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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/44—Oxidoreductases (1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
  • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
  • C12Y102/01036—Retinal dehydrogenase (1.2.1.36)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/542—Mucosal route oral/gastrointestinal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• tuberculosis tuberculosis, adenovirus
• genital Chlamydia , HIV-l, HPV
• protective immunity against mucosal pathogens requires the development of vaccine strategies capable of inducing mucosal immune responses (1, 2).
• diarrheal diseases constitute the second leading cause of death (after pneumonia) in young children of developing countries (3), and the design of tailored vaccines to the pathogen(s) and site of infection constitute a great challenge.
• modified polynucleotides encoding retinaldehyde dehydrogenase (RALDH).
• RALDH retinaldehyde dehydrogenase
• the modified polynucleotides may be used, for example, as components of vaccines.
• vaccines comprising modified polynucleotides encoding RALDH may be used, for example, to target mucosal tissues, leading to the generation of antigen-specific immunity in said tissues.
• the modified polynucleotides may comprise at least one pseudouridine in place of a uridine and/or at least one 5-methylcytosine in place of a cytosine.
• Other modifications, as described herein, are also possible.
• the modified polynucleotides can form the basis of new parenteral vaccine formulations that target mucosal tissues, bypassing the tolerogenic effects commonly associated with mucosal vaccinations as well as the toxicity mediated by other molecules, which also have mucosal imprinting properties, such as all -trans retinoic acid (ATRA).
• ATRA all -trans retinoic acid
• the vaccine which can, in some embodiments, induce mucosal receptors on antigen- specific T cells, may be used in cancer immunotherapy, particularly in regard to tumors developing at mucosal surfaces.
• novel modified polynucleotides e.g., mRNA
• a mRNA e.g., a mRNA
• the invention in some aspects, includes a variety of vaccines comprising the modified polynucleotides (e.g, mRNA) described herein.
• the vaccines may be used, for example, to treat infections (e.g., mucosal infections) or cancers (e.g., mucosal cancers).
• the modified polynucleotide may be used in a tolerogenic vaccine.
• Another aspect of the invention provides a kit comprising the modified polynucleotide and/or the vaccine described herein.
• the modified polynucleotides comprise an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein (e.g., human RALDH), wherein at least one uridine is pseudouridine, and/or at least one cytosine is 5-methylcytosine.
• ORF open reading frame
• RALDH retinaldehyde dehydrogenase
• the RALDH protein is selected from the group consisting of: retinaldehyde dehydrogenase 1 (RALDH 1) protein, retinaldehyde dehydrogenase 2 (RALDH2) protein, and retinaldehyde dehydrogenase 3 (RALDH3) protein.
• the RALDH protein is RALDH2.
• the RALDH protein is a human RALDH protein or variant thereof. Exemplary amino acid and nucleotide sequences of RADLH isoforms are provided herein.
• the open reading frame encodes two RALDH proteins selected from the group consisting of: retinaldehyde dehydrogenase 1 (RALDH 1) protein, retinaldehyde dehydrogenase 2 (RALDH2) protein, and retinaldehyde dehydrogenase 3 (RALDH3) protein.
• the open reading frame encodes retinaldehyde dehydrogenase 1 (RALDH 1) protein, retinaldehyde dehydrogenase 2 (RALDH2) protein, and retinaldehyde dehydrogenase 3 (RALDH3) protein.
• the invention provides a vaccine comprising at least one antigen, and at least one modified ribonucleic acid (e.g ., mRNA) polynucleotide comprising an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein.
• a vaccine comprising at least one antigen, and at least one modified ribonucleic acid (e.g ., mRNA) polynucleotide comprising an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein.
• mRNA modified ribonucleic acid
• ORF open reading frame
• RALDH retinaldehyde dehydrogenase
• the vaccine described herein further comprises an adjuvant (e.g., alum, AS03, AS04, MF59, or TLR agonists).
• the vaccine described herein further comprises retinal, retinol, b-carotene, or a combination thereof.
• the antigen may be, for example, a polynucleotide, protein, peptide, plasmid, virus, viral fragment, bacteria, bacterial fragment, fungi, fungal fragments and conjugate.
• the vaccine described herein is a mucosal vaccine.
• the antigen may be, for example, a viral or a bacterial pathogen, or a combination thereof.
• the vaccine may be a cancer vaccine
• the antigen may be a tumor antigen, e.g., a mucosal tumor antigen.
• exemplary mucosal tumor antigens include guanylyl cyclase C, sucrose isomaltase, CDX1, CDX2, mammoglobulin, small breast epithelial mucin, RAGE antigen, MUC1, and neoantigens.
• the mucosal tumor antigen is associated with a mucosal cancer selected from the group consisting of colon cancers, head and neck squamous cell carcinomas, lung cancers, cervical cancers, and pancreatic cancers.
• the vaccine may be formulated in a variety of different ways, for example, as a nanoparticle, microparticle, liposome, or hydrogel.
• the vaccine is formulated as a cationic lipid nanoparticle.
• the vaccine may further comprise a
• the invention provides a tolerogenic vaccine.
• Tolerogenic vaccines are used, for example, to reduce an immune response (induce tolerance) in the face of a pathological or unwanted activation of the normal immune response, which occurs, for example in autoimmune disorders.
• the tolerogenic vaccine in one aspect, comprises an antigen, an immunomodulatory agent, and at least one modified messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein.
• mRNA messenger ribonucleic acid
• ORF open reading frame
• RALDH retinaldehyde dehydrogenase
• exemplary immunomodulatory agents include, but are not limited to, mTOR inhibitors, HD AC inhibitors, MHC-peptide complexes, and antigen-laden erythrocytes.
• the vaccines or pharmaceutical compositions thereof described herein may be used in methods to induce antigen- specific immune responses (e.g ., a T cell response or a B cell response), for example, in the mucosal tissues of a subject.
• antigen-specific immune responses e.g ., a T cell response or a B cell response
• it may be used to immunize a subject against a pathogen (e.g., a mucosal pathogen).
• the vaccines or pharmaceutical compositions thereof may also be used to treat an infection (e.g., a mucosal infection) or a cancer (e.g., a mucosal cancer) in a subject.
• the vaccines or pharmaceutical compositions thereof may be administered parenterally, for example, by subcutaneous administration or intramuscular administration, or orally.
• the vaccine or pharmaceutical composition thereof may be administered as a single dose, or as a single dose followed by one or more subsequent booster doses.
• kits comprising: a polynucleotide described herein; a pharmaceutically acceptable excipient; a container; and instructions for using the kit. Further kits comprising any one of the vaccines or pharmaceutical
• compositions thereof are also described herein.
• Figures 1A-1D demonstrate that modRNA-RALDH2 induces the expression of gut-homing receptors on antigen-specific lymphocytes.
• Figure 1A Bone marrow-derived dendritic cells were cultured for 48 hours in the presence of 10 nM ATRA (ATRA), 20 pg modRNA-RALDH2 (modRNA), 20 pg modRNA-RALDH2 plus 1 pM retinol (modRNA + retinol), or left untouched (untreated). The expression of RALDH2 was detected by Western blot using a recombinant human RALDH2 sequence with an N-terminal His-tag as a positive control protein.
• Figures 1B-1C Purified ovalbumin- specific transgenic CD8 + T cells (OT-I) were cultured with wild-type splenocytes (APCs) and the indicated supplement.
