mRNA Expression and Delivery Systems

Abstract

The present disclosure provides an RNA expression system based on sequences from plant viruses, insect viruses, and flaviviruses that eliminates the need for expensive modifications. A method is provided for expressing and packaging RNA polynucleotides without the need for in vitro transcription and lipid nanoparticles. Also provided is a method for packaging RNA polynucleotides using synthetic polyanhydride nanoparticles that are stable at room temperature and suitable for delivery by nasal spray.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 267,703, filed February 8, 2022, which is incorporated by reference in its entirety.

[0002] Sequence table XML This application contains a Sequence Listing that has been submitted via electronic submission in XML file format, which is incorporated herein by reference in its entirety. The XML file, created on Feb. 7, 2023, is named P13653WO00.xml and is 58,819 bytes in size. [Background technology]

[0003] mRNA vaccines can be highly effective against viruses, as demonstrated by the mRNA vaccines targeting SARS-CoV-2. However, current technologies have several shortcomings, including: (1) the use of modified rNTPs such as pseudouridine and cap structures at the 5' end [e.g., m 7 (1) the need to modify the mRNA by using a poly(A) tail, a repeat sequence of 50 or more consecutive adenosine residues at the 3' end [poly(A) tail], all of which require expensive steps in production; (2) lack of thermal stability except at -20 to -80°C; (3) lipid nanoparticles whose chemical composition is known to provoke adverse immune responses; (4) complex purification systems in which up to 25% of the product is small RNA that does not make a protein and about which very little is known; and (5) the requirement for delivery by intramuscular injection, which requires trained medical professionals. Summary of the Invention

[0004] Provided is an RNA polynucleotide comprising a 5' untranslated region (5'UTR), a heterologous sequence encoding at least one polypeptide, and a 3' untranslated region (3'UTR), wherein the 5'UTR or the 3'UTR comprises a panicum mosaic virus-like cap-independent translation enhancer or an exoribonuclease-resistant RNA (xrRNA) element.

[0005] In certain embodiments, the 5'UTR or 3'UTR is derived from Thin paspalum asymptomatic virus (TPAV), Tomato Bushy Stunt Virus (TBSV), Sweet clover necrotic mosaic virus (SCNMV), Red clover necrotic mosaic virus (RCNMV), or Poppy mosaic virus (OPMV). In certain embodiments, the RNA polynucleotide does not comprise a 5' cap structure or a polyA tail. In certain embodiments, the RNA polynucleotide does not comprise modified nucleosides. In certain embodiments, the encoded polypeptide is an antigenic polypeptide. In certain embodiments, the polypeptide is an antibody light chain and a heavy chain.

[0006] Pharmaceutical compositions are provided that include any of the RNA polynucleotides disclosed herein and a pharma- ceutically acceptable excipient.

[0007] Exosomes for delivering any of the RNA polynucleotides disclosed herein are provided.

[0008] Stable polyanhydride compositions are provided that include a polyanhydride polymer and any of the RNA polynucleotides disclosed herein.Also provided are polyanhydride compositions that include a polyanhydride polymer and any of the exosomes disclosed herein.

[0009] Provided is an RNA polynucleotide comprising a 5'UTR, a heterologous multiple cloning site for inserting a sequence encoding a polypeptide, and a 3'UTR, wherein the 5'UTR or the 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer or an xrRNA element.

[0010] Methods are provided for producing a polypeptide of interest in a subject comprising administering to the subject any of the RNA polynucleotides, pharmaceutical compositions, polyanhydride compositions, or exosomes disclosed herein.

[0011] Methods are provided for delivering an RNA polynucleotide to a subject comprising administering to the subject any of the RNA polynucleotides, pharmaceutical compositions, polyanhydride compositions, or exosomes disclosed herein.

[0012] Methods of inducing an immune response in a subject are provided, comprising administering to the subject a composition comprising an RNA polynucleotide in an amount effective to produce an antigen-specific immune response in the subject, wherein the RNA polynucleotide comprises a 5'UTR, a sequence encoding at least one antigenic polypeptide, and a 3'UTR, wherein the 5'UTR or the 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer or an xrRNA element.

[0013] Provided is a method of delivering an antibody to a subject, comprising administering to the subject a composition comprising an RNA polynucleotide, wherein the RNA polynucleotide comprises a 5'UTR, a sequence encoding a light chain of the antibody, a sequence encoding a heavy chain of the antibody, at least one internal ribosome entry site (IRES), and a 3'UTR, wherein the 5'UTR or the 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer or an xrRNA element.

[0014] Provided is a method for producing exosomes for delivery of an RNA polynucleotide, the method comprising: transforming a cell with a polynucleotide construct expressing an RNA polynucleotide, the RNA polynucleotide comprising a 5' UTR, a sequence encoding at least one polypeptide, and a 3' UTR, wherein the 5' UTR or the 3' UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer or an xrRNA element; culturing the cell in a growth medium, wherein exosomes comprising the RNA polynucleotide are released outside the cell into the growth medium; removing the cells from the growth medium; and harvesting the exosomes comprising the RNA polynucleotide from the growth medium.

[0015] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

[0016] The following drawings form part of the present specification and are included to further demonstrate certain embodiments or various aspects of the present disclosure. In some cases, embodiments of the present disclosure can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may emphasize certain specific examples or certain aspects of the present disclosure. However, one of ordinary skill in the art will understand that parts of the examples or aspects can be used in combination with other examples or aspects of the present disclosure. [Brief description of the drawings]

[0017] [Figure 1]Cap-independent translation and stability elements in the 5' and 3' UTRs of mRNA vaccine transcripts are shown. Thick black lines: UTRs flanking the reporter (firefly luciferase (Fluc) or mKATE2) or vaccine antigen-encoding ORFs (thick boxes). RNA secondary structures of translation / stability elements are shown as wire diagrams with curved dashed lines indicating pseudoknot base pairing. Straight dashed lines indicate the approximate location where the element is inserted, and alternative structures map to the same site. The cap analog added to the 5' end (curved arrow) is in the box at the top left. Element names are the same as in the text, and lengths are indicated. A(60)-XA(60) indicates two 60 A tracts interrupted by 1 to 6 non-A bases. [Diagram 2] Expression of uncapped and untailed mRNA after mRNA transfection is shown. In vitro transcription (IVT) was used to generate mRNA by a commercial kit and transfected using mRNA transfection reagent. Cells were examined 48 hours after transfection. TPAV and xrRNA cassettes far outperformed b-actin and continued to express the reporter for at least 3 days. [Diagram 3] We show that polyanhydride nanoparticles provide room temperature stability for at least 2 days. xrRNA mRNA was added to the particles. 5 mg of particles were then degraded by incubation in water for 3 hours, and the RNA was added to a lipid transfection reagent, which was then added to HELA cells. The cells were examined after 48 hours. [Figure 4] We show that mRNA persists in polyanhydride nanoparticles. Nanoparticles containing xrRNA adjacent to mKATE (nano) were either dissolved for 4 hours or kept at room temperature for 26 hours and then dissolved in water for 4 hours. mRNA not contained in nanoparticles (naked) was subjected to similar conditions. RNA was reverse transcribed and then amplified by PCR for 40 cycles. Apparently, the nanoparticles confer some degree of thermal stability to the mRNA. [Diagram 5]Figure 1 shows the enhancement of mRNA delivery by copolymers. A549 cells were transfected with lipids or copolymers or H3N8 virus using horse HA mRNA (0.5 μg). They were then stained for HA followed by anti-mouse Dylight 555 (red). Lipids were optimized liposomes from Mirus Bio for mRNA transfection. Not only did the copolymer transfect more cells, but the red color appeared deeper, presumably meaning more protein was translated. Some cell loss in the copolymer is due to gelling at 37°C on top of the cells and removing the gel before staining. Transwells do not suffer this loss. Lipid negative controls (not shown) were also similar to the copolymer negative. One representative well is shown for both delivery methods with 12 wells tested for each. Arrows indicate HA positive cells. [Figure 6] Thermal stability in polyanhydride at room temperature is shown. RSV F mRNA was produced and lyophilized with spermidine. 80 μg was kept at room temperature in a closed microtube, while 100 μg was packaged in polyanhydride 20:80 and kept similarly. After 4 months, mRNA was added to liposomes according to the manufacturer's instructions and placed on HELA cells, or 3 mm of polyanhydride was crushed and added to HELA cells overnight. Cells were then intracellularly stained for RSV proteins followed by staining with anti-goat Alex 555. qRT-PCR results also confirmed that only polyanhydride-bound mRNA was intact after very long storage. [Figure 7]mRNA in extracellular vesicles (EVs) in transfected cells. A549 / T7 polymerase cells were transfected with mKATE / TPAV (5 μg) using DEA dextran. Cells were then shocked with 10% glycerol and the medium was removed. After 3 days, the supernatant was harvested by two rounds of centrifugation and 0.2 μM filtration. 100 μl of EVs were added to HELA cells (without T7) for 1 h, followed by washing and medium replacement. Cells began to bloom after 48 h and reached higher intensity after 72 h. [Figure 8] EVs examined by electron microscopy are shown. EVs were purified by ultracentrifugation (not shown) or a commercially available kit. They were then subjected to scanning electron microscopy. [Figure 9] EVs harvested from cells after DEA-dextran transfection are shown. A549 T7 cells were transfected with empty or TPAV mKATE PCR product in DEA-dextran carrier with glycerol shock. The supernatant was removed and cells were allowed to make EVs for 3 days in serum-free medium. These were harvested, spun down and examined under a nanosite microscope. The empty transfection on the left has small EV size that is not uniform. On the right, cells with TPAV PCR product and transcribed mRNA via T7 polymerase had larger EVs with a size typical of mRNA+EVs. The concentration of those on the right was 109 / ml in a total of 30 ml. As EVs and their contents are very stable, they were observed after 1 week at 4°C. [Figure 10] 1 shows the use of a self-cleaving cassette. A polycistronic system using a self-cleaving peptide resulted in high expression of both coding sequences. [Figure 11] Comparison of additional mRNA constructs expressed in T7 BHK cells. 1 mg of DNA transfected 18 hours after transfection is shown. SCNMV and TPAV carrying 3'xrRNA resulted in the most favorable expression of mCherry. [Figure 12]Hemagglutination inhibition (HAI) titers are shown for mice vaccinated once (rHA / 1 dose) or twice (rHA / 2 doses) with rHA derived from H3N8, with Alum or mRNA on a TPAV cassette for H3N8 HA in the correct (mRNA / 1 dose) or reverse orientation (reverse mRNA) in EVs, or with β-actin (UTR) capped and tailed H3N8 HA in liposomes (HK mRNA / 1 dose). [Figure 13] Naked mRNA transfection. T7-free A549 cells were transfected using Ribojuice liposomes with mock (left), a porcine cassette expressing mCherry (middle), or a commercial mRNA expressing mCherry (modified U / C, Arco capped, and tailed) (right). Cells were examined 18 hours after transfection. mCherry on brightfield image is shown. [Figure 14] Stability of mRNA in polyanhydride nanoparticles. Polyanhydride-placed mRNA constructs were resistant to heat-induced degradation after 7 days at 37° C. Conventional IVT-generated mRNA did not possess this level of thermal stability. [Figure 15A] Circular RNA constructs are shown. Figure 15A is a gel image showing two constructs that have been circularized and then digested with RNAse R, which degrades linear RNA. Figure 15B is a gel image showing a control that demonstrates that RNAse R degrades linear RNA. [Figure 15B] Circular RNA constructs are shown. Figure 15A is a gel image showing two constructs that have been circularized and then digested with RNAse R, which degrades linear RNA. Figure 15B is a gel image showing a control that demonstrates that RNAse R degrades linear RNA. [Figure 16A]TPAV single expression system. Figure 16A shows a TPAV mRNA cassette (SEQ ID NO: 1) containing a T7 promoter, 5'UTR from TPAV, cloning sites (BamHI and Xho) flanking the mKATE2 reporter gene, and the 3'UTR from TPAV. Figure 16B shows a TPAV mRNA cassette (SEQ ID NO: 2) that is the same as SEQ ID NO: 1, but with an RNA protein binding domain after the mCherry reporter gene (rather than mKATE) and between the Xho cloning sites before the 3'UTR, and a T7 polymerase terminator. [Figure 16B] TPAV single expression system. Figure 16A shows a TPAV mRNA cassette (SEQ ID NO: 1) containing a T7 promoter, 5'UTR from TPAV, cloning sites (BamHI and Xho) flanking the mKATE2 reporter gene, and the 3'UTR from TPAV. Figure 16B shows a TPAV mRNA cassette (SEQ ID NO: 2) that is the same as SEQ ID NO: 1, but with an RNA protein binding domain after the mCherry reporter gene (rather than mKATE) and between the Xho cloning sites before the 3'UTR, and a T7 polymerase terminator. [Figure 17] 1 shows an mRNA cassette (SEQ ID NO: 3) with xrRNA using Red Clover Necrotic Mosaic Virus (RCNMV). The cassette contains a T7 promoter, cloning sites adjacent to an mCherry reporter gene, a 3'UTR xrRNA sequence from RCNMV, and a T7 polymerase terminator. [Figure 18] The xrRNA leader, 5'TPAV UTR, 3'TPAV UTR mRNA cassette (SEQ ID NO: 17) is shown. The cassette contains a T7 polymerase promoter, xrRNA from Zika virus, 5'UTR TPAV, CDS of mCherry reporter gene, RNA protein binding domain, TPAV 3'UTR, T7 polymerase terminator, and cloning sites (AAGCT, CTCGAG, and CATATG). [Figure 19]Shown is the xrRNA, cricket paralysis virus IRES, 3'UTR TPAV mRNA expression cassette (SEQ ID NO: 18). The cassette contains a T7 polymerase promoter, a Zika virus-derived xrRNA, an IRES, an ATGless CDS reporter, an RNA protein binding domain, a 3'UTR TPAV, and a T7 polymerase terminator. [Figure 20] The xrRNA, TRIMV, 3'TPAV UTR mRNA cassette (SEQ ID NO: 19) is shown. The cassette contains a T7 polymerase promoter, xrRNA from Zika virus, TRIMV 5'IRES, CDS reporter, RNA protein binding domain, 3'UTR TPAV, and a T7 polymerase termination terminator. [Figure 21A] 21A shows an mRNA cassette based on Tomato bushy stunt virus (TBSV). Figure 21A shows a cassette (SEQ ID NO: 20) containing a T7 polymerase promoter, a T7 enhancer xrRNA from Zika virus, a CDS reporter, an RNA protein binding domain, a TBSV 3'UTR, a T7 polymerase terminator, and cloning sites (AAGCT, CTCGAG, and CATATG). Figure 21B shows a cassette (SEQ ID NO: 21) containing a T7 polymerase promoter, an xrRNA from Zika virus, a TBSV 5'UTR, a CDS reporter, a TBSV 3'UTR, and a T7 polymerase terminator. [Figure 21B] 21A shows an mRNA cassette based on Tomato bushy stunt virus (TBSV). Figure 21A shows a cassette (SEQ ID NO: 20) containing a T7 polymerase promoter, a T7 enhancer xrRNA from Zika virus, a CDS reporter, an RNA protein binding domain, a TBSV 3'UTR, a T7 polymerase terminator, and cloning sites (AAGCT, CTCGAG, and CATATG). Figure 21B shows a cassette (SEQ ID NO: 21) containing a T7 polymerase promoter, an xrRNA from Zika virus, a TBSV 5'UTR, a CDS reporter, a TBSV 3'UTR, and a T7 polymerase terminator. [Figure 22A]TPAV / head xrRNA cassette and TPAV / tail xrRNA cassette are shown. Figure 22A shows a TPAV / head xrRNA cassette (SEQ ID NO: 22) containing a T7 polymerase promoter, xrRNA from Red clover necrotic mosaic virus (RCNMV), an mCherry reporter gene, and a 3'UTR from TPAV. Figure 22B shows a TPAV / tail xrRNA cassette (SEQ ID NO: 23) containing a T7 polymerase promoter, a 5'UTR from RCNMV, an mCherry reporter gene, xrRNA from RCNMV, and a 3'UTR from TPAV. [Figure 22B] TPAV / head xrRNA cassette and TPAV / tail xrRNA cassette are shown. Figure 22A shows a TPAV / head xrRNA cassette (SEQ ID NO: 22) containing a T7 polymerase promoter, xrRNA from Red clover necrotic mosaic virus (RCNMV), an mCherry reporter gene, and a 3'UTR from TPAV. Figure 22B shows a TPAV / tail xrRNA cassette (SEQ ID NO: 23) containing a T7 polymerase promoter, a 5'UTR from RCNMV, an mCherry reporter gene, xrRNA from RCNMV, and a 3'UTR from TPAV. [Diagram 23]

