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WO2024228150A1 - Variants de csp optimisés et méthodes associées - Google Patents

Variants de csp optimisés et méthodes associées Download PDF

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Publication number
WO2024228150A1
WO2024228150A1 PCT/IB2024/054273 IB2024054273W WO2024228150A1 WO 2024228150 A1 WO2024228150 A1 WO 2024228150A1 IB 2024054273 W IB2024054273 W IB 2024054273W WO 2024228150 A1 WO2024228150 A1 WO 2024228150A1
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WO
WIPO (PCT)
Prior art keywords
plasmodium
rna
polyribonucleotide
sequence
csp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/IB2024/054273
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English (en)
Inventor
Thomas ZIEGENHALS
Charles JENNISON
Karla-Gerlinde SCHRAUT
Anja DOKIC
Svenja GROBE
Patricia DOS SANTOS MEIRELES
Ugur Sahin
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Biontech SE
Original Assignee
Biontech SE
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Filing date
Publication date
Priority claimed from PCT/IB2023/054622 external-priority patent/WO2024228044A1/fr
Application filed by Biontech SE filed Critical Biontech SE
Priority to AU2024265158A priority Critical patent/AU2024265158A1/en
Priority to CN202480029684.XA priority patent/CN121038807A/zh
Publication of WO2024228150A1 publication Critical patent/WO2024228150A1/fr
Priority to IL324297A priority patent/IL324297A/en
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • 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

  • Malaria is a mosquito-borne infectious disease caused by protozoan parasites of the Plasmodium genus. According to the World Health Organization, an estimated 3.4 billion people in 92 countries are at risk of being infected with the Plasmodium parasite and developing disease.
  • the present disclosure provide technologies (e.g., compositions, methods, etc.) for delivery of Plasmodium antigens (also referred to herein as "malaria antigens” or “Plasmodium antigens”).
  • Plasmodium antigens also referred to herein as "malaria antigens” or “Plasmodium antigens”
  • the present disclosure provides technologies (e.g., compositions, methods, etc.) for delivery of a Plasmodium circumsporozoite protein (CSP) antigen.
  • CSP Plasmodium circumsporozoite protein
  • the present disclosure identifies a problem with certain polyribonucleotides that encode a full-length Plasmodium CSP polypeptide (including a Plasmodium CSP secretory signal and Plasmodium CSP GPI anchor), in that the polyribonucleotides are characterized by an irregular peak (e.g., having a shoulder or a double peak), for example, when analyzed by electrophoresis (e.g., capillary electrophoresis, e.g., using a fragment analyzer).
  • electrophoresis e.g., capillary electrophoresis, e.g., using a fragment analyzer.
  • the present disclosure encompasses a recognition that such an irregular peak is inconsistent with standards of good manufacturing practices. For example, in some embodiments, RNA integrity cannot be confirmed if there is an irregular peak. In some embodiments, an irregular peak may disguise RNA degradation and/or a contaminant.
  • the present disclosure provides polyribonucleotides that encode
  • the present disclosure provides the insight that the nucleotide content within a portion of a polyribonucleotide, a portion of a polyribonucleotide encoding the N-terminal region of the Plasmodium CSP polypeptide, can affect peak shape.
  • the present disclosure provides polyribonucleotides that encode a full-length Plasmodium QSP polypeptide, where nucleotide content of the portion encoding the N-terminal region of the Plasmodium CSP polypeptide is within defined ranges.
  • provided polyribonucleotides have beneficial characteristics for manufacturing.
  • the present disclosure provides polyribonucleotides that encode a full-length Plasmodium CSP polypeptide whose integrity can be confirmed using capillary electrophoresis (e.g., a fragment analyzer).
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide.
  • a full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N-terminal domain.
  • a coding sequence has an adenine content that is between 35% and 42%, and a portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • the portion of the coding sequence that encodes the PiasmodiumCsP N-terminal domain has an adenine content that is between 35% and 45%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is between 36% and 42%. In some embodiments, the coding sequence has an adenine content that is between that is between 35% and 36.5%.
  • the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%. In some embodiments, the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium QSP N-terminal domain has a guanine content that is less than 27%.
  • the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide.
  • a full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N-terminal domain.
  • a coding sequence has a uracil content that is between 14% and 20%, and a portion of the coding sequence that encodes the Plasmodium QSP N-terminal domain has a uracil content that is at least 10%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is between 12% and 25%.