• ATRA ovalbumin- specific transgenic CD8 + T cells
• Upregulation of a4b7 and CCR9 was determined using flow cytometry (Figure IB) and represented as the geometric mean of the mean fluorescent intensity (GeoMFI; Figure 1C).
• Figure ID Wild-type C57BL/6J mice were randomly divided into groups (each group housed separately) and immunized subcutaneously at day 0 with 100 pg ovalbumin (Ova) and 5 pM CpG class B. At days 0 and 2, the corresponding group of mice received 5 mM ATRA, 5 mM retinol, 50 pg mod RNA, or 50 pg mod RNA plus 5mM retinol, administered subcutaneously at the same site of injection as the Ova/CpG. Fecal samples were collected throughout the duration of the experiment, and titers of anti-Ova IgA-specific antibodies were assessed. All mice were orally challenged by gavage with 10 mg Ova at day 57.
• nucleic acid and“nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides.
• polymeric nucleic acids e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage.
• “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides).
• “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues.
• polynucleotide can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides).
• “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule.
• a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides.
• the terms“nucleic acid,” “DNA,”“RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc.
• nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications.
• a nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated.
• modified polynucleotide refers to a polynucleotide (e.g., DNA, RNA) that comprises at least one modified nucleotide.
• the polynucleotide may comprise any of the nucleoside analogs, chemically modified bases, biologically modified bases, intercalated bases, modified sugars, isomers, and/or modified phosphate groups described herein.
• ORF open reading frame
• RNA refers to a continuous stretch of RNA beginning with a start codon (e.g., AUG) and ending with a stop codon (e.g., UAA, UAG, UGA) that encodes a protein.
• the protein encoded by the ORF is a retinaldehyde dehydrogenase (RALDH) protein.
• RALDH retinaldehyde dehydrogenase
• the ORF is codon-optimized.
• “codon- optimized polynucleotide” refers to a polynucleotide that comprises codons that do not match those of the wild-type polynucleotide, but that do not alter the translated amino acid sequence of the encoded protein.
• the optimized codons can be used, for example, to increase mRNA stability, reduce secondary structures, minimize tandem repeat codons or base runs (which may impair gene construction or expression), manipulate transcriptional and translational control regions, add or delete protein trafficking sequences, insert, delete, or shuffle protein domains, add or delete restriction sites, or match codon frequencies in target and host organisms (for proper folding and secondary structure). Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
• protein refers to a polymer of amino acid residues linked together by peptide (amide) bonds.
• the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
• a protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins.
• One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
• a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
• a protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide.
• a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
• a protein may comprise different domains, for example, a nucleic acid binding domain.
• a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA.
• Any of the proteins provided herein may be produced by any method known in the art.
• the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
• the protein is a RALDH protein.
• RALDH splice variants of RALDH
• RALDH proteins are enzymes that catalyze the synthesis of retinoic acid (RA) from retinaldehyde, b- carotene, and vitamin A (retinol).
• RA retinoic acid
• b- carotene retinol
• vitamin A vitamin A
• UTR untranslated region
• Each polynucleotide generally has a UTR flanking each terminus of the ORF: a 5' UTR which begins at the transcription start site and continues to the start codon but does not include the start codon, and a 3 ' UTR, which begins immediately following the stop codon and continues until the transcriptional termination signal.
• recombinant refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering.
• a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.
• the term“vaccine,” as used herein, refers to one or more agents administered to a subject in order to stimulate the production of antibodies and provide immunity against one or more diseases.
• an effective amount or“therapeutically effective amount,” as used herein, refers to an amount of a biologically active agent (e.g ., a vaccine) that is sufficient to elicit a desired biological response.
• a biologically active agent e.g ., a vaccine
• an effective amount of a vaccine comprising modified RNA encoding RALDH may refer to the amount of vaccine necessary to treat a given disease or disorder, e.g., to generate a therapeutically effective antigen- specific immune response in the subject.
• an agent e.g., a vaccine
• the effective amount of an agent may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific antigen used, disease targeted, and on the modified RNA being used.
• the terms“administer,”“administering,” or“administration,” as used herein refers to implanting, applying, absorbing, ingesting, injecting, or inhaling, the inventive polynucleotide (e.g., RNA), vaccine, or pharmaceutical composition thereof.
• inventive polynucleotide e.g., RNA
• vaccine or pharmaceutical composition thereof.
• the term“subject,” as used herein, refers to an individual organism, for example, an individual mammal.
• the subject is a human.
• the subject is a non-human mammal.
• the subject is a non-human primate.
• the subject is a rodent.
• the subject is a sheep, a goat, a cattle, a cat, or a dog.
• the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode.
• the subject is a research animal.
• the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.
• treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
• the terms“treatment,” “treat,” and“treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
• treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed.
• treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease.
• treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
• the present invention is based, at least in part, on the discovery that modified
• RNA encoding retinaldehyde dehydrogenase (modRNA-RALDH) can be used in vaccines to target immune response to mucosal tissues.
• modRNA-RALDH retinaldehyde dehydrogenase
• ATRA all -trans retinoic acid
• vaccines incorporating the modRNA-RALDH described herein are more efficacious than current mucosal tissue vaccines, which often have disagreeable tolerogenic effects.
• Many current mucosal vaccines require life-attenuated pathogens and/or potent adjuvants, resulting in suboptimal safety profiles.
• the vaccines described herein are able to robustly target mucosal tissues without such side effects. It was also surprisingly found that the mucosal immune surveillance conveyed by ATRA, for example, resulting from
• the modRNA-RALDH molecules provided herein may be formulated as pharmaceutical compositions and/or vaccines, and used to treat a number of diseases, including mucosal infections and cancers.
• modRNA modified mRNA
• modRNA-RALDH modified mRNA
• ATRA all -trans retinoic acid
• DCs dendritic cells exposed to modRNA-RALDH results in the upregulation of mucosal homing receptors on activated B and T cells.
• parenteral vaccine formulations containing modRNA- RALDH can generate antigen- specific immunity at mucosal tissues, without compromising the systemic immune surveillance, unlike other parenteral vaccines.
• This mucosal immune surveillance is of relevance in the context of mucosal vaccination strategies targeting a host of mucosal pathogens, including, but not limited to, Shigella, enterotoxigenic E. coll, rotavirus, Chlamydia, and HIV-1.
• modRNA- RALDH enhances the efficacy of parenterally (i.e., subcutaneously (SQ) or intra-muscularly (IM)) administered vaccines.
• Parenteral vaccines typically induce immune responses in skin- associated peripheral lymph nodes that drain the inoculation site and normally do not produce mucosa-tropic memory cells.
• DCs dendritic cells from gut-associated lymphoid structures (Peyer’s patches (PP) and mesenteric lymph nodes (mLN)) induce the up- regulation of a4b7 integrin and chemokine receptor CCR9 on T and B lymphocytes (4, 9-11). As a result, these cells acquire the capacity to home to the small intestine. DCs originating from spleen or peripheral lymph nodes (pLN) cannot induce a similar gut-homing phenotype.
• This intestinal imprinting has been shown to result from the exclusive capacity of DCs from PP and mLN, but not pLN, to express retinal dehydrogenases (RALDH (9)), enzymes that convert dietary vitamin A to all-trans retinoic acid (ATRA (9)).
