (DLSAM)).

24. A nanoparticle comprising: a nucleic acid sequence encoding an infectious agent antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

25. A nanoparticle comprising: a nucleic acid sequence encoding an infectious agent antigen polypeptide and at least one universal T cell epitope.

26. The nanoparticle according to any one of claims 24 to 25, wherein the at least one universal T cell epitope is as shown in Tables 1-5.

27. The nanoparticle according to any one of claims 24 to 26, wherein the infective agent is a virus.

28. The nanoparticle according to claim 27, wherein the virus is human metapneumovirus (hMPV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), measles virus (MeV), influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus, feline coronavirus (FCoV), or coronavirus or any combination thereof.

29. The nanoparticle according to any one of claims 24 to 28, wherein the mRNA encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide or any combination thereof.

30. The nanoparticle according to any one of claims 24 to 28, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide or any combination thereof.

31. The nanoparticle according to any one of claims 28 to 30, wherein the coronavirus is a human coronavirus.

32. The nanoparticle according to any one of claims 28 to 31, wherein the coronavirus is selected from HCoV-229E, HcoV-OC43, HcoV-HKU1, HcoV-NL, HcoV-NH, HcoV-NL63, SARS-CoV, SARS-CoV-2, and MERS-CoV.

33. The nanoparticle according to any one of claims 28 to 32, wherein the coronavirus is SARS-CoV-2.

34. The nanoparticle according to any one of claims 28 to 30, wherein the poxvirus is monkeypox.

35. The nanoparticle according to any one of claims 24 to 27, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide or any combination thereof.

36. A nanoparticle comprising: Nucleic acid sequences encoding feline coronavirus (FCoV) antigen polypeptides, and nucleic acid sequences encoding at least one universal T cell epitope.

37. A nanoparticle comprising: nucleic acid sequences encoding feline coronavirus (FCoV) antigen polypeptides and at least one universal T cell epitope.

38. A nanoparticle comprising: nucleic acid sequences encoding coronavirus antigen polypeptides, and nucleic acid sequences encoding at least one universal T cell epitope.

39. A nanoparticle comprising: nucleic acid sequences encoding coronavirus antigen polypeptides and at least one universal T cell epitope.

40. A nanoparticle comprising: nucleic acid sequences encoding herpes simplex virus (HSV-1 and / or HSV-2) antigen polypeptides, and nucleic acid sequences encoding at least one universal T cell epitope.

41. A nanoparticle comprising: nucleic acid sequences encoding herpes simplex virus (HSV-1 and / or HSV-2) antigen polypeptides and at least one universal T cell epitope.

42. The nanoparticle according to any one of claims 36 to 41, wherein the at least one universal T cell epitope is as shown in Tables 1-5.

43. The nanoparticle according to any one of claims 24 to 42, wherein the nucleic acid sequence encoding the pathogen antigen polypeptide elicits an antibody response in a subject.

44. The nanoparticle according to any one of claims 24 to 43, wherein the nucleic acid sequence encoding the pathogen antigen polypeptide elicits a T cell response in a subject.

45. The nanoparticle according to any one of claims 24 to 44, wherein the nucleic acid sequence encoding the pathogen antigen polypeptide elicits both an antibody response and a T cell response in a subject.

46. The nanoparticle according to any one of claims 24 to 45, wherein the nucleic acid sequence is DNA, RNA, any combination thereof (e.g., plasmid DNA, minicircle DNA, minimally immunogenic defined gene expression (MIDGE) and Doggybone, messenger RNA (mRNA), circular (cirRNA), self-amplifying RNA (saRNA, also known as SAM) or DNA-initiated SAM (DLSAM)).

47. A vaccine comprising: nucleic acid sequences encoding pathogen antigen polypeptides, and nucleic acid sequences encoding at least one universal T cell epitope.

48. A vaccine comprising: nucleic acid sequences encoding pathogen antigen polypeptides and at least one universal T cell epitope.

49. The vaccine according to any one of claims 47 to 48, wherein the at least one universal T cell epitope is as shown in Tables 1-5.

50. The vaccine according to any one of claims 47 to 48, wherein the pathogen is a virus.

51. The vaccine according to claim 50, wherein the virus is human metapneumovirus (hMPV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), measles virus (MeV), influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus, feline coronavirus (FCoV), or coronavirus or any combination thereof.

52. The vaccine according to any one of claims 47 to 50, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide or any combination thereof.

53. The vaccine according to any one of claims 47 to 50, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide or any combination thereof.

54. The vaccine according to any one of claims 51 to 53, wherein the coronavirus is a human coronavirus.

55. The vaccine according to any one of claims 51 to 54, wherein the coronavirus is selected from HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL, HCoV-NH, HCoV-NL63, SARS-CoV, SARS-CoV-2, and MERS-CoV.

56. The vaccine according to any one of claims 51 to 55, wherein the coronavirus is SARS-CoV-2.

57. The vaccine according to any one of claims 51 to 53, wherein the poxvirus is monkeypox.

58. The vaccine according to any one of claims 47 to 50, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide or any combination thereof.

59. A vaccine comprising: a nucleic acid sequence encoding a feline coronavirus (FCoV) antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

60. A vaccine comprising: a nucleic acid sequence encoding a feline coronavirus (FCoV) antigen polypeptide and at least one universal T cell epitope.