[0036] Figure 2 shows an mRNA cassette based on RCNMV (SEQ ID NO: 24). The cassette contains a T7 polymerase promoter, a 5'UTR from RCNMV, an mCherry reporter gene, and a 3'UTR from RCNMV. [Figure 24A] Dual expression systems are shown. Figure 24A shows a cassette (SEQ ID NO: 4) for the expression of an antibody. The cassette contains the heavy and light chains of an antibody without the CDR3 region flanked by the 3'UTR of xrRNA, and a forward IRES from poliovirus and a rear IRES from cricket paralysis virus. Figure 24B shows a similar construct, but without the antibody genes, a cassette for vaccine production (SEQ ID NO: 5). The forward CDS is based on eGFP and the rear is based on mCherry. Both proteins can be excised and replaced with the CDS of interest. [Figure 24B]Dual expression systems are shown. Figure 24A shows a cassette (SEQ ID NO: 4) for the expression of an antibody. The cassette contains the heavy and light chains of an antibody without the CDR3 region flanked by the 3'UTR of xrRNA, and a forward IRES from poliovirus and a rear IRES from cricket paralysis virus. Figure 24B shows a similar construct, but without the antibody genes, a cassette for vaccine production (SEQ ID NO: 5). The forward CDS is based on eGFP and the rear is based on mCherry. Both proteins can be excised and replaced with the CDS of interest. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] A brief description of the sequence SEQ ID NOs: 1 to 5 and 17 to 24 are sequences of mRNA expression cassettes.

[0019] SEQ ID NO:6 is the Zika virus xrRNA sequence.

[0020] SEQ ID NO:7 is the TPAV 5'UTR sequence.

[0021] SEQ ID NOs: 8 and 13 are TPAV 3'UTR sequences.

[0022] SEQ ID NO:9 is the poliovirus IRES sequence.

[0023] SEQ ID NO: 10 is the cricket paralysis virus IRES sequence.

[0024] SEQ ID NO:11 is the RNA protein binding domain sequence.

[0025] SEQ ID NO:12 is the Triticum Mosaic Virus (TRIMV) IRES sequence.

[0026] SEQ ID NO:14 is the Tomato bushy stunt virus (TBSV) 5'UTR sequence.

[0027] SEQ ID NOs:15 and 16 are the TBSV 3'UTR sequences.

[0028] SEQ ID NOs:25 and 36 are Red clover necrotic mosaic virus (RCNMV) xrRNA sequences.

[0029] SEQ ID NOs: 26 and 37 are the RCNMV 5'UTR sequences.

[0030] SEQ ID NOs:27 and 38 are RCNMV 3'UTR sequences.

[0031] SEQ ID NO:28 is the Sweet Clover Necrotic Mosaic Virus (SCNMV) 5'UTR sequence.

[0032] SEQ ID NO:29 is the swine fever virus 5'UTR sequence.

[0033] SEQ ID NO:30 is the Hepatitis C virus 3'UTR sequence.

[0034] SEQ ID NO:31 is the Potato leaf curl virus xrRNA sequence.

[0035] SEQ ID NO:32 is the bovine viral diarrhea virus 5'UTR sequence.

[0036] SEQ ID NO:33 is the poppy mosaic virus (OPMV) 5'UTR sequence.

[0037] SEQ ID NO:34 is the CHOP human mRNA sequence.

[0038] SEQ ID NO:35 is the Zika virus 3'UTR sequence. (Mode for carrying out the invention)

[0039] In order to make the present disclosure easier to understand, certain terms are defined first.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the embodiments of the present disclosure pertain.Many methods and materials similar to, modified or equivalent to those described herein can be used in the implementation of the embodiments of the present disclosure without undue experimentation, and preferred materials and methods are described herein.In describing and claiming the embodiments of the present disclosure, the following terms are used according to the definitions set forth below.

[0040] It is to be understood that all terms used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a," "an," and "the" may include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The word "or" means any one member of a particular list, and also includes any combination of members of that list. Additionally, all units, prefixes, and symbols may be denoted in their SI accepted form.

[0041] Numerical ranges recited herein are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of the disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges, fractions and individual numbers within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, and decimals and fractions, such as 1.2, 3.8, 1 1 / 2, and 4 3 / 4. This applies regardless of the breadth of the range.

[0042] The term "about" as used herein refers to the variation of numerical quantities that may occur through typical measurement techniques and devices for any quantifiable variable, including, but not limited to, mass, volume, time, and temperature. Furthermore, given the solid and liquid handling procedures used in the real world, there are certain inadvertent errors and variations that are likely due to differences in manufacture, source, or purity of ingredients used to make compositions or perform methods, etc. The term "about" also encompasses these variations. Whether or not modified by the term "about", the claims include the equivalents of the quantities.

[0043] The methods and compositions of the present disclosure may comprise, consist essentially of, or consist of the components and ingredients of the present disclosure as well as other components described herein. As used herein, "consisting essentially of" means that the methods, systems, devices, and compositions may include additional steps, components, or ingredients, but only if the additional steps, components, or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, devices, and compositions.

[0044] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms.

[0045] As used herein, the term "biocompatible" means compatibility with living cells, tissues, organs, or systems with little or no risk of damage, toxicity, or rejection by the immune system.

[0046] As used herein, the term "biodegradable" means capable of being broken down into harmless products by the action of living organisms.

[0047] As used herein, the term "conserved" refers to nucleotides or amino acid residues of a polynucleotide or polypeptide sequence, respectively, that do not vary at the same position in two or more sequences being compared. Relatively conserved nucleotides or amino acids are those that are conserved among related sequences to a greater extent than nucleotides or amino acids that appear elsewhere in the sequences.

[0048] In some embodiments, two or more sequences are said to be "fully conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Sequence conservation can apply to the entire length of an oligonucleotide or polypeptide, or to portions, regions, or features thereof.

[0049] As used herein, the term "circular" refers to the presence of a continuous loop. Circular molecules do not need to be circular, but only join to form an unbroken chain of subunits. Circular molecules such as the RNA polynucleotides of the present disclosure can be single units or multimers, or can include one or more components of a complex or higher order structure.

[0050] As used herein, "delivery" refers to the act or method of delivering a compound, substance, entity, moiety, cargo, or payload.

[0051] As used herein, "delivery agent" refers to any substance that facilitates, at least in part, the in vivo delivery of an RNA polynucleotide to a target cell.

[0052] As used herein, a "detectable label" refers to one or more markers, signals, or moieties that are bound to, incorporated into, or associated with another entity that is easily detected by methods known in the art, including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance, and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin, and haptens, quantum dots, and the like. Detectable labels can be located at any position in the peptides or proteins disclosed herein. They can be within an amino acid, peptide, or protein, or can be located at the N-terminus or C-terminus.

[0053] As used herein, the term "digest" means to divide into smaller fragments or components. When referring to polypeptides or proteins, digestion results in the production of peptides.

[0054] As used herein, the term "distal" means located away from the center or away from the point or area of ​​interest.

[0055] As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0056] As used herein, a "formulation" includes at least a polynucleotide and a delivery agent.

[0057] As used herein, a "fragment" refers to a portion. For example, a fragment of a protein can include a polypeptide obtained by digesting a full-length protein isolated from a cultured cell.

[0058] As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0059] The term "heterologous" refers to two biological components that are not found together in nature. The components can be a host cell, a gene, or a regulatory region such as a promoter. Heterologous components are not found together in nature, but they can function together, such as when a heterologous promoter is operably linked to a gene. Another example is when a coding sequence is heterologous to a non-translated region, such as the 5'UTR or 3'UTR, on the same polynucleotide.

[0060] As used herein, the term "in vitro" refers to events that take place not within a living organism (e.g., an animal, plant, or microorganism), but in an artificial environment, such as a test tube or reaction vessel, cell culture, petri dish, etc.

[0061] As used herein, the term "in vitro synthesis" refers to a cell-free method for the synthesis of mRNA.

[0062] As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0063] As used herein, the term "isolated" refers to a substance or entity that is separated from at least some of the components with which it is associated (either in nature or in an experimental environment). Isolated substances can have various levels of purity with reference to the substance with which they were associated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were originally associated. In some embodiments, an isolated agent is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. Substantially isolated: "Substantially isolated" means that the compound is substantially separated from the environment in which it was formed or detected. Partial separation may include, for example, compositions enriched in the compounds of the present disclosure. Substantial separation may include compositions containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds of the present disclosure or salts thereof. Methods for isolating compounds and their salts are routine in the art.

[0064] "Multiple cloning site" (MCS) refers to a nucleotide sequence that contains at least one unique restriction site, and more typically, a group of unique restriction sites, for cloning nucleic acid fragments into a vector or nucleic acid construct.

[0065] As used herein, "naturally occurring" means occurring in nature without artificial assistance.

[0066] As used herein, "non-human vertebrate" includes all vertebrates, except Homo sapiens, including wild and domestic species. Examples of non-human vertebrates include, but are not limited to, mammals such as alpacas, bantengs, bisons, camels, cats, cows, deer, dogs, donkeys, gayal, goats, guinea pigs, horses, llamas, mules, pigs, rabbits, reindeer, sheep, buffaloes, and yaks.

[0067] An "open reading frame" is a contiguous stretch of nucleotides beginning with a start codon (eg, methionine (ATG)) and ending with a stop codon (eg, TAA, TAG, or TGA) that encodes a polypeptide.

[0068] As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, and the like.

[0069] As used herein, "paratope" refers to the antigen-binding site of an antibody.

[0070] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0071] The phrase "pharmaceutical acceptable excipient" as used herein refers to any component other than the compounds described herein that has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Examples of excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, altitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0072] A "poly-A tail" is a region of an mRNA downstream, e.g., immediately downstream (i.e., 3'), of the 3'UTR that contains multiple consecutive adenosine monophosphates. A poly-A tail can contain 10-300 adenosine monophosphates. For example, a poly-A 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, a poly-A tail contains 50-250 adenosine monophosphates. In relevant biological contexts (e.g., within a cell, in vivo), the poly(A) tail functions, for example, in the cytoplasm, to protect the mRNA from enzymatic degradation and assists in transcription termination, transport of the mRNA from the nucleus, and translation.

[0073] As used herein, the term "prevent" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition, and / or reducing the risk of developing pathology associated with an infection, disease, disorder, and / or condition.

[0074] As used herein, the term "proximal" means located close to the center or to a point or area of ​​interest.

[0075] As used herein, "purify," "purified," and "purified" mean to make substantially pure or clear from undesirable components, contaminating substances, mixtures, or imperfections.

[0076] As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent similarity of polymer molecules to each other can be performed in the same manner as calculation of percent identity, except that percent similarity calculations take into account conservative substitutions as understood in the art.

[0077] As used herein, "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and, preferably, is capable of being formulated into an efficacious therapeutic agent.

[0078] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans).

[0079] The term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0080] As used herein, the term "therapeutically effective amount" means an amount of delivered agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0081] Nucleic Acids / Polynucleotides The compositions provided herein include at least one (one or more) ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one polypeptide. The term "nucleic acid" includes any compound and / or substance that includes a polymer of nucleotides (nucleotide monomers). These polymers are referred to as polynucleotides. Thus, the terms "nucleic acid" and "polynucleotide" are used interchangeably.

[0082] The nucleic acid may be or may include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (including LNA, LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2′-amino LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0083] In some embodiments, the polynucleotides of the present disclosure function as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-natural, 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 specified, the polynucleotide sequences described in this application recite a "T" of a representative DNA sequence, but when the sequence represents an RNA (e.g., mRNA), the "T" is replaced with a "U". Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number can also include the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each "T" in the DNA sequence is replaced with a "U".