• RALDH retinal dehydrogenases
• ATRA enzymes that convert dietary vitamin A to all-trans retinoic acid
• the addition of exogenous ATRA to pLN DCs enables these cells to efficiently upregulate gut-homing receptors on activated lymphocytes.
• Experiments with human lymphocytes and DCs isolated from the mLN vs. spleen yielded analogous results (11).
• the exposure to ATRA during parenteral immunization was shown to convey protection against Salmonella infections (12).
• mucosal immune surveillance conveyed by vaccine formulations containing ATRA is not limited to the small and large intestine, but parenteral ATRA exposure during immunization also generates antigen-specific T and B cells at other mucosal surfaces, such as the oral cavity (determined in the saliva), nasal cavity, and urogenital tract.
• vaccines were designed to deliver RALDH enzymes to pLN DCs in order to generate ATRA capable of mucosal imprinting of antigen- specific B and T cells, which would confer a mucosal homing phenotype upon the impacted B and T cells.
• modRNA-RALDH in parenteral vaccines does allow for mucosal imprinting while avoiding tissue exposure to toxic ATRA; administration of modRNA-RALDH results in the transient expression of RALDH and, in the presence of vitamin A, generation of ATRA by antigen-presenting cells (e.g DCs).
• modRNA-RALDH Antigen presentation by DCs exposed to modRNA-RALDH results in the upregulation of mucosal homing receptors on activated B and T cells. Therefore, the vaccine formulations described herein generate antigen- specific immunity at mucosal tissues without compromising systemic immune surveillance, unlike classic parenteral vaccines.
• modRNA in free form or packaged in cationic lipid nanoparticles, was found to have moderate adjuvant properties, act directly on cells with phagocytic capacity, and to be degraded intracellularly upon transient induction of protein expression.
• modRNA-RALDH can be readily administered either SQ or IM and can be combined with existing or new vaccine formulations to improve efficacy.
• the modRNA-RALDH described herein allows for the design of robust parenteral vaccine formulations targeting mucosal tissues, bypassing the tolerogenic effects commonly associated with mucosal vaccinations, as well as toxicity mediated by other molecules with mucosal imprinting properties.
• the induction of mucosal receptors on antigen- specific T cells by modRNA-RALDH means it may also be used in cancer immunotherapy, particularly in regard to tumors developing at mucosal surfaces.
• the present invention is directed to modified polynucleotides encoding RALDH protein (modRNA-RALDH), as well as pharmaceutical compositions and vaccines comprising the modRNA-RALDH.
• modRNA-RALDH modified polynucleotides encoding RALDH protein
• the invention also includes methods of using the modRNA-RALDH, for example, to treat various diseases, including viral infections and/or cancers.
• kits including the modRNA-RALDH are kits including the modRNA- RALDH.
• modified polynucleotides e.g ., modRNA
• modRNA an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein and functional fragments and variants thereof.
• ORF open reading frame
• RALDH retinaldehyde dehydrogenase
• the RALDH is a human RALDH.
• the RADLH is from a non-human source (e.g., Mus musculus, Rattus norvegicus, Drosophilia melanogaster, Bifidobacterium bifidum, Micromonas commode, Clostridioides difficile, Ralstonia solanacearum, Flavobacterium psychrophilum, Acinetobacter pittii, Clostridium botulinum, Bordetella bronchiseptica, and Shigella flexneri).
• a non-human source e.g., Mus musculus, Rattus norvegicus, Drosophilia melanogaster, Bifidobacterium bifidum, Micromonas commode, Clostridioides difficile, Ralstonia solanacearum, Flavobacterium psychrophilum, Acinetobacter pittii, Clostridium botulinum, Bordetella bronchiseptica, and Shigella
• RALDH2 RALDH2
• RALDH3 exemplary human RALDH ORF polynucleotide sequences
• the ORF of the modRNA comprises a sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any one of SEQ ID NOs: 1-3.
• the ORF of the modRNA may encode RALDH1,
• the ORF of the modRNA encodes RALDH1.
• the ORF of the modRNA encodes RALDH2. In one embodiment, the ORF of the modRNA encodes RALDH3. In another embodiment, the ORF of the modRNA encodes two of the following: RALDH1, RALDH2, and RALDH3. In other embodiments, the modRNA encodes RALDH1, RALDH2, and RALDH3.
• Exemplary sequences of human RALDH1, RALDH2, and RALDH3 are presented herein as SEQ ID NOs: 4-6.
• the ORF encodes a RALDH protein that is 100% identical to any one of SEQ ID NOs: 4-6.
• the ORF encodes a RALDH protein that is 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any one of SEQ ID NOs: 4-6.
• RALDH typically comprises three domains: a NAD + -binding domain
• the ORF of the modRNA may encode one or more domains of RALDH 1, RALDH2, and RALDH3, or a combination thereof.
• the ORF may comprise a first domain from RALDH 1, a second domain from RALDH2, and a third domain from RALDH3.
• the ORF encodes two domains from a first RALDH protein and a single domain from a second RALDH protein.
• the modRNA is a nucleic acid molecule that has undergone a molecular biological manipulation, i.e., non-naturally occurring nucleic acid molecule or genetically engineered nucleic acid molecule.
• “modRNA” refers to a nucleic acid sequence which is not naturally occurring, or can be made by the artificial combination of two otherwise separated segments of nucleic acid sequence, i.e., by ligating together pieces of DNA that are not normally continuous.
• the artificial combination may be accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques using restriction enzymes, ligases, and similar recombinant techniques as described by, for example, Sambrook et al, Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; (1989), or Ausubel et al, Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover) IREL Press, Oxford, (1985); each of which is incorporated herein by reference.
• Such manipulation may be done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site.
• it may be performed to join together nucleic acid segments of encoding different RALDH proteins or domains to generate a single genetic entity comprising a desired combination of RALDH not found in nature.
• Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, open reading frames, or other useful features may be incorporated by design.
• the modRNA may be modified in a number of ways.
• the modRNA comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) in addition to one or more of: nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine,
• the modRNA comprises at least one uridine that is pseudouridine and at least one cytosine that is 5-methylcytosine. Such modifications may increase transcription, reduce or eliminate immunogenicity, and/or reduce degradation of the polynucleotide, among other properties, compared to an unmodified polynucleotide.
• all of the uridine bases of the modRNA are pseudouridine. In other embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
• uridine bases of a modRNA 97%, 98%, 99%, or more of the uridine bases of a modRNA are pseudouridine.
• all of the cytosine bases of the modRNA are 5- methylcytosine. In other embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
• cytosine bases of a modRNA are 5-methylcytosine.
• the modRNA further comprises a 5' untranslated region
• UTR and a 3' UTR.
• UTRs have regulatory functions; for example, the 5' UTR typically has a role in translation initiation and may include a Kozak sequence.
• the consensus Kozak sequence, CCR(A/G)CCAUGG (where R is a purine (adenine or guanine) three bases upstream of a start codon (AUG), followed by another guanine) has been shown to be involved in ribosomal initiation of the translation of many genes.
• the polynucleotide is enzymatically capped, for example, with a 5' terminal cap, such as
• the 3' UTR may comprise a number of adenosines and uridines.
• the 3' UTR comprises a poly(A) sequence.