61. A vaccine comprising: A nucleic acid sequence encoding a coronavirus antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

62. A vaccine comprising: a nucleic acid sequence encoding a coronavirus antigen polypeptide and at least one universal T cell epitope.

63. A vaccine comprising: a nucleic acid sequence encoding a herpes simplex virus (HSV-1 and / or HSV-2) antigen polypeptide, and a nucleic acid sequence encoding at least one universal T cell epitope.

64. A vaccine comprising: a nucleic acid sequence encoding a herpes simplex virus (HSV-1 and / or HSV-2) antigen polypeptide and at least one universal T cell epitope.

65. The vaccine according to any one of claims 59 to 64, wherein the at least one universal T cell epitope is as shown in Tables 1-5.

66. The vaccine according to any one of claims 47 to 65, wherein the nucleic acid sequence encoding the infective agent antigen polypeptide elicits an antibody response in a subject.

67. The vaccine according to any one of claims 47 to 66, wherein the nucleic acid sequence encoding the infective agent antigen polypeptide elicits a T cell response in a subject.

68. The vaccine according to any one of claims 47 to 67, wherein the nucleic acid sequence encoding the infective agent antigen polypeptide elicits both an antibody response and a T cell response in a subject.

69. The vaccine according to any one of claims 47 to 68, wherein the nucleic acid sequence is DNA, RNA, any combination thereof (e.g., plasmid DNA, minicircle DNA, minimally immunogenic defined gene expression (MIDGE) and Doggybone, messenger RNA (mRNA), circular (circRNA), self-amplifying RNA (saRNA, also known as SAM) or DNA-initiated SAM (DLSAM)).

70. The vaccine according to any one of claims 47 to 69, wherein the universal T cell epitope provides broader protection against infective agent variants.

71. A method of treating, inhibiting, reducing, ameliorating, and / or preventing an infection caused by an infective agent in a subject, the method comprising administering to the subject a composition according to any one of claims 1 to 23, a nanoparticle according to any one of claims 24 to 46, or a vaccine according to any one of claims 47 to 70.

72. A method of activating T cells, B cells, or any combination thereof, the method comprising contacting T cells, B cells, or any combination thereof with a composition according to any one of claims 1 to 23, a nanoparticle according to any one of claims 24 to 46, or a vaccine according to any one of claims 47 to 70.

73. A method of stimulating the proliferation of T cells, B cells, or any combination, the method comprising contacting T cells, B cells, or any combination thereof with a composition according to any one of claims 1 to 23, a nanoparticle according to any one of claims 24 to 46, or a vaccine according to any one of claims 47 to 70.

74. A method of eliciting an immune response against an infectious agent in a subject, the method comprising administering to the subject a composition according to any one of claims 1 to 23, a nanoparticle according to any one of claims 24 to 46, or a vaccine according to any one of claims 47 to 70.

75. The method according to claim 74, wherein the immune response comprises a cellular immune response, a humoral immune response, or any combination thereof.

76. A method of enhancing an immune response generated by a nucleic acid vaccine, the method comprising: introducing a nucleic acid sequence encoding at least one universal T cell epitope into the nucleic acid vaccine.

77. A method of enhancing an immune response generated by a nucleic acid vaccine, the method comprising: introducing a nucleic acid sequence encoding at least one universal T cell epitope as shown in Tables 1-5 into the nucleic acid vaccine.

78. The method according to claim 77, wherein introducing the nucleic acid sequence encoding at least one universal T cell epitope expands the immune response against infectious agent variants.

79. The method according to any one of claims 76 to 78, wherein the nucleic acid sequence is DNA, RNA, any combination thereof (e.g., plasmid DNA, minicircle DNA, minimally immunogenic defined gene expression (MIDGE) and Doggybone, messenger RNA (mRNA), circular (cirRNA), self-amplifying RNA (saRNA, also known as SAM), or DNA-initiated SAM (DLSAM)).

80. The method according to any one of claims 71 to 79, wherein the infectious agent is a virus.

81. The method according to claim 80, wherein the virus comprises human metapneumovirus (hMPV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), measles virus (MeV), influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus, feline coronavirus (FCoV), or coronavirus, or any combination thereof.

82. The method according to any one of claims 71 to 81, wherein the mRNA encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide, or any combination thereof.

83. The method according to any one of claims 71 to 81, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or coronavirus antigen polypeptide, or any combination thereof.

84. The method according to any one of claims 81 to 83, wherein the coronavirus is a human coronavirus.

85. The method according to any one of claims 81 to 84, wherein the coronavirus is selected from HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL, HCoV-NH, HCoV-NL63, SARS-CoV, SARS-CoV-2, and MERS-CoV.

86. The method according to any one of claims 81 to 85, wherein the coronavirus is SARS-CoV-2.

87. The method according to any one of claims 81 to 83, wherein the poxvirus is monkeypox.

88. The method according to any one of claims 71 to 81, wherein the nucleic acid sequence encodes at least one human metapneumovirus (hMPV) antigen polypeptide, parainfluenza virus (PIV) antigen polypeptide, respiratory syncytial virus (RSV) antigen polypeptide, measles virus (MeV) antigen polypeptide, influenza virus antigen polypeptide, herpes simplex virus 1 (HSV1) antigen polypeptide, herpes simplex virus 2 (HSV2) antigen polypeptide, poxvirus antigen polypeptide, feline coronavirus (FCoV) antigen polypeptide, or any combination thereof.

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