  • the portion of the coding sequence that encodes the Plasmodium OS? N-terminal domain has a uracil content that is between 17% and 22%. In some embodiments, the coding sequence has a uracil content that is between that is between 15% and 16.5%.
  • the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%. In some embodiments, the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium QSP N-terminal domain has a guanine content that is less than 27%.
  • the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide.
  • a full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N-terminal domain.
  • a coding sequence has a guanine content that is between 15% and 19.5%, and a portion of the coding sequence encoding a Plasmodium CSP N-terminal domain has a guanine content that is less than 27%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a guanine content that is between 19% and 26%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a guanine content that is between 19.5% and 24.5%. In some embodiments, the coding sequence has a guanine content that is between that is between 18.5% and 19.5%.
  • the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%.
  • the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide.
  • a full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N-terminal domain.
  • a coding sequence has a cytosine content that is between 22% and 31%, and a portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is between 12% and 27% cytosine. In some embodiments, the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is between 15% and 22% cytosine. In some embodiments, the coding sequence has a cytosine content that is between that is between 28.5% and 30% cytosine.
  • the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%.
  • the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium QSP N-terminal domain has a guanine content that is less than 27%.
  • a full-length Plasmodium CSP polypeptide comprises a secretory signal, a Plasmodium CSP N-terminal domain, a Plasmodium CSP central domain, and a Plasmodium CSP C-terminal domain.
  • a full-length Plasmodium CSP polypeptide comprises a secretory signal.
  • a full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N-terminal domain.
  • a Plasmodium CSP N-terminal domain comprises an N-terminal region, an N-terminal end region, and a junction region.
  • a full-length Plasmodium CSP polypeptide comprises a Plasmodium OS? central domain.
  • a Plasmodium CSP central domain comprises a minor repeat region and a major repeat region.
  • a full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP C-terminal domain.
  • a Piasmodium CSP C-terminal domain comprises a C-terminal region and a transmembrane region.
  • a full-length Plasmodium CSP polypeptide comprises a secretory signal.
  • a secretory signal comprises or consists of a Plasmodium secretory signal, preferably a Plasmodium CSP secretory signal.
  • a full-length Plasmodium QSP polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid of SEQ ID NO: 1.
  • Plasmodium is Plasmodium falciparum, preferably Plasmodium falciparum isolate 3D7.
  • a portion of the coding sequence encoding the Plasmodium QSP N-terminal domain comprises or consists of a sequence according to any one of SEQ ID NOs: 11, 13, 15, and 17.
  • a portion of the coding sequence encoding the PiasmodiumCS? N-terminal domain comprises or consists of a sequence according to SEQ ID NO: 11.
  • a portion of the coding sequence encoding the Plasmodium CSP N-terminal domain comprises or consists of a sequence according to SEQ ID NO: 13.
  • a portion of the coding sequence encoding the PiasmodiumCS? N-terminal domain comprises or consists of a sequence according to SEQ ID NO: 15.
  • a portion of the coding sequence encoding the Plasmodium CSP N-terminal domain comprises or consists of a sequence according to SEQ ID NO: 17.
  • a coding sequence comprises or consists of a sequence according to any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, and 62. In some embodiments, a coding sequence comprises or consists of a sequence according to SEQ ID NO: 50. In some embodiments, a coding sequence comprises or consists of a sequence according to SEQ ID NO: 52. In some embodiments, a coding sequence comprises or consists of a sequence according to SEQ ID NO: 54. In some embodiments, a coding sequence comprises or consists of a sequence according to SEQ ID NO: 56. In some embodiments, a coding sequence comprises or consists of a sequence according to SEQ ID NO: 58. In some embodiments, a coding sequence comprises or consists of a sequence according to SEQ ID NO: 60. In some embodiments, a coding sequence comprises or consists of a sequence according to SEQ ID NO: 62.
  • an RNA construct comprises a polyribonucleotide provided herein.
  • an RNA construct comprises a 5' UTR.
  • a 5' UTR comprises or consists of a modified human alpha-globin 5'-UTR.
  • an RNA construct comprises a 3' UTR.
  • a 3' UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.
  • AES amino terminal enhancer of split
  • an RNA construct comprises a polyA tail sequence.