• the poly(A) tail enhances the expression level of the encoded protein.
• the 5' UTR and/or the 3' UTR may be heterologous or synthetic. In some embodiments, the 5' UTR may be heterologous, and the 3' UTR may be synthetic. In another embodiment, the 5' UTR may be synthetic, and the 3' UTR may be heterologous. Any UTR from any gene may be engineered into the modified polynucleotide (e.g ., RNA) described herein.
• modified polynucleotide e.g ., RNA
• the modRNA when the modRNA encodes more than one RALDH protein, the modRNA further comprises one or more ribosome-binding sites (RBS).
• RBS is a sequence of nucleotides upstream of the start codon of a mRNA transcript that recruits a ribosome during initiation of protein translation.
• IVSs Internal Ribosome Entry Sites
• 2A peptides one or more 2A peptides
• IRESs are elements that permit initiation of translation from an internal region of an mRNA, whereas 2A peptides cause the ribosome to skip the synthesis of a peptide bond located in the C-terminus of the 2A element, resulting in a separation between the end of the 2A sequence and the next peptide downstream.
• 2A peptides include, but are not limited to, T2A, P2A, E2A, and F2A.
• the modRNA may comprise further elements, such as natural regulatory elements
• control sequences or may be associated with heterologous sequences, including, promoters, enhancers, response elements, suppressors, signal sequences, and introns.
• the modRNA may be codon-optimized (e.g. the codon AGT being used instead of AGC for coding of the amino acid serine; TTT/TTC for phenylalanine; TTA/TTG/CTT/CTC/CTA/CTG for leucine; ATT/ATC/ATA for isoleucine; GTT/GTC/GTA/GTG for valine; TCT/TCC/TCA/TCG for serine; CCT/CCC/CCA/CCG for proline/ ACT/ ACC/AC A/ AC G for threonine; GCT/GCC/GCA/GCG for alanine;
• the codon AGT being used instead of AGC for coding of the amino acid serine
• TTT/TTC for phenylalanine
• TTA/TTG/CTT/CTC/CTA/CTG for leucine
• ATT/ATC/ATA for isoleucine
• GTT/GTC/GTA/GTG for
• TAT/TAC for tyrosine
• CAT/CAC for histidine
• CAA/CAG for glutamine
• AAT/AAC for asparagine
• AAA/AAG for lysine
• GAT/GAC for aspartic acid
• GAA/GAG for glutamic acid
• TGT/TGC for cysteine
• C GT/C GC/C G A/CGG/AG A/AGG for arginine
• the optimized codons can be used, for example, to increase mRNA stability, reduce secondary structures, minimize tandem repeat codons or base runs (which may impair gene construction or expression), manipulate transcriptional and translational control regions, add or delete protein trafficking sequences, insert, delete, or shuffle protein domains, add or delete restriction sites, or match codon frequencies in target and host organisms (for proper folding and secondary structure).
• Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
• the modRNA described herein may be synthesized by standard methods known in the art, e.g., by in vitro or in vivo transcription.
• DNA templates may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters, resulting in production of the desired RNA.
• RNA polymerase promoters such as the T7 or SP6 polymerase promoters
• cDNA constructs that synthesize RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines, resulting in the production of the desired RNA.
• the modRNA is provided as a component of a vaccine.
• the vaccine in some embodiments, comprises at least one antigen and at least one RNA polynucleotide (e.g., mRNA) comprising an ORF encoding a RALDH protein.
• the vaccine in a further embodiment, may include an adjuvant and/or a pharmaceutically acceptable excipient.
• the vaccine is formulated in a nanoparticle, microparticle, hydrogel, or liposome.
• the vaccine formulation includes at least one substrate (direct or indirect) of RALDH.
• the vaccine comprises at least one first RNA
• polynucleotide e.g., mRNA
• polynucleotide comprising an ORF encoding at least one antigenic polypeptide or immunogenic polypeptide fragment from one or more pathogens and at least one second RNA polynucleotide (e.g., mRNA) comprising an ORF encoding a RALDH protein.
• the second RNA polynucleotide comprises an ORF wherein at least one uridine is pseudouridine and/or at least one cytosine is 5-methylcytosine.
• the vaccine comprises at least one RNA
• polynucleotide e.g ., mRNA
• the vaccine may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more RNA
• polynucleotides e.g., mRNAs
• oligodetase e.g., adenosine triphosphate
• RALDH proteins e.g., adenosine triphosphate
• the vaccine comprises at least one RNA polynucleotide comprising an ORF encoding a RALDH 1 protein. In one embodiment, the vaccine comprises at least one RNA polynucleotide comprising an ORF encoding a RALDH2 protein. In one embodiment, the vaccine comprises at least one RNA polynucleotide comprising an ORF encoding a
• the vaccine comprises an RNA polynucleotide comprising an ORF encoding a RALDH 1 protein, and an RNA polynucleotide comprising an ORF encoding a RALDH2 protein. In other embodiments, the vaccine comprises at least one RNA polynucleotide comprising an ORF encoding a RALDH 1 protein, and at least one RNA polynucleotide comprising an ORF encoding a RALDH3 protein. In some embodiments, the vaccine comprises an RNA polynucleotide comprising an ORF encoding a RALDH 1 protein, and an RNA polynucleotide comprising an ORF encoding a RALDH2 protein. In other embodiments, the vaccine comprises at least one RNA polynucleotide comprising an ORF encoding a RALDH 1 protein, and at least one RNA polynucleotide comprising an ORF encoding a RALDH3 protein. In some embodiments, the
• the vaccine comprises at least one RNA polynucleotide comprising an ORF encoding a RALDH2 protein, and at least one RNA polynucleotide comprising an ORF encoding a RALDH3 protein.
• the vaccine comprises an RNA polynucleotide comprising an ORF encoding a RALDH 1 protein, an RNA polynucleotide comprising an ORF encoding a RALDH2 protein, and an RNA polynucleotide comprising an ORF encoding a RALDH3 protein.
• the ORF of the modRNA may encode one or more domains of RALDH 1, RALDH2, and RALDH3, or a combination thereof.
• the ORF may comprise a first domain from RALDH 1, a second domain from RALDH2, and a third domain from RALDH3.
• the ORF encodes two domains from a first RALDH protein and a single domain from a second RALDH protein.
• the ORF of the modRNA may encode one or more truncated RALDH proteins.
• the ORF of the modRNA may encode a truncated RALDH 1 protein, a truncated RALDH2 protein, a truncated RALDH3 protein, or any combination thereof.
• the vaccine further comprises an adjuvant.
• Adjuvants may be used, for example, to enhance an immune response and/or to improve the efficacy of a vaccine.
• Adjuvants include, but are not limited to, monophosphoryl lipid A (MPL), MF59 (squalene), adjuvant system (AS) 01, AS02, AS03, AS04, alum, CAF01, IC31® (Valneva Technologies), iscomatrix, and TLR agonists.
• TLR agonists include Pam3Cys, BCG, LPS, 852A, VTX-2337, Poly(LC), imidazoquinolines, and CpG.
• the adjuvant is alum.
• the adjuvant is one or more TLR agonists.
• the vaccine further comprises one or more components of the retinoic acid synthesis pathway.
• components include, but are not limited to, retinal, b-carotenes, and vitamin A.
• the vaccine comprises a combination of such components. In other embodiments, the vaccine comprises one of the components.