[0084] The basic components of an mRNA molecule typically include at least a coding region, a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and a polyA tail. The polynucleotides of the present disclosure may function as mRNAs, but may be distinguished from wild-type mRNAs in their functional and / or structural design features.

[0085] In certain embodiments, the RNA polynucleotide encodes at least two polypeptides. In some embodiments, the RNA polynucleotide encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 polypeptides. In some embodiments, the RNA polynucleotide encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 polypeptides. In some embodiments, the RNA polynucleotide encodes at least 100 or at least 200 polypeptides. In some embodiments, the RNA polynucleotide encodes 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50, or 2-100 polypeptides.

[0086] In certain embodiments, the polynucleotides encoding at least two polypeptides are separated from each other by a polynucleotide encoding a self-cleaving peptide. The self-cleaving peptide, first discovered in picornaviruses, is a peptide of 19-22 amino acids in length and is usually found between two proteins in some members of the picornavirus family. In some embodiments, the self-cleaving peptide is one or more 2A peptides. In some embodiments, the 2A self-cleaving peptide is selected from the F2A peptide (foot and mouth disease virus 2A peptide), the E2A peptide (equine rhinitis virus 2A peptide), the P2A peptide (porcine teschovirus 1 2A peptide), and the T2A peptide (Thosea asigna virus 2A).

[0087] The polynucleotides of the present disclosure are codon-optimized in some embodiments. Methods of codon optimization are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to match the codon frequency of the target organism and the host organism to ensure proper folding, to bias the GC content to increase mRNA stability or reduce secondary structures, to minimize tandem repeat codons or base runs that may impair gene assembly or expression, to customize transcriptional and translational control regions, to insert or remove protein transport sequences, to remove or add post-translational modification sites of the encoded protein (e.g., glycosylation sites), to add, remove, or shuffle protein domains, to insert or remove restriction sites, to modify ribosome binding sites and mRNA degradation sites, to adjust the translation rate to allow various domains of the protein to fold properly, or to reduce or eliminate problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.), and / or proprietary methods. In some embodiments, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0088] In some embodiments, the polynucleotide comprises 200 to 3,000 nucleotides. For example, the polynucleotide may 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.

[0089] "5' untranslated region (5'UTR)" refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.

[0090] "3' untranslated region (3'UTR)" refers to the region of an mRNA immediately downstream (ie, 3') from a stop codon (ie, a codon in the mRNA transcript that signals the end of translation) that does not encode a polypeptide.

[0091] UTRs are provided that contain a 3' cap-independent translation enhancer and / or an internal ribosome entry site derived from a plant virus. An exonuclease blocking structure at the 5' end and a virus-derived secondary structure at the 3' end are also provided. The UTR elements avoid the cost of capping, increase translation efficiency, and enhance RNA stability.

[0092] UTRs can contain cap-independent translational enhancers (CITEs). Numerous small (approximately 100-150 nt) viral-derived CITEs in the 5' regions of plant viral 3'UTRs have been characterized that confer efficient translation to uncapped mRNAs in plant cells.

[0093] In certain embodiments, the CITE is a Panicum mosaic virus-like CITE (PTE). The Panicum mosaic virus-like 3' CITE in the Thin Paspalum Asymptomatic Virus (TPAV) genome promotes highly efficient translation of uncapped mRNAs in mammalian cells. PTE functions by binding to the translation initiation factor eIF4E, whose 3D structure is well conserved between plants and animals. PTE binds tightly to eIF4E (Kd<100 nM). 7This is novel because it is the only known RNA structure that lacks G. eIF4E is part of the eIF4E / eIF4G / eIF4A heterotrimer known as eIF4F, which is key for recruiting ribosomes to the 5' cap of mRNAs. In addition to binding to eIF4F (through eIF4E), the PTE bases pair with the 5'UTR of mRNAs, presumably to allow eIF4F to recruit ribosomes to the 5' end. This circularizes the mRNA, forming a structure similar to that of the 5' cap-eIF4F-PABP-poly(A) tail interaction on cellular mRNAs. Thus, in addition to eliminating the need for a 5' cap, PTE-mediated translation does not require a poly(A) tail. Instead, viruses that carry a PTE (or any type of 3'CITE) rely on viral structures downstream of the CITE to block 3' to 5' exonuclease activity.

[0094] In certain embodiments, the UTR comprises the native TPAV 5' and 3' ends. This construct provides substantial expression in mammalian cells, and the virus must have native stability elements to prevent rapid degradation of its uncapped, non-polyadenylated RNA genome upon entry into the cell. At the 3' end downstream of the 3'CITE, all viruses of the TPAV family (Tombusviridae) have a bulging stem-loop, with the bulging bases pairing with the terminal GCCC, and these bases are likely buried in the center of three coaxially stacked helices, hiding the 3' end from exonucleases.

[0095] In certain embodiments, the UTRs are derived from a virus of the Tombusviridae family. Examples of suitable viruses of the Tombusviridae family include Amur mosaic virus, Angelonia flower splitting virus, Apple luteovirus 1, Apple associated luteovirus, Artichoke crinkle virus, Barley yellow dwarf virus, Bean leaf curl virus, Beet black scorch virus, and others. virus, Calibrachoa mottle virus, Cardamine chlorotic mottle virus, Carnation Italian ring virus, Carnation mottle virus, Carnation ring virus, Carrot mottle mimic virus, Carrot mottle virus, Cherry associated luteovirus, Clematis chlorotic mottle virus, Orchard grass mild mottle mosaic virus, Cowpea mottle virus, Cucumber Bulgarian latent virus, Cucumber leaf spot virus, Cucumber necrotic virus, Cymbidium ring virus, Eggplant mottle crinkle virus, Elderberry aureus virus 1, Elderberry latent virus, Ethiopian tobacco bushy top virus, Furculaea necrotic streak virus, Galinsoga mosaic virus, Grapevine Algerian latent virus, Groundnut rosette virus, Havel River virus, Hibiscus chlorotic ring virus, Honeysuckle ring virus, Ixeridium yellow mottle virus 2 virus 2), Japanese Iris necrotic ringspot virus, Jasmine virus H, Johnsongrass chlorotic streak mosaic virus, Leek white streak virus, Limonium malformation virus, Corn chlorotic mottle virus, Corn necrotic streak virus, Corn white line mosaic virus, Melon necrotic spot virus, Pepper Morocco virus, Neckar virus, Nectarine stem pitting associated virus, Nutzcarpan vein clearing virus, Oat chlorotic dwarf virus, Olive latent virus 1, Olive mild mottle mosaic virus, Poppy mosaic virus, Panicum mosaic virus, Ominaeshi mild mottle virus, Pea pleur mosaic virus 2, Pea stem necrosis virus, Pelargonium chlorotic ring pattern virus (Pelargoniumchlorotic ring pattern virus, Pelargonium flower break virus, Pelargonium leaf cigar virus, Pelargonium line pattern virus, Pelargonium necrotic spot virus, Pelargonium ring pattern virus, Petunia asteroid mosaic virus, Potato necrotic virus, Pothos latent virus, Red clover necrotic mosaic virus, Rugosa rosa leaf deformed virus, Rose spring dwarf associated virus, Saguaro virus, Sitke waterborne virus, Soybean dwarf virus, Soybean yellow mottle mosaic virus, Sweet clover necrotic mosaic virus, Pale paspalum asymptomatic virus, Tobacco bushy top virus, Tobacco mottle virus, Tobacco necrotic virus A, Tobacco necrotic virus D, Tomato bushy stunt virus, Tralespevirus gompholobii, Tralespevirus lespedezae, turnip crinkle virus, and yam spherical virus. The UTR may include an IRES. In certain embodiments, the IRES is an intergenic region internal ribosome entry site (IGR IRES). These relatively compact (≦200nt) structures allow highly efficient internal initiation of translation of the second ORF in the genome of dicistroviruses that infect invertebrates. They are much smaller than the 500nt+ IRES of picorna and hepacivirus types I, II, and III IRES, and are likely stronger than host "IRESs" that occur only in the 5'UTR on capped mRNAs and usually function only in certain cell cycle stages or under stress conditions. The X-ray crystal structure of the IGR IRES reveals that the 3' portion of the structure mimics the anticodon stem loop of a tRNA base paired to a codon in the mRNA. This allows the IGR IRES to "trick" the 80S ribosome into "immediate elongation" and translate the downstream RNA sequence, i.e., this translation does not require initiation factors and a start codon. This suggests that host innate immune mechanisms such as PKR, which blocks the formation of the ribosomal preinitiation complex, can block the IGR IRES.It offers the important advantage of not affecting IRES-mediated translation.

[0096] Examples of IGR IRES sequences that may be used in accordance with the present disclosure include, but are not limited to, those from Cricket Paralysis Virus (CrPV), Aphid Lethal Paralysis Virus, Rhodospirillus hircus virus (RhPV), and other dicistroviruses.

[0097] The UTRs may contain elements that further protect the RNA polynucleotide from exonucleotide degradation.

[0098] In certain embodiments, the UTR contains tRNA-like structures (TLS) present at the 3' end of many plant virus genomes. These multifunctional pseudo-knot structures interact with host CTP:ATP nucleotidyl transferases that add CCA to the 3' end of pre-tRNA in a non-templated manner, thus functioning as telomerases to ensure an intact 3' end. Host tRNA synthetases aminoacylate the 3' end as they do on tRNA, further blocking exonucleases. TLS are also known to enhance translation.

[0099] In certain embodiments, the UTR comprises a stable stem-loop structure present at the 3' end of a flavivirus (e.g., Zika virus, West Nile virus).

[0100] In certain embodiments, the UTR comprises an xrRNA sequence. These short (50-70 nt) but complex RNA structures have evolved to block and sequester the host exonuclease Xrn I, the primary nuclease for turnover of mRNA after decapping. Xrn I also degrades certain RNA virus genomes in a 5' to 3' direction until it reaches a stalled xrRNA structure, leaving the 3' portion of the genome (usually the 3' UTR) intact. xrRNAs are present in the 3' UTR of flaviviruses and generate non-coding short flaviviral RNAs (sfRNAs) that perform a variety of proviral regulatory and immunosuppressive functions.

[0101] Examples of suitable flaviviruses include Apoivirus, Aroa virus, Bagaza virus, Banzi virus, Bouboui virus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadgets Gully virus, Irheus virus, Israeli turkey meningoencephalitis virus, Japanese encephalitis virus, Japanese encephalitis virus group, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Cocobera virus, Koutango virus, virus, Kyasanur Forest disease virus, Langat virus, Loopingir virus, Meaban virus, Modoc virus, Modoc virus group, Montana myositis-leukoencephalitis virus, mosquito-borne viruses, Murray Valley encephalitis virus, Untaya virus, Untaya virus group, Omsk hemorrhagic fever virus, Phnom Penh bat virus, Powassan virus, Rio Bravo virus, Rio Bravo virus group, Royal Farm virus, Saboya virus, St. Louis encephalitis virus, Sal Vieja virus, San Perlita virus, Saumarez Reef virus, Seaborne tick-borne virus group, Sepik virus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virusvirus, Uganda S virus, Ustu virus, Wesselsbron virus, West Nile virus, Yaoundé virus, Yellow fever virus, Yokose virus, and Zika virus.

[0102] In some embodiments, the xrRNA is located at the 5' end of the 5'UTR, immediately upstream of the IRES, or in a stem loop complementary to the 3'CITE. Because of its structure, the xrRNA can block ribosome entry and scanning at the 5' end, since the 40S subunit prefers an unstructured 5'UTR for optimal scanning to the start codon. This is not an issue for constructs with an IGR IRES, since the ribosome does not enter and scan directly at the IRES, but CITE-mediated translation requires ribosome scanning from the 5' end. The xrRNA structures at sites predicted to be 5'UTRs of subgenomic mRNAs encoding viral proteins are known, and these xrRNAs are expected not to block ribosome entry for translation. Given the similarity of xrRNA structures of plant and animal viruses, and the fact that yeast Xrn I is blocked by plant and animal virus xrRNAs, xrRNAs are predicted to function in any field of organisms.

[0103] In certain embodiments, a UTR comprises a polynucleotide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

[0104] The RNA polynucleotides of the present disclosure may include a 5' cap. In certain embodiments, the RNA polynucleotides are uncapped (i.e., do not contain a 5' cap). The 5' cap structure of an mRNA is involved in nuclear export, increases the stability of the mRNA, binds to mRNA cap-binding protein (CBP), and is responsible for the stability and translational competence of the mRNA in the cell through the association of CBP with poly(A) binding protein to form mature circular mRNA species. The cap also assists in the removal of the 5' proximal intron during mRNA splicing.

[0105] Endogenous mRNA molecules may be 5'-end capped, which creates a 5'ppp-5' triphosphate bond between the terminal guanosine cap residue of the mRNA and the 5'-terminal transcribed sense nucleotide. This 5'-guanylate cap may then be methylated to create an N7-methyl-guanylate residue. The ribose sugar of the terminal and / or pre-terminal transcribed nucleotide at the 5' end of the mRNA may also optionally be 2'-O-methylated. 5' decapping via hydrolysis and cleavage of the guanylate cap structure may target nucleic acid molecules, such as mRNA molecules, for degradation.

[0106] 5' cap structures include those described in International Patent Publication Nos. WO 2008 / 016473 and WO 2011 / 015347, each of which is incorporated by reference in its entirety.

[0107] The RNA polynucleotides of the present disclosure may include a poly-A tail. In certain embodiments, the RNA polynucleotides are non-polyadenylated (i.e., do not include a poly-A tail). During RNA processing, a long chain of adenosine nucleotides (a poly-A tail) is typically added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Poly-A polymerase then adds a chain of adenosine nucleotides to the RNA. A process called polyadenylation adds a poly-A tail that is 100-250 residues long.