  • an RNA construct comprises in 5' to 3' order: (i) a 5' UTR that comprises or consists of a modified human alpha-globin 5'-UTR; (ii) a polyribonucleotide of any one of claims 1-15; (iii) a 3' UTR that comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA; and (iv) a polyA tail sequence.
  • AES amino terminal enhancer of split
  • an RNA construct comprises a 5' cap.
  • a composition comprises one or more polyribonucleotides as provided herein. In some embodiments, a composition comprises one or more RNA constructs as provided herein.
  • a composition comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.
  • PLX polyplexes
  • LPLX lipidated polyplexes
  • one or more polyribonucleotides are fully or partially encapsulated within lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.
  • a pharmaceutical composition comprises a composition as provided herein and at least one pharmaceutically acceptable excipient.
  • a pharmacuetical composition provided herein is for use in the treatment and/or prevention of a malaria infection that comprises administering one or more doses of the pharmaceutical composition to a subject.
  • a method comprises administering one or more doses of the pharmaceutical composition as provided herein to a subject.
  • the present disclosure provides use of a pharmaceutical composition as described herein in the treatment of a malaria infection, use of a pharmaceutical composition as described herein in the prevention of a malaria infection, and use of a pharmaceutical composition as described herein in inducing an antimalaria immune response in a subject.
  • a combination comprises a first pharmaceutical composition and a second pharmaceutical composition.
  • a first pharmaceutical composition comprises a first polyribonucleotide, where the first polyribonucleotide is a polyribonucleotide as described herein.
  • a second pharmaceutical composition comprises a second polyribonucleotide, where the second polyribonucleotide encodes a second polypeptide, and the second polypeptide comprises one or more Plasmodium T-cell antigens.
  • the present disclosure also provides methods comprising administering a combination provided herein to a subject.
  • the present disclosure provides host cells comprising polyribonucleotides described herein and host cells comprising RNA constructs described herein.
  • FIG. 1 depicts an electropherogram of an exemplary full-length CSP construct, RNA construct 23, that was in vitro translated at pH 7.0.
  • FIG. 2 depicts an electropherogram of an exemplary full-length CSP construct, RNA construct 23, that was in vitro translated at pH 8.35.
  • FIG. 3 depicts an electropherogram of an exemplary full-length CSP construct, RNA construct 23, that was in vitro translated at pH 7.0 and purified using various techniques: tangential flow filtration (TFF), magnetic bead purification, or oligo-dT affinity chromatography.
  • TMF tangential flow filtration
  • magnetic bead purification or oligo-dT affinity chromatography.
  • FIGS. 4A-4E depict electropherograms of RNA construct 23 at different time points in a forced degradation analysis: 0 minutes (FIG. 4A), 5 minutes (FIG. 4B), 10 minutes (FIG. 4C), 15 minutes (FIG. 4D), and 20 minutes (FIG. 4E).
  • FIG. 5 depicts an HPLC chromatogram of a sample of RNA construct 23. Three fractions of eluant were collected corresponding to the portions of the chromatogram labeled Fraction 1, Fraction 2 and Fraction 3.
  • FIG. 6A depicts an HPLC chromatogram of a further HPLC analysis of each of the fractions from FIG. 5.
  • FIG. 6B depicts an HPLC chromatogram of a further HPLC analysis of pooled fractions and unfractioned sample.
  • FIG. 7 depicts fragment analyzer electropherograms for each of the fractions from FIG. 5.
  • FIG. 8 depicts fragment analyzer electropherograms for samples of the pooled fractions and unfractioned sample.
  • FIG. 9 depicts fragment analyzer electropherograms of RNA construct 23 and nucleotide optimized variants 6 and 7 thereof.
  • FIG. 10 depicts fragment analyzer electropherograms of nucleotide optimized variants 8 and 9 of RNA construct 23.
  • FIGS. 11A-11B depict in vitro expression of non -formulated RNA construct 23 ("23") and different nucleotide optimized variants thereof ("V4" to "V13") in HEK293T cells.
  • FIG. 11A shows transfection rate as measured by percentage of HEK293T population that is positive for presence of expressed protein, providing a measure of efficiency of RNA delivery into the cell.
  • FIG. 11B shows total expression as measured by median fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bar, intracellular staining) and non-permeabilized cells show only surface expressed protein (grey bar, surface staining). NT refers to non-transfected.