• the vaccine is a mucosal vaccine.
• the mucosal vaccine may be targeted to different mucosal tissues, including, but not limited to, the oral cavity, the nasal cavity, the eye, the respiratory tract, the intestine, and the urogenital tract.
• the vaccine is monovalent. In other embodiments, the vaccine is multivalent, and is capable of protecting against more than one strain of the same microorganism and/or more than one microorganism.
• Such vaccines may include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different antigenic polypeptides/polynucleotides and/or immunogenic polypeptide/polynucleotide fragments from one or more pathogens ( e.g ., mucosal pathogens).
• the at least one antigen may comprise a
• polynucleotide protein, peptide, plasmid, virus, bacteria, bacterial fragment, fungus, fungal fragment and/or a conjugate.
• Other antigens are known in the art, and are contemplated herein.
• the pathogens are viral or bacterial pathogens, or a combination thereof.
• exemplary bacterial pathogens include a Bacillus species, a Bartonella species, a Bordetella species, a Borrelia species, a
• the bacterial pathogen is a Chlamydia species, for example, Chlamydia trachomatis.
• Exemplary viral pathogens include, but are not limited to, the following:
• the mucosal vaccine includes a recombinant or purified antigen or a mixture of antigens from one or more mucosal pathogens.
• antigens from mucosal pathogens include, but are not limited to: capsular polysaccharides (e.g ., Streptococcus species), chlamydia protease-like activity factor (CPAF; e.g.,
• Chlamydia species Chlamydia species), Cwp66 and Cwp84 ( Clostridium species), fimbrial adhesins (e.g., Escherichia species), flagellin (e.g., Escherichia species, Salmonella species,
• glycan polymers contained within a bacterial lipopolysaccharide e.g., O-antigen; Escherichia species, Salmonella species, Shigella species
• glycoconjugates e.g., Escherichia species, Salmonella species, Shigella species,
• Streptococcus species heat-labile toxin subunits (LT; e.g., Escherichia species), glycoprotein VP7 (Rotavirus), hemagglutinin antigens (e.g., Influenza A virus), major outer membrane protein (MOMP; e.g., Chlamydia species), matrix proteins (e.g., Influenza A virus), neuroaminidase (e.g., influenza A virus), outer membrane proteins (OMP; e.g., Chlamydia species, Treponema species), periplasmic protein CjaA (Campylobacter species), plasmid glycoprotein 3 (Pgp3; e.g., Chlamydia species), polymorphic membrane proteins (Pmps; e.g., Chlamydia species), pneumococcal surface protein A (PspA;
• Streptococcus species pneumolysin (Streptococcus species), S -layer proteins (SLP; e.g., Clostridium species), spike protein VP4 (Rotavirus), spike protein VP8 (Rotavirus), virus like particles (VLP; e.g., Norwalk virus, Influenza virus).
• SLP S -layer proteins
• VLP virus like particles
• the mucosal vaccine includes one or more species of inactivated bacteria, one or more species of live attenuated bacteria, or one or more strains of attenuated or inactivated viruses.
• the vaccine in some embodiments, is formulated in a hydrogel.
• the vaccine in some embodiments, is formulated as a particle (e.g., nanoparticle, microparticle).
• the modRNA-RALDH is formulated in the nanoparticle.
• the antigen e.g., the polynucleotide encoding at least one antigenic polypeptide or immunogenic polypeptide fragment from one or more mucosal pathogens
• an adjuvant is formulated in the nanoparticle.
• all three components are formulated in the same nanoparticle. In other embodiments, all three components are formulated in separate nanoparticles.
• two of the components are formulated in a nanoparticle and the third component is either formulated in a separate nanoparticle or not formulated in a nanoparticle.
• the modRNA-RALDH and the antigen e.g., the polynucleotide encoding at least one antigenic polypeptide or
• immunogenic polypeptide fragment from one or more pathogens are formulated in the same nanoparticle, and an adjuvant is not formulated in a nanoparticle.
• the vaccine formulated in a nanoparticle is on the surface of the nanoparticle (e.g., covalently or non-covalently associated), encapsulated within the nanoparticle (e.g., covalently or non-covalently associated), or both on the surface of the nanoparticle (e.g., covalently or non-covalently associated) and encapsulated within the nanoparticle (e.g., covalently or non-covalently associated).
• each component separately may be present on the surface of the nanoparticle (e.g ., covalently or non-covalently associated), encapsulated within the nanoparticle (e.g., covalently or non-covalently associated), or both on the surface of the nanoparticle (e.g., covalently or non-covalently associated) and encapsulated within the nanoparticle (e.g., covalently or non-covalently associated).
• the nanoparticle in some embodiments, can be composed of polymer and/or non-polymer molecules. Accordingly, the nanoparticle can be protein-based. The nanoparticle, in some embodiments, is macromolecular. In some embodiments, the nanoparticle is composed of amino acids.
• a nanoparticle can be, but is not limited to, one or a plurality of lipid-based nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, and/or nanoparticles that are developed using a combination of nanomaterials, such as lipid-polymer nanoparticles.
• the nanoparticle is composed of one or more polymers.
• polymers include, but are not limited to, polysaccharides (e.g., alginate, dextran, chitosan, agarose, and pullulan), polypeptides (e.g., albumin, gelatin, lectin, legumine, and viciline), polyesters, polyethers, and polyamides.
• polysaccharides e.g., alginate, dextran, chitosan, agarose, and pullulan
• polypeptides e.g., albumin, gelatin, lectin, legumine, and viciline
• polyesters e.g., polyethers, and polyamides.
• the one or more polymers is a water soluble, non-adhesive polymer.
• polymer is polyethylene glycol (PEG) or polyethylene oxide (PEO).
• PEG polyethylene glycol
• PEO polyethylene oxide
• the polymer is polyalkylene glycol or polyalkylene oxide.
• the one or more polymers is a biodegradable polymer.
• the one or more polymers is a biocompatible polymer that is a conjugate of a water soluble, non-adhesive polymer and a biodegradable polymer.
• the biodegradable polymer is polylactic acid (PLA), poly(glycolic acid) (PGA), or poly(lactic acid/glycolic acid) (PLGA).
• the nanoparticle is composed of PEG-PLGA polymers.
• the nanoparticle comprises one or more cationic lipids.
• polymers can be cationic polymers.
• cationic polymers are able to condense and/or protect negatively charged strands of nucleic acids (e.g., DNA, RNA, or derivatives thereof).
• Amine-containing polymers such as poly(lysine) (Zauner et al, 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al, 1995, Bioconjugate Chem., 6:7; both of which are incorporated herein by reference),
• the nanoparticle is a cationic lipid nanoparticle, such as those described in WO 2015/164674.
• lipid nanoparticle compositions and methods of making them are described, for example in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578.
• the nanoparticle is formed by self-assembly.
• Self- assembly refers to the process of the formation of a nanoparticle using components that will orient themselves in a predictable manner forming nanoparticles predictably and
• the nanoparticles are formed using amphiphilic biomaterials which orient themselves with respect to one another to form nanoparticles of predictable dimension, constituents, and placement of constituents.
• the amphiphilic biomaterials may have attached to them at least one of the vaccine components such that when the nanoparticles self assemble, there is a reproducible pattern of localization and density of the agents on/in the nanoparticle.