[0108] The RNA polynucleotide of the present disclosure may include modified nucleosides. In certain embodiments, the RNA polynucleotide does not include modified nucleosides. "Nucleoside" refers to a compound that contains a sugar molecule (e.g., pentose or ribose) or its derivative in combination with an organic base (e.g., purine or pyrimidine) or its derivative (also referred to as "nucleobase"). "Nucleotide" refers to a nucleoside that has a phosphate group. Modified nucleotides can be synthesized by any useful method, such as, for example, chemical, enzymatic, or recombinant, and can include one or more modified or non-natural nucleosides.

[0109] Polypeptides Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides may be single molecules or multi-molecular complexes, such as dimers, trimers, or tetramers. Polypeptides may also include single-chain or multi-chain polypeptides, such as antibodies, that may be associated or linked to each other. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term "polypeptide" may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of the corresponding naturally occurring amino acid.

[0110] A "polypeptide variant" is a molecule whose amino acid sequence differs from a native or reference sequence. An amino acid sequence variant may have substitutions, deletions, insertions, or a combination of any two or three of the foregoing at certain positions in the amino acid sequence compared to the native or reference sequence. Typically, a variant has at least 50% identity to the native or reference sequence. In some embodiments, a variant shares at least 80% identity or at least 90% identity with the native or reference sequence.

[0111] "Orthologs" refer to genes in different species that have evolved from a common ancestral gene by speciation. Orthologs usually retain the same function during evolution. Identification of orthologs is important for reliable prediction of gene function in newly sequenced genomes.

[0112] "Analog" is meant to include polypeptide variants that differ by one or more amino acid changes, e.g., substitution, addition, or deletion of an amino acid residue, that still retain one or more of the properties of a parent or starting polypeptide.

[0113] The present disclosure provides several types of polynucleotide or polypeptide-based compositions, including variants and derivatives. These include, for example, substitution, insertion, 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 or starting molecule.

[0114] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions, and / or additions, deletions, and covalent modifications with respect to a reference sequence, particularly the polypeptide sequences disclosed herein, are included within the scope of this disclosure. For example, a sequence tag or one or more amino acids such as lysine can be added to the peptide sequence (e.g., 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 enable biotinylation. Alternatively, amino acid residues located in the carboxy and amino acid terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Certain amino acids (e.g., C-terminus or N-terminus residues) can alternatively be deleted depending on the use of the sequence, for example, expression of the sequence as part of a larger sequence that is soluble or linked to a solid support.

[0115] "Substitutional variant," when referring to a polypeptide, is one in which at least one amino acid residue of a native or starting sequence has been removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid has been substituted in the molecule, or multiple, where two or more (e.g., 3, 4, or 5) amino acids have been substituted in the same molecule.

[0116] 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 of similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another non-polar residue. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, the replacement of a basic residue, such as lysine, arginine, or histidine, with another, or the replacement of one acidic residue, such as aspartic acid or glutamic acid, with another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include substitutions of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or substitution of a polar residue for a non-polar residue.

[0117] A "feature" when referring to a polypeptide or polynucleotide is defined as a distinct amino acid sequence-based or nucleotide-based component of a molecule, respectively. Features of a polypeptide encoded by a polynucleotide include surface signatures, local conformational shapes, folds, loops, semi-loops, domains, semi-domains, sites, termini, and any combination or combinations thereof.

[0118] The term "domain," as used herein when referring to a polypeptide, refers to a motif in a polypeptide having one or more identifiable structural or functional features or characteristics (e.g., binding ability, serving as a site for protein-protein interaction).

[0119] As used herein when referring to a polypeptide, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" in reference to amino acid-based embodiments. As used herein when referring to a polynucleotide, the term "site" is used synonymously with "nucleotide" in reference to nucleotide-based embodiments. A site represents a position within a peptide or polypeptide or polynucleotide that can be modified, manipulated, altered, derivatized, or changed within a polypeptide- or polynucleotide-based molecule.

[0120] As used herein, the term "terminus" or "terminus" refers to the end of a polypeptide or polynucleotide, respectively. Such termini are not limited to only the first or last site of a polypeptide or polynucleotide, but may include additional amino acids or nucleotides in the terminal region. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid having a free amino group (NH2)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). Proteins are composed of multiple polypeptide chains (multimers, oligomers) held together, in some cases, by disulfide bonds or non-covalent forces. These proteins have multiple N-terminus and C-terminus. Alternatively, the termini of a polypeptide may be modified, in some cases, to begin or end with a non-polypeptide-based moiety, such as an organic conjugate.

[0121] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the polypeptide of interest. For example, provided herein is any protein fragment (meaning at least one amino acid residue shorter than the reference polypeptide sequence but otherwise identical) of a reference protein having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids or more. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 (contiguous) amino acids that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein may be utilized in accordance with the present disclosure. 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 or referenced herein. In another example, any protein comprising a stretch of 20, 30, 40, 50 or 100 amino acids that are more than 80%, 90%, 95% or 100% identical to any of the sequences described herein, where the protein has a stretch of 5, 10, 15, 20, 25 or 30 amino acids that are less than 80%, 75%, 70%, 65%-60% identical to any of the sequences described herein may be utilized in accordance with the present disclosure.

[0122] A polypeptide or polynucleotide molecule of the present disclosure may share a certain degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or reference polynucleotide), such as a molecule described in the art (e.g., an engineered or designed molecule or a wild-type molecule). The term "identity" as known in the art refers to a relationship between two or more polypeptide or polynucleotide sequences, 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 of two or more amino acid or nucleic acid residues. Identity measures the percent of identical matches between two or more smaller sequences, with gap alignment (if any) accommodated by a particular mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be readily calculated by known methods. "% identity" as applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. Identity is dependent on the calculation of percent identity, but may vary due to gaps and penalties introduced in the calculation. In general, 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 to that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.Tools for such alignment include those in 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 common local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences," J. Mol. Biol. 147: 195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins," J. Mol. Biol. 48: 443-453). Recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed which purportedly produces global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, specifically in the definition of "identity" below.

[0123] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymer 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 as determined by alignment of matching residues are referred to as homologous. Homology is a qualitative term that describes the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. In some embodiments, polymer molecules are considered to be "homologous" to each other if their sequences 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 (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical over at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by their 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 their ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical over at least one stretch of at least 20 amino acids.

[0124] Homology means that the compared sequences have diverged in evolution from a common origin. The term "homolog" refers to a first amino acid or nucleic acid sequence (e.g., a gene (DNA or RNA) or protein sequence) that is related to a second amino acid or nucleic acid sequence by descent from a common ancestral sequence. The term "homolog" can apply to the relationship between genes and / or proteins that have been separated by the event of speciation, or to the relationship between genes and / or proteins that have been separated by the event of gene duplication. An "ortholog" is a gene (or protein) from different species that has evolved from a common ancestral gene (or protein) by speciation. Typically, an ortholog retains the same function during evolution. A "paralog" is a gene (or protein) that is related by duplication within a genome. An ortholog retains the same function during evolution, whereas a paralog evolves a new function even though it is related to the original function.

[0125] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent identity of two polynucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequence 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 corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as 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, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in 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. Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs.Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0126] The polypeptide can be an antigenic polypeptide. In certain embodiments, the antigenic polypeptide is a viral or bacterial antigen. In some embodiments, the virus is an adenovirus, herpes simplex type 1, herpes simplex type 2, encephalitis virus, papillomavirus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, influenza ... These include strains of exanthema virus, hepatitis E virus, human immunodeficiency virus (HIV), influenza virus, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D, rotavirus, orbivirus, Coltivirus, Banna virus, human enterovirus, Hantavirus, West Nile virus, Middle East respiratory syndrome coronavirus, Japanese encephalitis virus, Vesicular exanthernavirus, and Eastern equine encephalitis.

[0127] In certain embodiments, the virus is an influenza A or influenza B strain, or a combination thereof. In some embodiments, the influenza A or influenza B strain is associated with an avian, porcine, equine, canine, human, or non-human primate. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or a fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof.

[0128] In some embodiments, the composition comprises at least one polynucleotide having an open reading frame encoding an HA protein or immunogenic fragment thereof (e.g., at least one of H1-H18), an NP protein or immunogenic fragment thereof, an NA protein or immunogenic fragment thereof, an M1 protein or immunogenic fragment thereof, an M2 protein or immunogenic fragment thereof, an NS1 protein or immunogenic fragment thereof, and an NS2 protein or immunogenic fragment thereof obtained from an influenza virus.

[0129] The present disclosure encompasses compositions comprising multiple RNA polynucleotides, each encoding a single antigenic polypeptide, and compositions comprising a single RNA polynucleotide encoding two or more antigenic polypeptides (e.g., as a fusion polypeptide). Thus, a composition comprising an RNA polynucleotide having an open reading frame encoding a first antigenic polypeptide and an RNA polynucleotide having an open reading frame encoding a second antigenic polypeptide encompasses (a) a composition comprising a first RNA polynucleotide encoding the first antigenic polypeptide and a second RNA polynucleotide encoding the second antigenic polypeptide, and (b) a composition comprising a single RNA polynucleotide encoding the first and second antigenic polypeptides (e.g., as a fusion polypeptide). In some embodiments, a composition of the present disclosure comprises 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more RNA polynucleotides (or a single RNA polynucleotide encoding 2-10 or more different antigenic polypeptides), each having an open reading frame encoding a different antigenic polypeptide.

[0130] A single RNA polynucleotide may encode two polypeptides. In certain embodiments, the two polypeptides include the light and heavy chains of an antibody. As used herein, the term "antibody" encompasses both intact antibodies and antibody fragments. Typically, an intact "antibody" is an immunoglobulin that specifically binds to a particular antigen. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgE, IgA, and IgD. Typically, an intact antibody is a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these corresponding regions on the light and heavy chains, respectively. Each variable region can be further subdivided into hypervariable (HV) and framework (FR) regions. The hypervariable regions contain three regions of hypervariable sequences called complementarity determining regions (CDR1, CDR2, and CDR3), separated by four framework regions (FR1, FR2, FR2, and FR4), which form a beta-sheet structure and act as a scaffold to hold the HV regions in place. The C-terminus of each heavy and light chain defines a constant region consisting of one domain (CL) for the light chain and three domains (CH1, CH2, and CH3) for the heavy chain. The light chain of an immunoglobulin can be further classified into isotypes kappa and lambda.

[0131] In some embodiments, the term "intact antibody" or "fully assembled antibody" is used in reference to an antibody that contains two heavy chains and two light chains, optionally associated by disulfide bonds, as occurs in naturally produced antibodies. In some embodiments, an antibody according to the present disclosure is an antibody fragment.

[0132] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, an antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, triabodies, tetrabodies, linear antibodies; single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of amino acid residues that mimic the hypervariable region. In many embodiments, an antibody fragment contains sufficient amino acid sequence of a parent antibody that the fragment binds to the same antigen as the parent antibody, and in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs).

[0133] Any antibody known in the art can be used, and standard methods can be used to generate antibodies against the desired antigen. The antibody can be a monoclonal antibody, a polyclonal antibody, a mixture or cocktail of antibodies, a human or humanized antibody, a chimeric antibody, or a bispecific antibody.

[0134] Exemplary antibodies include anti-chemokine (CC motif) ligand 2 (CCL2), anti-lysyl oxidase-like 2 (LOXL2), anti-Flt-1, anti-TNF-α, anti-interleukin-2Rα receptor (CD25), anti-TGF β, anti-B cell activator, anti-alpha-4 integrin, anti-BAGE, anti-β-catenin / m, anti-Bcr-abl, anti-C5, anti-CA125, anti-CAMEL, anti-CAP-1, anti-CASP-8, anti-CD4, anti-CD19, anti-CD20, anti-CD22, anti-CD25, anti-CDC27 / m, anti-CD30, anti-CD33, anti-CD52 , anti-CD56, anti-CD80, anti-CDK4 / m, anti-CEA, anti-CT, anti-CTL4, anti-Cyp-B, anti-DAM, anti-EGFR, anti-ErbB3, anti-ELF2M, anti-EMMPRIN, anti-EpCam, anti-ETV6-AML1, anti-HER2, anti-G250, anti-GAGE, anti-GnT-V, anti-Gp100, anti-HAGE, anti-HER-2 Antibodies include, but are not limited to, anti-neu, anti-HLA-A*0201-R170I, anti-IGF-1R, anti-IL-2R, anti-IL-5, anti-MC1R, anti-myosin / m, anti-MUC1, anti-MUM-1, anti-MUM-2, anti-MUM-3, anti-proteinase-3, anti-p190 minor bcr-abl, anti-Pml / RARα, anti-PRAMS, anti-PSA, anti-PSM, anti-PSMA, anti-RAGE, anti-RANKL, anti-RU1 or RU2, anti-SAGE, anti-SART-1 or SART-3, anti-survivin, anti-TEL / AML1, anti-TPI / m, anti-TRP-1, anti-TRP-2, anti-TRP-2 / INT2, and anti-VEGF or anti-VEGF receptor.

[0135] Therapeutic and prophylactic compositions Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, or diagnosis of infectious diseases in humans and other animals. The compositions can be used as therapeutic or prophylactic agents. They can be used in medicine to prevent and / or treat infectious diseases.

[0136] In some embodiments, a composition containing an RNA polynucleotide described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide.

[0137] The composition may induce translation of a polypeptide (e.g., an antigen or immunogen) in a cell, tissue, or organism. In some embodiments, such translation occurs in vivo, although such translation may occur ex vivo, in culture, or in vitro. In some embodiments, the cell, tissue, or organism is contacted with an effective amount of a composition containing an RNA polynucleotide having at least one translatable region encoding an antigenic polypeptide.

[0138] An "effective amount" of a composition is provided based, at least in part, on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide and other components, and other determinants. In general, an effective amount of a composition provides for the induction or enhancement of an immune response as a function of antigen production in cells. Increased antigen production can be demonstrated by increased cell transfection (percentage of cells transfected with RNA), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., as demonstrated by an increased duration of protein translation from a modified polynucleotide), or a change in the antigen-specific immune response of the host cell.

[0139] In some embodiments, compositions according to the present disclosure (including polynucleotides and their encoded polypeptides) may be used to treat infectious diseases.