  • FIGS. 12A-12B depict in vitro expression of RNA constructs in HEK293T cells using MessengerMax as transfection reagent.
  • FIG. 12A shows total expression as measured by median fluorescence of the total HEK293T population for both transfected and non-transfected cells.
  • FIG. 12B shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein.
  • RNA construct 23 is labeled "23"; different nucleotide optimized variants are labeled "V4" to "V14"; NT refers to non-transfected; control refers to an irrelevant RNA control.
  • FIGS. 13A-13C depict in vitro expression of RNA construct 23 (labeled "ERMA 23”) and nucleotide optimized variant 7 RNA construct (labeled "ERMA 23-7”) in HEK293T cells.
  • FIG. 13A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein.
  • FIG. 13B shows total expression as measured by median fluorescence of the total HEK293T population for both transfected and non-transfected cells.
  • FIG. 13C shows percentage of viable cells that are positive for presence of expressed protein, with non-transfected cells serving as a control.
  • FIGS. 14A-14B depict titers of antibodies elicited against /’XZSP after immunization of mice with a pharmaceutical composition comprising RNA construct 23 (labeled "ERMA 23") or variant 7 (labeled "ERMA 23-7").
  • FIG. 14A shows endpoint titers against full-length /TCSP on day 21, pre-boost.
  • FIG. 14B shows endpoint titers against full-length A’/CSP on day 35 after boost.
  • FIGS. 15A-15B depict epitope specificity of antibodies elicited upon immunization of mice with a pharmaceutical composition comprising RNA construct 23 (labeled “ERMA 23”) or variant 7 (labeled "ERMA 23-7”).
  • FIG. 15A shows a diagram depicting localization of peptides using in multiplex assay in central region of A’/CSP sequence.
  • FIG. 15B shows binding to epitopes by calculating AUC of 8-point dilution of immune serum samples (left bars (dots) are RNA construct 23 (labeled "ERMA 23”); rights bars (diagonal stripes) are variant 7 (labeled "ERMA 23- 7").
  • FIGS. 16A-16C depict pro-inflammatory response from T cells after immunization of mice with a pharmaceutical composition comprising RNA construct 23 (labeled "ERMA 23") or variant 7 (labeled "ERMA 23-7”).
  • FIG. 16A shows production of IFNy in mouse splenocytes after immunization with a pharmaceutical composition comprising RNA construct 23 (labeled "ERMA 23”) or variant 7 (labeled "ERMA 23-7”) and stimulation with A’/CSP peptides.
  • FIG. 16B shows production of IFNy in combination with IL-2 in mouse splenocytes after immunization with a pharmaceutical composition comprising RNA construct 23 (labeled "ERMA 23”) or variant 7 (labeled “ERMA 23-7”) and stimulation with A’XZSP peptides.
  • FIG. 16C shows production of IFNy in combination with IL-2 and TNFa in mouse splenocytes after immunization with a pharmaceutical composition comprising RNA construct 23 (labeled "ERMA 23”) or variant 7 (labeled "ERMA 23-7”) and stimulation with /7CSP peptides.
  • FIGS. 17A-17B depict titers of antibodies elicited against A’/CSP after immunization of mice with a pharmaceutical composition comprising variant 7 (labeled "ERMA 23-7") or vehicle.
  • FIG. 17A shows endpoint titers (reciprocal serum titer) against full-length A’/CSP pre-boost on Day 21.
  • FIG. 17B shows endpoint titers (reciprocal serum titer) against full-length /7CSP after boost on Day 35.
  • FIG. 18 depicts epitope specificity of antibodies elicited upon immunization of mice with a pharmaceutical composition comprising variant 7 (labeled "ERMA 23-7”) or vehicle.
  • FIG. 19 depicts T-cell induction following administration of a pharmaceutical composition comprising 1 pg variant 7 (labeled "ERMA 23-7") or DMSO.
  • FIGS. 20A-20B depict binding specificity of antibodies generated from mice immunized with variant 7 (labeled "ERMA 23-7") to CSP protein in Plasmodium falciparum sporozoite lysates.
  • FIG. 20A shows binding between antibodies in the sera of immunized mice and CSP protein in the sporozoite (spz) lysates represented as area under the curve (AUC) created when plotting dilution steps versus luminescence signal.