• the nanoparticle has a positive zeta potential. In some embodiments, the nanoparticle has a net positive charge at neutral pH. In some
• the nanoparticle comprises one or more amine moieties at its surface.
• the amine moiety is a primary, secondary, tertiary, or quaternary amine.
• the amine moiety is an aliphatic amine.
• the nanoparticle comprises an amine-containing polymer.
• the nanoparticle comprises an amine-containing lipid.
• the nanoparticle comprises a protein or a peptide that is positively-charged at neutral pH.
• the nanoparticle is a latex particle.
• the nanoparticle with the one or more amine moieties on its surface has a net positive charge at neutral pH.
• the nanoparticles have mean geometric diameter that is less than 500 nm.
• the nanoparticles have mean geometric diameter that is greater than 50 nm but less than 500 nm.
• the mean geometric diameter of a population of nanoparticles is about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm.
• the mean geometric diameter is between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, the mean geometric diameter is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some embodiments, the mean geometric diameter is between 75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of a population of nanoparticles have a diameter that is less than 500 nM.
• 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of a population of nanoparticles have a diameter that is greater than 50 nm but less than 500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of a population of nanoparticles have a diameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm.
• 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of a population of nanoparticles have a diameter that is between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of a population of nanoparticles have a diameter that is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm.
• the vaccine is a tolerogenic vaccine.
• the vaccine comprises a modRNA-RALDH, an immunomodulatory agent, and at least one antigen (e.g ., from one or more mucosal pathogens).
• Immunomodulatory agents are modRNAs capable of reducing an immune response (e.g., promoting a tolerogenic immune response), such as a T cell and/or B cell response. For example, such agents may reduce the number or proliferation of T and/or B cells, and/or may increase the number of T regulatory cells and/or B regulatory cells.
• Exemplary immunomodulatory agents include, but are not limited to, GM-CSF, TNF-a, mTOR inhibitors, HDAC inhibitors, MHC-peptide complexes, and antigen-laden erythrocytes.
• the vaccine is a cancer vaccine. In such embodiment,
• the vaccine comprises a modRNA-RALDH, at least one tumor antigen, and at least one adjuvant.
• the tumor antigen may be a polynucleotide encoding at least one tumor antigen (e.g., mucosal antigen) or immunogenic polypeptide fragment thereof.
• exemplary mucosal tumor antigens include, but are not limited to, the following: guanylyl cyclase C, sucrose isomaltase, CDX1, CDX2, mammoglobulin, small breast epithelial mucin, RAGE antigens, and MUC1.
• the tumor antigen is one or more neoantigens (e.g., antigens encoded by tumor- specific mutated genes).
• the cancer vaccine described herein may further comprise administering to the subject an inhibitor of a checkpoint molecule, an activator of a co-stimulatory receptor, or an inhibitor of an innate immune cell target.
• checkpoint molecules include, but are not limited to, PD-l, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, and A2aR.
• co-stimulatory receptors include, but are not limited to, 0X40, GITR, CD137, CD40, CD27, and ICOS.
• innate immune cell targets include, but are not limited to, KIR, NKG2A, CD96, TLR, and IDO.
• the cancer vaccine is administered in addition to a cell therapy, such as antigen-pulsed antigen-presenting cells (e.g ., antigen-pulsed dendritic cells or antigen-loaded T cells or B cells, or PBMC), gene therapy (e.g., SOZ vectors), or with CAR T cell therapy or CAR NK cell therapy.
• a cell therapy such as antigen-pulsed antigen-presenting cells (e.g ., antigen-pulsed dendritic cells or antigen-loaded T cells or B cells, or PBMC), gene therapy (e.g., SOZ vectors), or with CAR T cell therapy or CAR NK cell therapy.
• a cell therapy such as antigen-pulsed antigen-presenting cells (e.g ., antigen-pulsed dendritic cells or antigen-loaded T cells or B cells, or PBMC), gene therapy (e.g., SOZ vectors), or with CAR T cell therapy or CAR NK cell therapy.
• compositions comprising at least one modRNA-RALDH molecule, at least antigen (e.g., the polynucleotide encoding at least one antigenic polypeptide or immunogenic polypeptide fragment from one or more pathogens), and optionally, a pharmaceutically acceptable excipient.
• at least one modRNA-RALDH molecule e.g., the polynucleotide encoding at least one antigenic polypeptide or immunogenic polypeptide fragment from one or more pathogens
• a pharmaceutically acceptable excipient e.g., the polynucleotide encoding at least one antigenic polypeptide or immunogenic polypeptide fragment from one or more pathogens.
• the vaccine is administered in an effective amount, e.g., a therapeutically effective amount or prophylactically effective amount.
• a pharmaceutical composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art.
• routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example by epidermal administration (e.g., by injection or infusion).
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
• the vaccine is administered subcutaneously or intramuscularly. In certain embodiments, the vaccine is administered orally.
• the pharmaceutical composition or vaccine may be coated in a material to protect the modRNA from the action of acids and other natural conditions that may inactivate the modRNA (e.g., gastric acid).
• the vaccine is formulated within a nanoparticle, as described above.
• compositions agents include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
• solvents diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
• compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the active ingredient into association with the excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
• compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
• a“unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
• the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
• Relative amounts of the vaccine, the pharmaceutically acceptable carrier, and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
• the composition may comprise between 0.1% and 100% (w/w) active ingredient.
• the vaccine or pharmaceutical composition may comprise a buffering agent.
• buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D- gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alg
• Liquid dosage forms for parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing
• the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
• adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
• the conjugates of the invention are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.
• Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation can be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in l,3-butanediol.
• the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid are used in the preparation of injectables.
• the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
• the active ingredient can be prepared with carriers that will protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,
• compositions can be administered with medical devices known in the art.
• a pharmaceutical composition of this disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
• a needleless hypodermic injection device such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
• Examples of well-known implants and modules useful in the present disclosure include: U.S. Pat. No. 4,487,603, which discloses an implantable micro infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S.
• Pat. No. 4,447,233 which discloses a medication infusion pump for delivering medication at a precise infusion rate
• U.S. Pat. No. 4,447,224 which discloses a variable flow implantable infusion apparatus for continuous drug delivery
• U.S. Pat. No. 4,439,196 which discloses an osmotic drug delivery system having multi-chamber compartments
• U.S. Pat. No. 4,475,196 which discloses an osmotic drug delivery system.
• compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of ah sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation. General considerations in the formulation and/or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 2l st ed., Lippincott Williams & Wilkins, 2005.
• the exact amount of the vaccine required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like.
• the desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
• the desired dosage can be delivered using multiple administrations (e.g ., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
• the vaccine is administered as a single dose.
• the vaccine is administered as a single dose, followed by 1, 2, 3, 4, 5, 6 7, 8, 9, 10, or more booster doses.
• the booster dose does not comprise all of the elements of the vaccine.
• the booster dose may comprise only the modRNA-RALDH component of the vaccine.
• the booster dose may only comprise the modRNA-RALDH and the adjuvant.
• an effective amount of a vaccine for administration to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of the active ingredient per unit dosage form.
• the vaccine may be administered parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
• the vaccine may be administered orally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
• dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
• the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
• the vaccines can be administered in combination with one or more additional therapeutically active agents.