[0140] The compositions may be administered prophylactically or therapeutically, as part of an active immunization scheme, to healthy individuals, or early in infection, during the incubation stage or after the onset of symptoms during active infection, hi some embodiments, the amount of the composition of the present disclosure provided to a cell, tissue, or subject may be an amount effective for immunoprophylaxis.

[0141] The composition may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or booster. As used herein, when referring to a prophylactic composition such as a vaccine, the term "booster" refers to an additional administration of a prophylactic (vaccine) composition. A booster (or booster vaccine) may be administered following an earlier administration of the prophylactic composition. The time between the first dose of the prophylactic composition and the booster can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week In some embodiments, the time between the first administration of the prophylactic composition and the booster may be, but is not limited to, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In some embodiments, the time between the first administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0142] In some embodiments, the compositions may be administered intramuscularly, intradermally, or intranasally.

[0143] The compositions may be utilized in a variety of settings, depending on the prevalence of infection or the extent or level of unmet medical need. By way of non-limiting example, the compositions may be utilized to treat and / or prevent a variety of infectious diseases.

[0144] Provided herein are pharmaceutical compositions, optionally in combination with one or more pharma- ceutically acceptable excipients.

[0145] The composition may be formulated or administered alone or in combination with one or more other components. For example, the composition (e.g., a vaccine composition) may include other components, including, but not limited to, an adjuvant. In some embodiments, the compositions do not include an adjuvant (they are adjuvant-free).

[0146] The composition may be formulated or administered in combination with one or more pharma- ceutically acceptable excipients. In some embodiments, the composition includes at least one additional active agent, such as, for example, a therapeutic active agent, a prophylactic active agent, or a combination of both. The composition may be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams&Wilkins, 2005, which is incorporated herein by reference in its entirety.

[0147] In some embodiments, the compositions are administered to a human, human patient, or subject. For purposes of this disclosure, the term "active ingredient" generally refers to an RNA polynucleotide (e.g., an mRNA polynucleotide) that encodes a polypeptide.

[0148] The formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the step of bringing into association an active ingredient (e.g., an RNA polynucleotide) with an excipient and / or one or more other accessory ingredients, and then, as necessary and / or desired, dividing, shaping, and / or packaging the product into the desired single or multiple dosage units.

[0149] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100%, e.g., from 0.5 to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) active ingredient.

[0150] Compositions may be formulated using one or more excipients to increase stability, increase cell transfection, allow sustained or delayed release (e.g., from a depot formulation), alter biodistribution (e.g., targeting to specific tissues or cell types), increase translation of the encoded protein in vivo, and / or alter the release profile of the encoded protein (antigen) in vivo. Conventional excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, and preservatives.

[0151] In some embodiments, the compositions of the present disclosure may be used as a method for preventing an infection (e.g., a viral infection) in a subject, the method comprising administering to the subject at least one composition provided herein. In some embodiments, the compositions of the present disclosure may be used as a method for treating an infection (e.g., a viral infection) in a subject, the method comprising administering to the subject at least one composition provided herein. In some embodiments, the compositions of the present disclosure may be used as a method for reducing the incidence of an infection (e.g., a viral infection) in a subject, the method comprising administering to the subject at least one composition provided herein. In some embodiments, the compositions of the present disclosure may be used as a method for inhibiting the spread of an infection from an infected first subject to an uninfected second subject, the method comprising administering to at least one of the first subject and the second subject at least one composition provided herein.

[0152] Methods for eliciting an immune response in a subject are provided. The methods include administering to the subject at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, thereby inducing an immune response in the subject. In some embodiments, the methods may include exosome-mediated delivery of the RNA polynucleotide.

[0153] A prophylactically effective dose is a therapeutically effective dose that prevents infection by the virus at a clinically tolerated level, hi some embodiments, a therapeutically effective dose is the dose described in the vaccine package insert.

[0154] Exosome composition In certain embodiments, the RNA polynucleotide may be encapsulated by an exosome. The terms "exosome," "microvesicle," and "extracellular vesicle" are used interchangeably herein. They generally refer to extracellular vesicles ranging in size from 30 to 200 nm, e.g., from 50 to 100 nm. In some embodiments, the extracellular vesicles may range in size from 20 to 300 nm, e.g., from 30 to 250 nm, e.g., from 50 to 200 nm.

[0155] The phrase "encapsulated by exosomes" or grammatical variations thereof is used interchangeably herein with the phrase "exosome composition" to refer to exosomes in which a lipid bilayer surrounds an RNA polynucleotide.

[0156] In certain embodiments, the present disclosure provides exosomes for use in delivering RNA polynucleotides to cells. The present disclosure also provides pharmaceutical compositions comprising the exosomes.

[0157] The exosomes may contain RNA polynucleotides for delivery to cells. In some embodiments, the delivery may be performed ex vivo. In some embodiments, the delivery may be performed in vivo. In certain embodiments, the pharmaceutical composition is in a form suitable for injection.

[0158] A method of producing exosomes comprising an RNA polynucleotide is provided, the method comprising: transforming a cell with a polynucleotide construct expressing the RNA polynucleotide; culturing the cell in a growth medium, where the exosomes comprising the RNA polynucleotide are released outside the cell into the growth medium; removing the cell from the growth medium; and harvesting the exosomes comprising the RNA polynucleotide from the growth medium. In certain embodiments, the harvesting is by ultracentrifugation. The cell can be an A549 cell. In certain embodiments, the cell constitutively expresses T7 RNA polymerase, and the polynucleotide construct comprises a T7 promoter.

[0159] Exosomes containing RNA polynucleotides that can be harvested and used to directly transfect other cells or packaged into polyanhydride nanoparticles.

[0160] Polyanhydride Polymers In some embodiments, the composition comprising the RNA polynucleotide is formulated in a nanoparticle. Conventional mRNA vaccination, especially those in cutting edge clinical trials (i.e. Moderna or CureVac), is based on liposomal transfection of cells after subQ / IM injection of the vaccine. The mRNA then transfects local cells near the injection site or transfects antigen-presenting cells recruited to the injection site (liposomal vaccines are known to be expressed in the liver, not locally). The liposome needs to be protected from RNAses before transfecting permissive cells, and then needs to be endocytosed into the cell to have any chance of becoming a protein; the true mechanism of escape from the endosome is not known, but is presumed to be some sort of export pore.

[0161] The present disclosure provides alternatives to liposome-mediated vehicles that enhance transfection efficiency, better protect against thermal degradation on the shelf and in the body, or increase subsequent immune profiles after vaccination.

[0162] In some embodiments, a composition comprising an RNA polynucleotide is formulated in polyanhydride particles.

[0163] The terms "polyanhydride particles" and "polyanhydride nanospheres" both refer to microparticles and nanoparticles made from the polyanhydride polymers described herein. The polyanhydride polymers of the particles are typically copolymers, such as random mixtures of anhydride oligomers (condensation prepolymers). Polyanhydride particles may be abbreviated as "PA particles," which may be microparticles or nanoparticles. Nanoparticles may also be referred to as polyanhydride nanospheres (PANS).

[0164] The group "alkyl" refers to a linear or branched hydrocarbon radical or diradical that is optionally unsaturated and optionally substituted with a functional group as described herein. The alkyl group may contain from 1 to about 20 carbon atoms. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 3-pentyl, hexyl, heptyl, octyl, or decyl. In one embodiment, the alkyl is preferably a (C1-C6) alkyl. In another embodiment, the alkyl is preferably a (C1-C4) alkyl.

[0165] In embodiments where the alkyl group is unsaturated, the alkyl is an alkenyl group or an alkynyl group. The alkenyl can be, for example, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl. The alkenyl can be unsubstituted or substituted. The alkynyl can be, for example, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-octynyl, etc. The alkynyl can be unsubstituted or substituted.

[0166] The term "aryl" refers to an aromatic hydrocarbon derived from a parent aromatic ring system. An aryl can be linked to another group at a saturated or unsaturated carbon atom of the parent ring system. An aryl group can have from 6 to about 14 carbon atoms. An aryl group can have a single ring (e.g., phenyl) or multiple condensed (fused) rings, in which at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. An aryl can be unsubstituted or substituted as described herein.

[0167] The term "halo" refers to fluoro, chloro, bromo, and iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0168] The term "substituted" is intended to indicate that one or more (e.g., 1, 2, 3, 4, or 5, in some embodiments 1, 2, or 3, and in other embodiments 1 or 2) hydrogen atoms on the group designated using the term "substituted" are replaced with a selection from the substituents listed below, or with a suitable group known to one of ordinary skill in the art, provided that the normal valence of the designated replacement atom is not exceeded, and that the substitution results in a stable compound. Suitable substituents include, for example, alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, acylamino, nitro, difluoromethyl, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, and cyano. Suitable substituents also include, for example, -X, -R, -OR, -SR, -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, NC(=O)R, -C(=O)R, -C(=O)NRR, -S(=O)2OH, -S(=O)R, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -OP(=O)(OR)2, -P(=O)(OR)2, Examples include -P(=O)(OH), -C(=O)R, -C(=O)X, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(NR)NRR, where each X is independently a halogen ("halo"): F, Cl, Br, or I, and each R is independently H, an alkyl, aryl, heterocycle, or protecting group, or a cation or anion thereof. As will be readily understood by one of ordinary skill in the art, when a substituent is keto (i.e., =O) or thioxo (i.e., =S), etc., two hydrogen atoms on the substituted atom are thus replaced.

[0169] In the case of any of the above groups containing one or more substituents, it is to be understood that such groups do not of course include any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. In addition, the compounds of the present disclosure include all stereochemical isomers arising from the substitution of these compounds.

[0170] The term "dibasic acid" refers to any group that contains two carboxylic acid (-C(=O)OH) groups. Dibasic acids can be aliphatic or aromatic dicarboxylic acids. Aliphatic dicarboxylic acids are any alkyl groups substituted with two (or more) carboxylic acid groups. Aromatic dicarboxylic acids are any compounds that contain at least one aryl group and two (or more) carboxylic acids. The two carboxylic acid groups can be on the same aryl group or they can be on different aryl groups. When the two carboxylic acid groups are on different aryl groups, the aryl groups can be linked by a single bond and thus by other groups, for example, alkyl groups. The alkyl groups that link the aryl groups can be optionally substituted and can be optionally interrupted between the carbon and other groups as defined herein.

[0171] The term "polymer" refers to a molecule of one or more repeating monomer residue units covalently bonded together by one or more repeating chemical functional groups. The term includes all polymer forms, such as linear, branched, star, random, block, graft, etc. Includes homopolymers formed from a single monomer, copolymers formed from two or more monomers, terpolymers formed from three or more polymers, and other polymers formed from more than three monomers. Different forms of polymers may also have more than one repeating covalently bonded functional group.

[0172] The term "polyanhydride" refers to a polymer derived from the condensation of carboxylic acids or carboxylic acid derivatives such that the repeat units of the resulting polymer are linked by anhydride (-C(=O)-OC(=O)-) groups. Polyanhydrides can be prepared by condensing dibasic acids or by condensing anhydride prepolymers, as described herein.

[0173] The term "carboxylic acid anhydride" refers to a compound containing an anhydride (-C(=O)-OC(=O)-) group. Carboxylic acid anhydrides typically contain only one anhydride group per molecule. Carboxylic acid anhydrides can be formed by the condensation of two carboxylic acids. Carboxylic acid anhydrides that can be used in conjunction with the methods described herein include bis-alkyl carboxylic acid anhydrides, bis-aryl carboxylic acid anhydrides, and mixed anhydrides. Examples include, but are not limited to, acetic anhydride, trifluoroacetic anhydride, and benzoic anhydride. Mixed anhydrides such as acetic benzoic anhydride, which is the condensation product of acetic acid and benzoic acid, can also be used.

[0174] As used herein, an "acyl" group is a group such as a (C1-C4) alkyl group that terminates in a carbonyl radical at its point of attachment to another group. An "acyloxy" group is a substituent such as a (C1-C4) alkyl group that terminates in a carboxyl radical at its point of attachment to another group.

[0175] The term "acylated" refers to the conversion of a hydroxyl group to an acyloxy group. Acylation can be carried out by contacting the hydroxyl group or a hydroxyl-containing group with a carboxylic acid anhydride.

[0176] As used herein, a "prepolymer" is a monomer, oligomer, or mixture thereof that can be converted to a polymer (e.g., a long chain polyanhydride). Diacid prepolymers are typically acylated on their terminal carboxy groups. The prepolymer can be, for example, a bis(carboxylic acid acetyl ester), or anhydride oligomers thereof. In some embodiments, the prepolymer can be a 1,ω-(4-acetoxycarbonylphenoxy)alkane, or anhydride oligomers thereof. The phenoxy group of the 1,ω-(4-acetoxycarbonylphenoxy)alkane can have an ortho, meta, or para substitution pattern.

[0177] As used herein, a "homopolymer" is a polymer composed of repeat units of one type of monomer. A "copolymer" is a polymer composed of repeat units of two or more different types of monomer. In a random copolymer, the configuration of the repeat units is random.

[0178] The polyanhydrides used to prepare the particles of the present disclosure may be prepared as described herein or by methods known to those skilled in the art. Several examples of methods for the preparation of polyanhydrides are provided below. A wide range of suitable diacids may be used to prepare the polyanhydrides. The diacid may be a diacid-substituted straight or branched alkane, optionally interrupted by about 1 to about 5 -Ph-, -O-, -CH=CH-, and / or -N(R)- groups, where R is H, phenyl, benzyl, or (C1-C6) alkyl. In one embodiment, the alkane of the diacid is a C2-C6 alkyl group. 12 (alkyl). In another embodiment, the alkane can be C4-C8 (alkyl). In addition, the alkane group of the dibasic acid can be optionally interrupted by about 1 to about 12 -OCH2CH2O- groups, e.g., poly(ethylene glycol) segments. The alkane group can also be optionally substituted with 1, 2, or 3 (C1-C6) alkyl, (C1-C6) alkenyl, trifluoromethyl, trifluoromethoxy, or oxo groups, or combinations thereof.