• the vaccines can be administered in combination with additional therapeutically active agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
• the therapy employed may achieve a desired effect for the same disorder (for example, a compound can be administered in combination with an anti-cancer agent, etc.), and/or it may achieve different effects (e.g ., control of adverse side-effects, e.g., emesis controlled by an anti-emetic).
• the vaccine can be administered concurrently with, prior to, or subsequent to one or more additional therapeutically active agents.
• each agent will be administered at a dose and/or on a time schedule determined for that agent.
• the additional therapeutically active agent utilized in this combination can be administered together in a single composition or administered separately in different compositions.
• the particular combination to employ in a regimen will take into account compatibility of the active ingredient with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
• additional therapeutically active agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
• Exemplary additional therapeutically active agents include, but are not limited to, cancer therapies, antibiotics, anti-viral agents, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or non-steroidal anti-inflammatory agents, antihistamine, immunosuppressant agents, anti-neoplastic agents, antigens, additional vaccines, antibodies, decongestant, sedatives, opioids, pain-relieving agents, analgesics, anti pyretics, hormones, prostaglandins, progestational agents, anti-glaucoma agents, ophthalmic agents, anti-cholinergic s, anti-depressants, anti-psychotics, hypnotics, tranquilizers, anti- convulsants/anti-epileptics (e.g ., Neurontin, Lyrica, valproates (e.g., Depacon), and other neurostabilizing agents), muscle relaxants, anti-spasmodics, muscle contractants, channel
• Therapeutically active agents include small organic molecules such as drug compounds (e.g., compounds approved by the Food and Drugs Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, minerals, and cells.
• drug compounds e.g., compounds approved by the Food and Drugs Administration as provided in the Code of Federal Regulations (CFR)
• CFR Code of Federal Regulations
• the additional therapeutic agent is a cancer therapy.
• Cancer therapies include, but are not limited to, surgery and surgical treatments, radiation therapy, and administration of additional therapeutic cancer agents (e.g., biotherapeutic and chemotherapeutic cancer agents).
• additional therapeutic cancer agents e.g., biotherapeutic and chemotherapeutic cancer agents.
• biotherapeutic cancer agents include, but are not limited to, interferons, cytokines (e.g., tumor necrosis factor, interferon a, interferon g), vaccines, hematopoietic growth factors, monoclonal serotherapy, immuno stimulants and/or
• immunodulatory agents e.g., IL-l, 2, 4, 6, or 12
• immune cell growth factors e.g., GM- CSF
• antibodies e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)).
• chemotherapeutic cancer agents include, but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g.
• anti-estrogens e.g. tamoxifen, raloxifene, and megestrol
• LHRH agonists e.g. goscrclin and leuprolide
• anti-androgens e.g. flutamide and bicalutamide
• photodynamic therapies e.g. vertoporfin (BPD-MA), phthalocyan
• cyclophosphamide ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan
• nitrosoureas e.g. carmustine (BCNU) and lomustine (CCNU)
• alkylsulphonates e.g. busulfan and treosulfan
• triazenes e.g.
• Taxoprexin polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT- 2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-l (paclitaxel bound to the erbB2-recognizing peptide EC-l), and glucose-conjugated paclitaxel, e.g., 2 -paclitaxel methyl 2- glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g.
• etoposide etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C
• anti-metabolites DHFR inhibitors (e.g. methotrexate,
• dichloromethotrexate trimetrexate, edatrexate
• IMP dehydrogenase inhibitors e.g.
• mycophenolic acid mycophenolic acid, tiazofurin, ribavirin, and EICAR
• ribonuclotide reductase inhibitors e.g. hydroxyurea and deferoxamine
• uracil analogs e.g. 5-fluorouracil (5-FU)
• floxuridine doxifluridine, ratitrexed, tegafur-uracil, capecitabine
• cytosine analogs e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine
• purine analogs e.g. mercaptopurine and
• Vitamin D3 analogs e.g. EB 1089, CB 1093, and KH 1060
• isoprenylation inhibitors e.g. lovastatin
• dopaminergic neurotoxins e.g., l-methyl-4-phenylpyridinium ion
• cell cycle inhibitors e.g. staurosporine
• actinomycin e.g., actinomycin D
• dactinomycin e.g. dactinomycin
• bleomycin e.g. bleomycin A2, bleomycin B2, peplomycin
• anthracycline e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone
• MDR inhibitors e.g. verapamil
• Ca 2+ ATPase inhibitors e.g.
• thapsigargin imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTINTM, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib
• HERCEPTIN® bevacizumab
• AVASTIN® bevacizumab
• RITUXAN® rituximab
• cetuximab ERBITUX®
• panitumumab VECTIBIX®
• ranibizumab nilotinib
• TASIGNA® sorafenib
• NEXAVAR® everolimus
• AFINITOR® alemtuzumab
• CAMPATH® gemtuzumab ozogamicin
• MYLOTARG® gemtuzumab ozogamicin
• TORISEL® temsirolimus
• ENMD-2076 PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOKTM), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g ., bortezomib (VELCADE)), mTOR inhibitors
• the additional pharmaceutical agent is an
• the immunotherapy is useful in the treatment of a cancer.
• exemplary immunotherapies include, but are not limited to, T-cell therapies, interferons, cytokines (e.g., tumor necrosis factor, interferon a, interferon g), vaccines, hematopoietic growth factors, monoclonal serotherapy, immuno stimulants and/or
• the immunotherapy is a T-cell therapy.
• the T-cell therapy is chimeric antigen receptor T cells (CAR-T).
• the immunotherapy is an antibody.
• the antibody is an anti-PD-l antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-OX40 antibody, an anti-GITR antibody, an anti-LAG-3 antibody, an anti-CD 137 antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti- CD28H antibody, an anti-CD30 antibody, an anti-CD39 antibody, an anti-CD40 antibody, an anti-CD47 antibody, an anti-CD48 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD96 antibody, an anti-CD 160 antibody, an anti-CD200 antibody, an anti-CD244 antibody, an anti-ICOS antibody, an anti-TNFRSF25 antibody, an anti-TMIGD2 antibody, an anti-DNAMl antibody, an anti-BTLA antibody, an anti-LIGHT antibody, an anti-TIGIT antibody, an anti- VISTA antibody, an anti-HVEM antibody, an anti-Siglec antibody, an anti- GAL
• (butrophylins) antibody an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-B7-H5 antibody, an anti-B7-H6 antibody, an anti-KIR antibody, an anti-LIR antibody, an anti-ILT antibody, an anti-MICA antibody, an anti-MICB antibody, an anti-NKG2D antibody, an anti- NKG2A antibody, an anti-TGFp antibody, an anti-TGFpR antibody, an anti-CXCR4 antibody, an anti-CXCLl2 antibody, an anti-CCL2 antibody, an anti-IL-lO antibody, an anti- IL-13 antibody, an anti-IL-23 antibody, an anti-phosphatidylserine antibody, an anti- neuropilin antibody, an anti-GalCer antibody, an anti-HER2 antibody, an anti-VEGFA antibody, an anti-VEGFR antibody, an anti-EGFR antibody, or an anti-Tie2 antibody.
• the antibody is pembrolizumab, nivolumab, pidilizumab, ipilimumab, tremelimumab, durvalumab, atezolizumab, avelumab, PF-06801591, utomilumab, PDR001, PBF-509, MGB453, LAG525, AMP-224, INCSHR1210, INCAGN1876, INCAGN1949, samalizumab, PF-05082566, urelumab, lirilumab, lulizumab, BMS-936559, BMS-936561, BMS-986004, BMS-986012, BMS-986016, BMS-986178, IMP321, IPH2101, IPH2201, varilumab, ulocuplumab, monalizumab, MEDI0562, MEDI0680, MEDI1873, MEDI6383,
• the vaccines or pharmaceutical compositions described herein can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, and transplantation (e.g., stem cell
• the additional therapeutically active agent is an anti inflammatory agent.
• anti-inflammatory agents include, but are not limited to, aspirin; ibuprofen; ketoprofen; naproxen; etodolac (LODINE ® ); COX-2 inhibitors such as celecoxib (CELEBREX ® ), rofecoxib (VIOXX ® ), valdecoxib (BEXTRA ® parecoxib, etoricoxib (MK663), deracoxib, 2-(4-ethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)- pyrazolo[ 1 ,5-b] pyridazine, 4-(2-oxo-3-phenyl-2,3-dihydrooxazol-4- yl)benzenesulfonamide, darbufelone, flosulide, 4-(4-cyclohexyl-2-methyl-5
• anti-inflammatory agents include naproxen, which is commercially available in the form of EC-NAPROSYN ® delayed release tablets,
• NAPROSYN ® , ANAPROX ® and ANAPROX ® DS tablets and NAPROSYN ® suspension from Roche Labs CELEBREX ® brand of celecoxib tablets, VIOXX ® brand of rofecoxib, CELESTONE ® brand of betamethasone, CUPRAMINE ® brand penicillamine capsules, DEPEN ® brand titratable penicillamine tablets, DEPO-MEDROL brand of
• methylprednisolone acetate injectable suspension methylprednisolone acetate injectable suspension, ARAVATM leflunomide tablets,
• AZULFIDIINE EN-tabs ® brand of sulfasalazine delayed release tablets, FELDENE ® brand piroxicam capsules, CATAFLAM ® diclofenac potassium tablets, VOLTAREN ® diclofenac sodium delayed release tablets, VOLTAREN ® -XR diclofenac sodium extended release tablets, or ENBREL ® etanerecept products.
• a method of treating a disease, disorder, or condition selected from the group consisting of proliferative disease (e.g., cancer, benign tumors), autoimmune disease, and infectious disease (e.g., bacterial infections, viral infections) comprising administering an effective amount of a modRNA of the present disclosure to a subject in need thereof.
• the treatment induces an antigen- specific immune response, such as a T cell and/or B cell response.
• the treatment induces an antigen-specific tolerogenic immune response; e.g., reduces a T and/or B cell response.
• the modRNA of the present disclosure is useful in the treatment of a proliferative disease.
• exemplary proliferative diseases include, but are not limited to, cancers and benign neoplasms.
• the proliferative disease is cancer.
• Exemplary cancers include, but are not limited to, acoustic neuroma,
• adenocarcinoma adrenal gland cancer
• anal cancer angiosarcoma (e.g., lymphangio sarcoma, lymphangio-endotheliosarcoma, hemangio sarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e
• lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary medias
• HL Hodgkin lymphoma
• NHL non-Hodgkin lymphoma
• NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large
• Wilms tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung),
• liver cancer e.g., hepatocellular cancer (HCC), malignant hepatoma
• lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
• LMS leiomyosarcoma
• mastocytosis e.g., systemic mastocytosis
• MDS myelodysplastic syndrome
• MMD mesothelioma
• MPD myeloproliferative disorder
• PV polycythemia Vera
• ET essential thrombocytosis
• ALM agnogenic myeloid metaplasia
• myelofibrosis MF
• chronic idiopathic myelofibrosis CML
• chronic neutrophilic leukemia CML
• hypereosinophilic syndrome HES
• neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
• NF neurofibromatosis
• neuroendocrine cancer e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget’s disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), mela
• the cancer is a mucosal cancer, such as colon cancer, head and neck squamous cell carcinoma, lung cancer, cervical cancer, and
• gastrointestinal cancer examples include, but are not limited to, esophageal cancer, stomach (gastric) cancer, and pancreatic cancer.
• the modRNA of the present disclosure is useful in the treatment of an autoimmune disease.
• autoimmune diseases include, but are not limited to, arthritis (e.g., including rheumatoid arthritis, spondyloarthopathies, gouty arthritis, degenerative joint diseases such as osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behcet’s disease, haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriatic, and juvenile arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin condition ( e.g ., psoriasis, eczema, bums, dermatitis, pruritus (itch)), enuresis
• arthritis e.g.
• eosinophilic gastrointestinal disorders e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis
• gastritis diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD)
• IBD inflammatory bowel disease
• IBS inflammatory bowel syndrome
• gastroprokinetic agent e.g., ileus, postoperative ileus and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD
• eosinophilic esophagitis eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis
• gastritis diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD)
• IBD inflammatory bowel disease
• IBS inflammatory bowel syndrome
• the modRNA of the present disclosure is useful in the treatment or prevention of an infectious disease (e.g., bacterial infection, viral infection).
• an infectious disease e.g., bacterial infection, viral infection.
• the modRNA is useful in treating or preventing a bacterial infection (e.g., Chlamydia).
• the modRNA is useful in treating or preventing a viral infection (e.g., HIV-l).
• kits provided may comprise a provided composition and a container (e.g., a vial, ampoule, bottle, syringe, and/or dispenser package, or other suitable container).
• a container e.g., a vial, ampoule, bottle, syringe, and/or dispenser package, or other suitable container.
• provided kits may optionally further include a second container comprising a suitable aqueous carrier for dilution or suspension of the provided composition for preparation of administration to a subject.
• contents of provided formulation container and solvent container combine to form at least one unit dosage form.
• a single container may comprise one or more compartments for containing a provided composition, and/or appropriate aqueous carrier for suspension or dilution.
• a single container can be appropriate for modification such that the container may receive a physical modification so as to allow combination of compartments and/or components of individual compartments.
• a foil or plastic bag may comprise two or more compartments separated by a perforated seal which can be broken so as to allow combination of contents of two individual compartments once the signal to break the seal is generated.
• a pharmaceutical pack or kit may thus comprise such multi-compartment containers including a provided composition and appropriate solvent and/or appropriate aqueous carrier for suspension.
• instructions for use are additionally provided in such kits of the invention.
• Such instructions may provide, generally, for example, instructions for dosage and administration.
• instructions may further provide additional detail relating to specialized instructions for particular containers and/or systems for administration.
• instructions may provide specialized instructions for use in conjunction and/or in combination with additional therapy.
• Flow cytometry ( Figure IB) and the geometric mean of the mean fluorescent intensity (GeoMFI; Figure 1C) indicate that the modRNA-RALDH2 transfected DCs induce upregulation of the two gut-homing receptors to the same extent as free ATRA, demonstrating that modRNA-RALDH-exposed DCs have the capacity to activate T cells and provide them with ATRA, which results in a signal to upregulate mucosal homing receptors.
• mice were orally challenged by gavage with 10 mg of Ova at day 57.
• modRNA- RALDH2 in a prime-boost vaccine protocol was sufficient to induce sustained and stable antigen-specific mucosal immunity for at least two months.
• Articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context.
• the disclosure of a group that includes“or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

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