[0179] In one embodiment, the prepolymer can be prepared as shown in Scheme 1: [ka] An "organic group" is any organic group capable of linking two carboxylic acid moieties, where R is alkyl or aryl, and n is from 1 to about 12. Examples of suitable organic groups include C2-C 12 (Alkyl) group, -PhO-C 2- C 12 Examples of suitable carboxylic acid anhydrides include, but are not limited to, PEG groups having 1 to about 12 PEG units, such as (alkyl)-OPh- groups, and 3,6-dioxaoctane groups. A molar excess of carboxylic acid anhydride may be used. About 2 to about 30 molar equivalents of carboxylic acid anhydride may be used. Alternatively, about 5 to about 20 molar equivalents of carboxylic acid anhydride may be used. In one embodiment, 6 molar equivalents of carboxylic acid anhydride are used. In another embodiment, 18 molar equivalents of carboxylic acid anhydride are used. The carboxylic acid anhydride may be, for example, acetic anhydride, trifluoroacetic anhydride, benzoic anhydride, combinations thereof, and / or derivatives thereof.

[0180] The prepolymer may also be prepared as shown in Scheme 2: [ka] where n is 1 to about 12. Other carboxylic acid anhydrides, such as, but not limited to, benzoic anhydride, can be used to form the end groups of the prepolymer. The central aliphatic group can optionally be substituted or interrupted as described herein.

[0181] The dibasic acid may also be a 1,ω-bis(carboxy)alkane. As will be appreciated by those of skill in the art, an alternative nomenclature for a 1,ω-bis(carboxy)alkane is a 1,ω-alkane diacid, which has two additional carbons in the alkane moiety compared to the corresponding bis(carboxy)alkane.

[0182] The prepolymer may also be prepared as shown in Scheme 3: [ka] where n is 1 to about 12. Carboxylic acid anhydrides other than acetic anhydride may be used to form the end groups of the prepolymer. The central aliphatic group, the aryl group, or both, may be optionally substituted in any combination. The central aliphatic group may also be interrupted by oxygen, for example, to have a poly(ethylene glycol) chain.

[0183] Thus, a dibasic acid can be two aryl groups, each substituted with a carboxy group, where the aryl groups are linked by a straight or branched chain alkane, and are optionally interrupted by about 1 to about 5 -Ph-, -O-, -CH=CH-, and / or -N(R)- groups, where R is H, phenyl, benzyl, or (C1-C6) alkyl. In some embodiments, one or both of the aryl groups can be omitted, and the carboxy groups are linked by an alkyl chain. In one embodiment, the alkane is a C2-C6 alkyl group. 12 (alkyl). In another embodiment, the alkane can be C4-C8 (alkyl). In another embodiment, the alkane can be one or more PEG groups. In addition, the alkane group linking the carboxylic acid substituted aryl group can be optionally interrupted by 1 to about 12 -OCH2CH2O- groups, e.g., poly(ethylene glycol) segments. The alkane group linking the carboxylic acid substituted aryl group can also be optionally substituted with 1, 2, or 3 (C1-C6) alkyl, (C1-C6) alkenyl, trifluoromethyl, trifluoromethoxy, or oxo groups, or combinations thereof.

[0184] The dibasic acid may be a 1,ω-bis(4-carboxyphenoxy)alkane. In one embodiment, the alkane is (C 10) alkane. In another embodiment, the alkane can be C4-C8 (alkyl). In certain specific embodiments, the alkane can be ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and branched isomers thereof. In one embodiment, the dibasic acid is 1,6-bis(4-carboxyphenoxy)hexane. In another embodiment, the dibasic acid is 1,6-bis(carboxy)octane. In another embodiment, the dibasic acid can be 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane. Mixtures of any of these dibasic acids can be used in conjunction with the microwave-assisted methods described herein.

[0185] The polyanhydrides may be prepared by condensation methods known in the art or by irradiating the prepolymer with a sufficient amount of microwave radiation to polymerize the prepolymer. A sufficient amount of microwave radiation may typically be generated by a conventional microwave oven set at 1100 watts for about 1 to about 30 minutes. More often, a sufficient amount of microwave radiation may be generated in about 1 to about 20 minutes. The resulting polyanhydrides may be homopolymers or copolymers, depending on the nature of the prepolymer composition used in the reaction.

[0186] Polyanhydrides can also be prepared by forming a prepolymer in situ from a dibasic acid. The dibasic acid can be converted to a prepolymer by irradiating the dibasic acid in the presence of a carboxylic acid anhydride. The prepolymer can be prepared, for example, by irradiating a mixture of (a) a carboxylic acid anhydride and (b) an aromatic dicarboxylic acid, an aliphatic dicarboxylic acid, or a mixture thereof with an amount of microwave radiation effective to form a prepolymer. One suitable carboxylic acid anhydride is acetic anhydride. Other suitable carboxylic acid anhydrides include, for example, trifluoroacetic anhydride and benzoic anhydride.

[0187] The end groups of the polyanhydrides prepared according to the methods described herein typically have terminal acyl groups. Some hydrolysis of the polyanhydride may occur during the reaction or during isolation of the polyanhydride. Thus, some end groups of such polyanhydrides may be carboxylic acid groups. Thus, the methods of the present disclosure include the preparation of polyanhydrides that terminate with acyl groups, carboxylic acid groups, or a combination thereof.

[0188] Polyanhydrides can be prepared, for example, as shown in Scheme 4: [ka] wherein "organic group" is any organic group that links two carboxylic acid moieties, R is alkyl or aryl, n is 1 to about 12, and m is about 5 to about 200.

[0189] Polyanhydrides can also be prepared as shown in Scheme 5: [ka] wherein n is 1 to about 12, and m is about 5 to about 200. In other embodiments, m can be about 10 to about 100, or about 10 to about 50. As will be appreciated by one of ordinary skill in the art, the value of m is typically greater than the value of n. End groups other than acetate can be used, and the central aliphatic group can be optionally substituted, optionally interrupted (e.g., for PEG groups), or both, as described herein.

[0190] Polyanhydrides can also be prepared as shown in Scheme 6: [ka] where n is 1 to about 12 and m is about 5 to about 100. In other embodiments, m can be about 10 to about 50, or about 15 to about 35. End groups other than acetate can be used, and the central aliphatic group, aryl group, or both can be optionally substituted in any combination. The central aliphatic group can also be optionally interrupted by oxygen, for example, to have a poly(ethylene glycol) chain.

[0191] A method for preparing polyanhydride microparticles or nanoparticles includes irradiating one or more diacids, where the one or more diacids include aromatic dicarboxylic acids, aliphatic dicarboxylic acids, or mixtures thereof, with microwave radiation in the presence of a carboxylic acid anhydride to acylate the one or more diacids to obtain at least one prepolymer, and irradiating the prepolymer with microwave radiation to polymerize the prepolymer to obtain the polyanhydride as a homopolymer or copolymer.

[0192] The prepolymer may be composed of a dicarboxylic acid ("diacid") acylated at both acid moieties. The prepolymer may be a single acylated diacid unit (monomer) or may have up to about 12 condensed diacid units. A mixture of different diacids may be used. A mixture of diacids may result in a random copolymer. One or more diacids may optionally be a diacid-substituted C2-C carboxylate, interrupted by about 1 to about 5 -Ph-, -O-, -CH=CH-, and / or -N(R)- groups. 12 The diacids may include straight or branched chain alkanes, where R is H, phenyl, benzyl, or (C1-C6) alkyl. The one or more diacids may also be optionally interrupted by about 1 to about 12 -OCH2CH2O- groups. The one or more diacids may also be optionally substituted with 1, 2, or 3 trifluoromethyl, trifluoromethoxy, (C1-C6) alkyl, (C1-C6) alkenyl, or oxo groups, or combinations thereof.

[0193] The at least one dibasic acid may be a 1,ω-bis(carboxy)alkane. The at least one dibasic acid may also be a 1,ω-bis(4-carboxyphenoxy)alkane. The alkane may be, for example, a (C3-C8)alkane. Specific examples of alkanes include hexane and octane. The dibasic acid may be 1,6-bis(4-carboxyphenoxy)hexane. Alternatively, the dibasic acid may be 1,6-bis(carboxy)octane (sebacic acid). The at least one prepolymer may also include a bis(carboxylic acid acetyl ester), or an anhydride oligomer thereof. The at least one prepolymer may also include a 1,ω-(4-acetoxycarbonylphenoxyl)alkane, or an anhydride oligomer thereof, or 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane, or an anhydride oligomer thereof.

[0194] The carboxylic anhydride may be a bis-alkyl carboxylic anhydride, a bis-aryl carboxylic anhydride, an alkyl-aryl carboxylic anhydride, or a mixture thereof. The carboxylic anhydride may be, for example, acetic anhydride, trifluoroacetic anhydride, or benzoic anhydride. A molar excess of the carboxylic anhydride may be used. The excess carboxylic anhydride may be removed after the prepolymer is formed.

[0195] In various embodiments, the polymer of the microparticles and / or nanoparticles described herein can be poly-sebacic anhydride (SA), poly-1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride, or poly-1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) anhydride. In other embodiments, the polymer of the microparticles and / or nanoparticles described herein can be a copolymer of sebacic anhydride (SA) and 1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride, or a copolymer of 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) anhydride and 1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride. The ratio of SA to CPH, or CPTEG to CPH, can be any integer from about 1:19 to about 19:1. In certain embodiments, the ratio of CPTEG to CPH is about 20:80. An example of the structure of the SA:CPH copolymer is as follows: [ka] Each block (indicated by single or double brackets) has an M of about 5,000 to about 50,000 g / mol, such as about 10,000 to about 25,000 g / mol, or about 15,000 to about 20,000 g / mol. n The anhydride copolymers may be block or random copolymers, or a combination thereof. CPTEG:CPH copolymers also include a polymer having an M of about 5,000 to about 50,000 g / mol, such as about 10,000 to about 25,000 g / mol, or about 15,000 to about 20,000 g / mol. n The polymer may be prepared to form a polymer that may contain a number of repeating units sufficient to provide a polymer having the formula:

[0196] The polyanhydride particles described herein can carry an effective amount of an RNA polynucleotide. In some embodiments, exosomes containing an RNA polynucleotide can be encapsulated in the polyanhydride polymer.

[0197] Mode of administration The composition may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited to, intradermal, intramuscular, intranasal, and / or subcutaneous administration. The present disclosure provides a method comprising administering a composition comprising an RNA polynucleotide to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The composition comprising an RNA polynucleotide is typically formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the composition comprising an RNA polynucleotide may be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective dose level, prophylactically effective dose level, or appropriate imaging dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound used; the particular composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; the duration of treatment; drugs used in combination or concomitantly with the particular compound used; and similar factors well known in the medical arts.

[0198] In some embodiments, a composition comprising an RNA polynucleotide is administered at a dose of between 0.0001 mg / kg and 100 mg / kg, 0.001 mg / kg and 0.05 mg / kg, 0.005 mg / kg and 0.05 mg / kg, 0.001 mg / kg and 0.005 mg / kg, 0.05 mg / kg and 0.5 mg / kg of a subject's body weight per day, one or more times per day, per week, per month, etc. to achieve a desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be administered at dosage levels sufficient to deliver 0.01 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, or 1 mg / kg to 25 mg / kg, (see, e.g., the unit dose ranges set forth in WO 2013 / 078199, the contents of which are incorporated herein by reference in their entirety). The desired dosage may be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, etc. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are used, split administration regimens such as those described herein may be used. In certain embodiments, the composition comprising the RNA polynucleotide may be administered at a dosage level sufficient to deliver 0.0005mg / kg to 0.01mg / kg, for example, about 0.0005mg / kg to about 0.0075mg / kg, for example, about 0.0005mg / kg, about 0.001mg / kg, about 0.002mg / kg, about 0.003mg / kg, about 0.004mg / kg, or about 0.005mg / kg.

[0199] In some embodiments, compositions comprising an RNA polynucleotide may be administered once or twice (or more) at a dosage level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg.

[0200] In some embodiments, the composition comprising an RNA polynucleotide is administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months, 0 and 6 months, 0 and 9 months, 0 and 12 months, 0 and 18 months, 0 and 2 years, 0 and 5 years, or 0 and 10 years) at 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.25 mg, 0.400 mg, 0.600 mg, 0.800 mg, 0.900 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.300 mg, 0.400 mg, 0.500 mg, 0.600 mg, 0.7 ...250 mg, 0.300 mg, 0. A total dose of 25 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg, or at a dosage level sufficient to deliver such a total dose. Higher and lower dosages and frequencies of administration are encompassed by the present disclosure. For example, a composition containing an RNA polynucleotide can be administered three or four times.

[0201] In some embodiments, a composition comprising an RNA polynucleotide may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months, 0 and 6 months, 0 and 9 months, 0 and 12 months, 0 and 18 months, 0 and 2 years, 0 and 5 years, or 0 and 10 years) at a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg, or at a dosage level sufficient to deliver that total dose.

[0202] Compositions comprising the RNA polynucleotides described herein may be formulated into dosage forms described herein, such as intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, intranasal, and subcutaneous).

[0203] Embodiment The following numbered embodiments also form part of this disclosure.

[0204] 1. An RNA polynucleotide comprising a 5' untranslated region (5'UTR), a heterologous sequence encoding at least one polypeptide, and a 3' untranslated region (3'UTR), wherein the 5'UTR or the 3'UTR comprises a cap-independent translation enhancer or an exoribonuclease-resistant RNA (xrRNA) element.

[0205] 2. The RNA polynucleotide of embodiment 1, wherein the 5'UTR or 3'UTR comprises an xrRNA element.

[0206] 3. The RNA polynucleotide of embodiment 1 or embodiment 2, wherein the xrRNA element comprises SEQ ID NO: 6, 25, 31, or 36.

[0207] 4. The RNA polynucleotide of any one of embodiments 1 to 3, wherein the 5'UTR or 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer.

[0208] 5. The RNA polynucleotide of any one of embodiments 1 to 4, wherein the 5'UTR or 3'UTR is derived from Thin paspalum asymptomatic virus (TPAV), Tomato bushy stunt virus (TBSV), Sweet clover necrotic mosaic virus (SCNMV), Red clover necrotic mosaic virus (RCNMV), or Poppy mosaic virus (OPMV).

[0209] 6. The RNA polynucleotide of any one of embodiments 1-5, wherein the 5'UTR or 3'UTR comprises a polynucleotide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

[0210] 7. The RNA polynucleotide of any one of embodiments 1-6, wherein the 5'UTR comprises SEQ ID NO: 7, 14, 26, 28, 29, 32, 33, or 37, and the 3'UTR comprises SEQ ID NO: 8, 13, 15, 16, 27, 30, 35, or 38.

[0211] 9. The RNA polynucleotide of any one of embodiments 1-7, wherein the RNA polynucleotide comprises an internal ribosome entry site (IRES).

[0212] 9. The RNA polynucleotide of any one of embodiments 1-8, wherein the IRES comprises SEQ ID NO: 9, 10, 12, or 34.

[0213] 10. The RNA polynucleotide of any one of embodiments 1-9, wherein the RNA polynucleotide comprises an RNA protein binding domain.

[0214] 11. The RNA polynucleotide of any one of embodiments 1 to 10, wherein the RNA polynucleotide does not comprise a 5' cap structure or a polyA tail.

[0215] 12. The RNA polynucleotide of any one of embodiments 1-11, wherein the RNA polynucleotide does not comprise modified nucleosides.

[0216] 13. The RNA polynucleotide of any one of embodiments 1-12, wherein at least one polypeptide comprises an antigenic polypeptide.

[0217] 14. The RNA polynucleotide of embodiment 13, wherein the antigenic polypeptide is derived from an influenza virus or a coronavirus.

[0218] 15. The RNA polynucleotide of any one of embodiments 1-12, wherein at least one polypeptide comprises a light chain and a heavy chain of an antibody.

[0219] 16. The RNA polynucleotide of any one of embodiments 1-15, wherein the RNA polynucleotide is a circular RNA polynucleotide.

[0220] 17. A polyanhydride composition comprising a polyanhydride polymer and an RNA polynucleotide of any one of embodiments 1-16, wherein the polyanhydride polymer encapsulates the RNA polynucleotide.

[0221] 18. The composition of embodiment 17, wherein the polyanhydride polymer comprises a copolymer of 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis-(p-carboxyphenoxy)hexane (CPH).

[0222] 19. The composition of embodiment 18, wherein the CPTEG:CPH ratio is from about 20:80 to about 50:50.

[0223] 20. An exosome for delivering an RNA polynucleotide, the exosome comprising an RNA polynucleotide comprising a 5'UTR, a sequence encoding at least one polypeptide, and a 3'UTR, wherein the 5'UTR or the 3'UTR comprises a cap-independent translation enhancer or an xrRNA element.

[0224] 21. The exosome of embodiment 20, wherein the 5'UTR or 3'UTR comprises an xrRNA element.

[0225] 22. The exosome of embodiment 20 or embodiment 21, wherein the xrRNA comprises SEQ ID NO: 6, 25, 31, or 36.

[0226] 23. The exosome of any one of embodiments 20 to 22, wherein the 5'UTR or 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer.

[0227] 24. The exosome of any one of embodiments 20 to 23, wherein the 5'UTR or 3'UTR is derived from TPAV, TBSV, SCNMV, RCNMV, or OPMV.

[0228] 25. The exosome of any one of embodiments 20-24, wherein the 5'UTR or 3'UTR comprises a polynucleotide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

[0229] 26. The exosome of any one of embodiments 20 to 25, wherein the 5'UTR comprises SEQ ID NO: 7, 14, 26, 28, 29, 32, 33, or 37, and the 3'UTR comprises SEQ ID NO: 8, 13, 15, 16, 27, 30, 35, or 38.

[0230] 28. The exosome of any one of embodiments 20 to 26, wherein the RNA polynucleotide comprises an IRES.

[0231] 29. The exosome of any one of embodiments 20 to 27, wherein the IRES comprises SEQ ID NO: 9, 10, 12, or 34.

[0232] 30. The exosome of any one of embodiments 20 to 28, wherein the RNA polynucleotide comprises an RNA protein binding domain.

[0233] 31. The exosome of any one of embodiments 20 to 29, wherein the RNA polynucleotide does not comprise a 5' cap structure or a polyA tail.

[0234] 32. The exosome of any one of embodiments 20 to 30, wherein the RNA polynucleotide does not contain modified nucleosides.

[0235] 33. The exosome of any one of embodiments 20-31, wherein at least one polypeptide comprises an antigenic polypeptide.

[0236] 34. The exosome of embodiment 33, wherein the antigenic polypeptide is derived from an influenza virus or a coronavirus.

[0237] 35. The exosome of any one of embodiments 20 to 34, wherein at least one polypeptide comprises a light chain and a heavy chain of an antibody.

[0238] 36. A polyanhydride composition comprising a polyanhydride polymer and an exosome of any one of embodiments 20 to 35, wherein the polyanhydride polymer encapsulates the exosome.

[0239] 37. The composition of embodiment 36, wherein the polyanhydride polymer comprises a copolymer of CPTEG and CPH.

[0240] 38. The composition of embodiment 37, wherein the CPTEG:CPH ratio is from about 20:80 to about 50:50.

[0241] 39. A pharmaceutical composition comprising an RNA polynucleotide according to any one of embodiments 1 to 16 and a pharma- ceutically acceptable excipient.

[0242] 40. An RNA polynucleotide comprising a 5'UTR, a heterologous multiple cloning site for inserting a sequence encoding a polypeptide, and a 3'UTR, wherein the 5'UTR or the 3'UTR comprises a cap-independent translation enhancer or an xrRNA element.

[0243] 41. A DNA polynucleotide encoding the RNA polynucleotide of any one of embodiments 1-16 or 40.

[0244] 42. A method for producing a polypeptide of interest in a subject, the method comprising administering to the subject an RNA polynucleotide of any one of embodiments 1-16, a composition of any one of embodiments 17-19 or 36-39, or an exosome of any one of embodiments 20-35.

[0245] 43. A method for delivering an RNA polynucleotide to a subject, the method comprising administering to the subject an RNA polynucleotide of any one of embodiments 1-16, a composition of any one of embodiments 17-19 or 36-39, or an exosome of any one of embodiments 20-35.

[0246] 44. A method for inducing an immune response in a subject, the method comprising administering to the subject a composition comprising an RNA polynucleotide in an amount effective to produce an antigen-specific immune response in the subject, the RNA polynucleotide comprising a 5'UTR, a sequence encoding at least one antigenic polypeptide, and a 3'UTR, wherein the 5'UTR or the 3'UTR comprises a cap-independent translation enhancer or an xrRNA element.

[0247] 45. The method of embodiment 44, wherein the 5'UTR or 3'UTR comprises an xrRNA element.

[0248] 46. ​​The method of embodiment 44 or embodiment 45, wherein the xrRNA element comprises SEQ ID NO: 6, 25, 31, or 36.

[0249] 47. The method of any one of embodiments 44 to 46, wherein the 5'UTR or 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer.

[0250] 48. The method of any one of embodiments 44-47, wherein the 5'UTR and / or the 3'UTR is from TPAV, TBSV, SCNMV, RCNMV, or OPMV.

[0251] 49. The method of any one of embodiments 44-48, wherein the 5'UTR or 3'UTR comprises a polynucleotide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

[0252] 50. The method of any one of embodiments 44 to 49, wherein the 5'UTR comprises SEQ ID NO: 7, 14, 26, 28, 29, 32, 33, or 37, and the 3'UTR comprises SEQ ID NO: 8, 13, 15, 16, 27, 30, 35, or 38.

[0253] 51. The method of any one of embodiments 44 to 50, wherein the RNA polynucleotide comprises an IRES.

[0254] 52. The method of any one of embodiments 44-51, wherein the IRES comprises SEQ ID NO: 9, 10, 12, or 34.

[0255] 53. The method of any one of embodiments 44-52, wherein the RNA polynucleotide comprises an RNA protein binding domain.

[0256] 54. The method of any one of embodiments 44 to 53, wherein the RNA polynucleotide does not comprise a 5' cap structure or a polyA tail.

[0257] 55. The method of any one of embodiments 44-54, wherein the RNA polynucleotide does not contain modified nucleosides.

[0258] 56. The method of any one of embodiments 44-55, wherein the antigenic polypeptide is derived from an influenza virus or a coronavirus.

[0259] 57. The method of any one of embodiments 44-56, wherein the composition comprises a pharma- ceutically acceptable excipient.

[0260] 58. The method of any one of embodiments 44 to 57, wherein the composition comprises exosomes.

[0261] 59. The method of any one of embodiments 44-58, wherein the composition comprises a polyanhydride polymer.

[0262] 60. The method of embodiment 59, wherein the polyanhydride polymer comprises a copolymer of CPTEG and CPH.

[0263] 61. The method of embodiment 60, wherein the CPTEG:CPH ratio is from about 20:80 to about 50:50.

[0264] 62. The method of any one of embodiments 44-61, wherein the composition is administered to the subject intramuscularly, intranasally, or intradermally.

[0265] 63. The method of any one of embodiments 44-62, wherein the composition is administered intranasally to the subject.

[0266] 64. The method of any one of embodiments 44-63, wherein the subject is a human.

[0267] 65. A method of delivering an antibody to a subject, the method comprising administering to the subject a composition comprising an RNA polynucleotide, the RNA polynucleotide comprising a 5'UTR, a sequence encoding a light chain of the antibody, a sequence encoding a heavy chain of the antibody, at least one IRES, and a 3'UTR, wherein the 5'UTR or the 3'UTR comprises a cap-independent translation enhancer or an xrRNA element.

[0268] 66. The method of embodiment 65, wherein the 5'UTR or 3'UTR comprises an xrRNA element.

[0269] 67. The method of embodiment 65 or embodiment 66, wherein the xrRNA element comprises SEQ ID NO: 6, 25, 31, or 36.

[0270] 68. The method of any one of embodiments 65 to 67, wherein the 5'UTR or 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer.

[0271] 69. The method of any one of embodiments 65-68, wherein the 5'UTR and / or the 3'UTR is derived from TPAV, TBSV, SCNMV, RCNMV, or OPMV.

[0272] 70. The method of any one of embodiments 65-69, wherein the 5'UTR or 3'UTR comprises a polynucleotide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

[0273] 71. The method of any one of embodiments 65 to 70, wherein the 5'UTR comprises SEQ ID NO: 7, 14, 26, 28, 29, 32, 33, or 37, and the 3'UTR comprises SEQ ID NO: 8, 13, 15, 16, 27, 30, 35, or 38.

[0274] 72. The method of any one of embodiments 65 to 71, wherein the RNA polynucleotide comprises two IRES, the IRES comprising SEQ ID NO: 9 and SEQ ID NO: 10.

[0275] 73. The method of any one of embodiments 65-72, wherein the RNA polynucleotide comprises an RNA protein binding domain.

[0276] 74. The method of any one of embodiments 65 to 73, wherein the RNA polynucleotide does not comprise a 5' cap structure or a polyA tail.

[0277] 75. The method of any one of embodiments 65-74, wherein the RNA polynucleotide does not contain modified nucleosides.

[0278] 76. The method of any one of embodiments 65-75, wherein the composition comprises a pharma- ceutically acceptable excipient.

[0279] 77. The method of any one of embodiments 65-76, wherein the composition comprises exosomes.

[0280] 78. The method of any one of embodiments 65-77, wherein the composition comprises a polyanhydride polymer.

[0281] 79. The method of embodiment 78, wherein the polyanhydride polymer comprises a copolymer of CPTEG and CPH.

[0282] 80. The method of embodiment 79, wherein the CPTEG:CPH ratio is from about 20:80 to about 50:50.

[0283] 81. The method of any one of embodiments 65-80, wherein the composition is administered to the subject intramuscularly, intranasally, or intradermally.

[0284] 82. The method of any one of embodiments 65-81, wherein the subject is a human.

[0285] 83. A method for producing exosomes for delivery of an RNA polynucleotide, the method comprising: transforming a cell with a polynucleotide construct expressing an RNA polynucleotide, the RNA polynucleotide comprising a 5' UTR, a sequence encoding at least one polypeptide, and a 3' UTR, wherein the 5' UTR or the 3' UTR comprises a cap-independent translation enhancer or an xrRNA element; culturing the cell in a growth medium, wherein exosomes comprising the RNA polynucleotide are released outside the cell into the growth medium; removing the cells from the growth medium; and harvesting the exosomes comprising the RNA polynucleotide from the growth medium.

[0286] 84. The method of embodiment 83, wherein the harvesting is by ultracentrifugation.

[0287] 85. The method of embodiment 83 or embodiment 84, wherein the cell is an A549 cell.

[0288] 86. The method of any one of embodiments 83 to 85, wherein the polynucleotide construct comprises a promoter.

[0289] 87. The method of embodiment 86, wherein the promoter is a T7 promoter and the cells constitutively express T7 RNA polymerase.

[0290] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0291] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0292] Example 1: Alternative mRNA Expression Cassettes A series of expression cassettes were tested that function to stabilize mRNAs by flanking the coding region with untranslated regions that confer some protection from host endonucleases. Expression of fluorescent reporters flanked by beta-actin (an abundant housekeeping protein in cells) untranslated regions (UTRs) similar to those on the Moderna vaccine mRNAs, but without capping or tailing, was transient (less than 24 hours) unless continuously transcribed by intracellular T7 RNA polymerase. In contrast, fluorescent reporter proteins persisted in cells when using exonuclease-resistant RNA structures (xrRNAs), as well as thin Paspalum Asymptomatic Virus (TPAV) and Aphid Lethal Paralysis Virus (ALPV) UTRs flanking the coding region of the reporter gene, suggesting that the mRNAs flanked by these UTRs persisted and were efficiently translated despite lacking caps and poly(A) tails.

[0293] The goal of mRNA vaccines is to express a protein (antigen) in cells so as to mount a long-lived antibody response against the protein. These alternative expression cassettes appear to be superior to conventional mRNA constructs that contain "housekeeping gene" UTRs + cap + tailing. The most preferred expression cassettes tested so far are those containing UTRs from TPAV.

[0294] For specific methodology, in vitro transcribed (IVT) expressed mRNA was generated using a T7 RNA polymerase expression kit, and mRNA was harvested by phenol / chloroform and ethanol / salt purification. 1 μg of mRNA was transfected into cells using Mirus Biotech's mRNA transfection reagent. A similar transfection using only a PCR fragment containing the T7 promoter, 5' and 3' UTR, and reporter coding region was also transfected into A549 cells, which constitutively express T7 polymerase, by Lipofectamine.

[0295] A diagram of an exemplary expression cassette is shown in Figure 1. Figure 2 shows the accumulation of mKATE fluorescent reporter protein following transfection with mRNA containing the indicated UTRs.

[0296] Example 2: Thermally stable polyanhydride nanoparticles Incorporation of mRNA into polyanhydride nanoparticles mRNA was successfully incorporated into polyanhydride nanoparticles (based on a 20:80 CPTEG:CPH formulation) using an xrRNA expression cassette. CPTEG is specifically 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane, while CPH is 1,6-bis-(p-carboxyphenoxy)-hexane, which can be blended in different ratios to vary the release kinetics and protection from water and oxygen. Specifically, 800 μg of mRNA was produced by conventional IVT expression using the Lucigen T7 Mega Kit. The RNA was purified by phenol / chloroform extraction, salt and glycogen carrier. The RNA pellet was resuspended in pure water with 0.1% spermine (Sigma). The RNA was then lyophilized and encapsulated in 20:80 CPTEG / CPH. The mRNA-containing nanoparticles (5 mg) were then dissolved in water and the RNA was subjected to reverse transcription and then PCR using mKATE primers to find the product. Some of the RNA was also blended with lipofectamine and placed on A549 cells, and fluorescent protein was found in the cells after 24 hours. The same approach was repeated but omitting the nanoparticles at room temperature with similar findings. Figure 3 shows that the mRNA extracted from the nanoparticles can still be transfected into cells. Figure 4 shows that the mRNA extracted from the nanoparticles is likely protected from thermal degradation at room temperature. For comparison, the mRNA in both the Moderna and Pfizer / BioNtech vaccines has a room temperature shelf life of about 4 hours before degrading.

[0297] mRNA transfection in copolymers results in higher transfection efficiency than lipid transfection We tested whether the copolymers could transfect cells as well as lipids designed to optimally deliver mRNA into cells, and found that the copolymers were able to deliver horse HA mRNA into cells with enhanced delivery relative to lipids (Figure 5).

[0298] mRNA in polyanhydride carriers is heat stable and can be transfected after months on the shelf Formulations of 40 μg of mRNA encoding the RSV F protein were left on the shelf for four months, one in polyanhydride 20:80 and one without polyanhydride. Only the one in polyanhydride was stable (Figure 6). Higher transfection efficiencies can be obtained, but quickly getting the mRNA back out of 20:80 can be difficult as it is designed to dissolve much more slowly. Similar studies were performed at 37°C with mRNA and RNA viruses and found to be stable only in the polyanhydride material.

[0299] Example 3: Extracellular vesicles The TPAV / mKATE expression cassette was PCR amplified from a commercially synthesized plasmid with overlapping oligos. The PCR product contains a T7 promoter (5'-TAATACGACTCACTATAG-3') in front of the 5'UTR. The PCR product (5 μg) was transfected into A549 cells previously engineered to continuously express T7 polymerase (A549-T7 cells) by DEA-dextran and 10% glycerol shock. The transfection reagent was rinsed and replaced with standard medium without serum after 2 h. After 3 days, the supernatant was harvested, centrifuged at 3000 g, filtered twice through a 0.2 μm filter, and 100 μl of the 40 ml was transferred back into a 6-well plate containing HELA cells without T7 polymerase. The cells were observed for 4 days, and the fluorescence of mKATE was observed after 2 days (Figure 7). The brightness of the red fluorescence and the number of fluorescent cells increased over the entire period.

[0300] The EVs were spun down using high-speed ultracentrifugation and the final product was diluted in 100 μl of water. It was then diluted 500-fold and imaged by scanning electron microscopy. At the same time, the EVs were imaged by a NanoSight particle imager (Figure 8). mKATE mRNA was detected in the EVs harvested by the commercial kit, suggesting that the transcript was incorporated and protected from degradation for one week before the EVs were harvested from pre-cleared cell culture supernatants stored at 4 °C (Figure 9).

[0301] Example 4: RNA protein binding domains Additional TPAV and xrRNA systems have been developed that incorporate RNA protein binding domains (SEQ ID NO: 11) that target either lipid membranes or MVP protein. These enhance the incorporation of mRNA into exosomes. The inclusion of T7 termination sequences at the ends of the expression cassettes ensures that uncut DNA plasmids can be transfected into cells for transcription without the need to cleave and transfect linearized plasmids. The use of MVP protein binding domains also allows for capture of RNA using a recombinant version of the protein immobilized on a column for capture of RNA following lysis of cells that have undergone transcription via T7 polymerase.

[0302] Example 5: Dual Expression System A dual expression system was developed incorporating an IRES sequence from poliovirus and another IRES from cricket paralysis virus. This system allows dual expression of two proteins. Expression of two proteins for a vaccine is possible. The inclusion of heavy and light chain antibody sequences with multiple cloning sites also allows the antibody to be cloned by the CDR3 region and ligated in frame with the antibody (one for the light chain and one for the heavy chain). This then allows dual expression of the complete heavy and light chains in target cells in vaccinated animals / humans for the generation of therapeutic antibodies that are made and secreted in the body.

[0303] A system has also been developed that uses the Cricket Paralysis Virus IRES for downstream production of heavy chain antibodies and upstream using the TPAV / xrRNA system for light chain antibodies, with the aim of using this mRNA to generate therapeutic antibodies in vivo.

[0304] Example 6: Additional Expression Systems Several new expression systems were developed for testing. The first system uses the Cricket Paralysis Virus IRES (SEQ ID NO: 18) in conjunction with other codes. The second uses the Wheat Mosaic Virus (TRIMV) IRES (SEQ ID NO: 19) in conjunction with other codes. Two additional systems were developed that use either both or a single UTR from Tomato Bushy Stunt Virus (SEQ ID NOs: 20 and 21).

[0305] Example 7: Self-Cutting Cassette A polycistronic system using a self-cleaving peptide was tested. The use of a double 2A, TA2 peptide placed between two coding sequences on the mRNA construct allowed for nearly 1:1 expression levels of both proteins (Figure 10). This is unusual as the traditional use of an IRES between coding sequences often leads to poor expression of downstream genes. Thus, mRNA constructs can be designed to make one, two, or more proteins from the same mRNA construct. The double peptide ensures that more of the proteins are separate proteins rather than fusions. This system is an option for expressing more than one flu protein in the same APC and reducing production costs versus multiple mRNA constructs.

[0306] Example 8: Comparison of additional mRNA constructs Additional mRNA constructs were expressed in T7 BHK cells. Expression levels from the various constructs indicated high levels of expression of the reporter protein. Among those tested, SCNMV and TPAV with 3'xrRNA resulted in the most favorable protein expression of mCherry (Figure 11) and mRNA for incorporation into EVs (not shown). These data demonstrate the ability to tailor individual UTRs to genes of interest to ensure high levels of expression.

[0307] The mRNA construct has a 10 bp leader before the xrRNA in the 5'UTR, therefore the lower expression shown may be from the xrRNA gaining access to the transcript, rather than being blocked by the xrRNA starting immediately at the 5' end. Future studies will optimize the 5'xrRNA to compare with the 3'xrRNA UTR.

[0308] Additional mRNA constructs are summarized in Table 1. [Table 1]

[0309] Example 9: mRNA Vaccination Mice were vaccinated once or twice with rHA from H3N8, with H3N8 HA in the correct or reverse orientation in EVs, or with Alum or mRNA on a TPAV cassette for H3N8 HA capped and tailed with β-actin (UTR) in liposomes. Hemagglutination inhibition (HAI) was assessed (Figure 12). The mRNA vaccine was superior to Flu HA protein in eliciting neutralizing antibodies. These data strongly indicate that the mRNA construct can confer protection from flu challenge.

[0310] Example 10: Naked mRNA transfection A549 cells without T7 were transfected with mock, a pig cassette expressing mCherry, or a commercially available mRNA expressing mCherry (modified U / C, Arco capped, and tailed) using Ribojuice liposomes. Actual expression of mRNA compared to commercially available mRNA with modified bases, 5' cap, and poly(A) tail (Figure 13). These data suggest that the mRNA constructs of the present disclosure should express their genes at high levels when injected into animals, and therefore should elicit an immune response.

[0311] Example 11: Stability of mRNA in polyanhydride nanoparticles Thermal stability assays demonstrated that polyanhydride-placed mRNA constructs exhibited resistance to degradation from heat after 7 days at 37° C. (FIG. 14). Conventional IVT-generated mRNA did not possess this level of thermal stability. Thus, mRNA should be stable at room temperature when placed within polyanhydride nanoparticles or rods.

[0312] Example 12: Circular RNA constructs The mRNA constructs of the present disclosure can be circularized with RNA ligase 1 by removing the 5' triphosphate and adding a single phosphate group with a kinase (Figure 15). The use of circular RNA further alleviates the need to include modified bases and also allows for digestion of dsRNA and short RNA products by using T4 RNA ligase 1 which only ligates linear ssRNA (not dsRNA as with T4 RNA ligase 2). This allows for skipping a step in the manufacturing process to remove dsRNA and short products.

Claims

1. 1. An RNA polynucleotide comprising a 5' untranslated region (5'UTR), a heterologous sequence encoding at least one polypeptide, and a 3' untranslated region (3'UTR), wherein the 5'UTR or the 3'UTR comprises a panicum mosaic virus-like cap-independent translation enhancer or an exoribonuclease-resistant RNA (xrRNA) element.

2. The RNA polynucleotide of claim 1 , wherein the 5′UTR or the 3′UTR comprises an xrRNA element.

3. 2. The RNA polynucleotide of claim 1, wherein the xrRNA element comprises SEQ ID NO: 6, 25, 31, or 36.

4. 2. The RNA polynucleotide of claim 1, wherein the 5'UTR or the 3'UTR comprises a Panicum mosaic virus-like cap-independent translation enhancer.

5. 2. The RNA polynucleotide of claim 1, wherein the 5'UTR or the 3'UTR is derived from Thin paspalum asymptomatic virus (TPAV), Tomato Bushy Stunt Virus (TBSV), Sweet clover necrotic mosaic virus (SCNMV), Red clover necrotic mosaic virus (RCNMV), or Poppy mosaic virus (OPMV).

6. 2. The RNA polynucleotide of claim 1, wherein the 5'UTR or the 3'UTR comprises a polynucleotide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

7. 2. The RNA polynucleotide of claim 1, wherein the 5'UTR comprises SEQ ID NO: 7, 14, 26, 28, 29, 32, 33, or 37 and the 3'UTR comprises SEQ ID NO: 8, 13, 15, 16, 27, 30, 35, or 38.

8. The RNA polynucleotide of claim 1 , wherein the RNA polynucleotide comprises an internal ribosome entry site (IRES).

9. The RNA polynucleotide of claim 1 , wherein the IRES comprises SEQ ID NO: 9, 10, 12, or 34.

10. The RNA polynucleotide of claim 1 , wherein the RNA polynucleotide comprises an RNA protein binding domain.

11. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide does not comprise a 5' cap structure or a polyA tail.

12. The RNA polynucleotide of claim 1 , wherein the RNA polynucleotide does not contain modified nucleosides.

13. The RNA polynucleotide of claim 1 , wherein the at least one polypeptide comprises an antigenic polypeptide.

14. 14. The RNA polynucleotide of claim 13, wherein the antigenic polypeptide is derived from an influenza virus or a coronavirus.

15. The RNA polynucleotide of claim 1 , wherein the at least one polypeptide comprises a light chain and a heavy chain of an antibody.

16. 16. A polyanhydride composition comprising a polyanhydride polymer and an RNA polynucleotide according to any one of claims 1 to 15, wherein the polyanhydride polymer encapsulates the RNA polynucleotide.

17. 17. The composition of claim 16, wherein the polyanhydride polymer comprises a copolymer of 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis-(p-carboxyphenoxy)hexane (CPH).

18. 18. The composition of claim 17, wherein the CPTEG:CPH ratio is from about 20:80 to about 50:

50.

19. 16. A composition for delivery of an RNA polynucleotide, comprising exosomes, wherein the exosomes comprise the RNA polynucleotide of any one of claims 1 to 15.

20. An RNA polynucleotide according to any one of claims 1 to 15; and a pharmaceutically acceptable excipient.

21. 16. A composition comprising the RNA polynucleotide of any one of claims 1 to 15, a composition comprising an exosome comprising the RNA polynucleotide of any one of claims 1 to 15, a polyanhydride composition comprising the RNA polynucleotide of any one of claims 1 to 15, or a pharmaceutical composition comprising the RNA polynucleotide of any one of claims 1 to 15, for producing a polypeptide of interest in a subject, for delivering an RNA polynucleotide to a subject, for inducing an immune response in a subject, or for delivering an antibody to a subject.

22. 22. The composition, polyanhydride composition, or pharmaceutical composition of claim 21, wherein the composition, polyanhydride composition, or pharmaceutical composition is administered to the subject intramuscularly, intranasally, or intradermally.

23. 22. The composition, polyanhydride composition, or pharmaceutical composition of claim 21, wherein the subject is a human.

24. 1. A method for producing exosomes for delivery of an RNA polynucleotide, the method comprising: Transforming a cell with a polynucleotide construct that expresses an RNA polynucleotide according to any one of claims 1 to 15; culturing the cells in a growth medium, wherein exosomes containing the RNA polynucleotide are released into the extracellular growth medium; removing the cells from the growth medium; and collecting the exosomes comprising the RNA polynucleotide from the growth medium.

25. 25. The method of claim 24, wherein said harvesting is by ultracentrifugation.

26. 26. The method of claim 25, wherein the cells are A549 cells.

27. 25. The method of claim 24, wherein the polynucleotide construct comprises a promoter.

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