  • FIG. 20B shows binding of murine anti-CSP mAb3SP2 used as a positive control.
  • structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure.
  • the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure.
  • provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and/or as a mixture.
  • all tautomeric forms of provided compounds are within the scope of the disclosure.
  • structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure.
  • agent may refer to a physical entity.
  • an agent may be characterized by a particular feature and/or effect.
  • therapeutic agent refers to a physical entity has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.
  • amino acid refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally-occurring amino acid.
  • an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid.
  • Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide can contain a structural modification as compared with the general structure above.
  • an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure.
  • such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
  • such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
  • the term "amino acid" may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
  • Antigen refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor ⁇ e.g., when presented by an MHC molecule) or to an antibody.
  • Two events or entities are "associated" with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
  • two or more entities are physically "associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non -covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
  • Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)).
  • the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
  • administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
  • combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.
  • Comparable- refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • the term “corresponding to” refers to a relationship between two or more entities.
  • the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition).
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • a residue in an appropriate reference polymer may be identified as “corresponding to” a residue in an appropriate reference polymer.
  • residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190 th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids.
  • sequence alignment strategies including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids in accordance with the present disclosure.
  • software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, Scala
  • corresponding to may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity).
  • a gene or protein in one organism may be described as "corresponding to"a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.
  • Dosing regimen- may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • Encode- refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids.
  • a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme).
  • An RNA molecule can encode a polypeptide (e.g., by a translation process).
  • a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system.
  • a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen.
  • a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.
  • a gene product can be a transcript, e.g., a polyribonucleotide as provided herein.
  • a gene product can be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and/or (4) post -translational modification of a polypeptide or protein.
  • homolog refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be "homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and/or as having "polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution.
  • Identity refers to the overall relatedness between polynucleotide molecules ⁇ e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polynucleotide molecules ⁇ e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.
  • Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non -identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence.
  • the nucleotides at corresponding positions are then compared.
  • the percent identity between the 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, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • an assessed value achieved in a subject may be "increased" relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.).
  • comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance).
  • the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.
  • the term "increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.
  • in order refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide.
  • in order refers to the order of features moving from the N-terminal-most of the features to the C- terminal-most of the features along the polypeptide. "In order” does not mean that no additional features can be present among the listed features.
  • IVT in vitro transcription-.
  • IVT in vitro transcription
  • the transcription i.e., the generation of RNA
  • IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (e.g., T7, T3 or SP6 polymerase)).
  • Isolated means altered or removed from the natural state.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • RNA lipid nanoparticie As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticie comprises at least one cationic amino lipid.
  • an RNA lipid nanoparticie comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer- conjugated lipid (e.g., PEG-conjugated lipid).
  • RNA lipid nanopartides as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm.
  • RNA lipid nanopartides can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm.
  • an average size of lipid nanopartides is determined by measuring the average particle diameter.
  • RNA lipid nanopartides may be prepared by mixing lipids with RNA molecules described herein.
  • Neutralization refers to an event in which binding agents such as antibodies bind to a biological active site of a parasite such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.
  • nucleic acid refers to a polymer of at least 10 nucleotides or more.
  • a nucleic acid is or comprises DNA.
  • a nucleic acid is or comprises RNA.
  • a nucleic acid is or comprises peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a nucleic acid is or comprises a single stranded nucleic acid.
  • a nucleic acid is or comprises a double-stranded nucleic acid.
  • a nucleic acid comprises both single and doublestranded portions.
  • a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphoroth ioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a "peptide nucleic acid".
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues.
  • natural residues e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil.
  • a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2- thiocytidine, methylated bases, intercalated bases, and combinations thereof).
  • a nucleoside analog
  • a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2 1 -deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
  • compositions comprising: [0083] Pharmaceutically effective amount.
  • pharmaceutically effective amount or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses.
  • a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., malaria).
  • such inhibition may comprise slowing down the progress of a disease (e.g., malaria) and/or interrupting or reversing the progress of the disease (e.g., malaria).
  • a desired reaction in a treatment of a disease may be or comprise delay er prevention of the onset of a disease (e.g., malaria) or a condition (e.g., a malaria associated condition).
  • an effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on disease (e.g., malaria) or a condition (e.g., a malaria associated condition) to be treated, the severity of such a disease (e.g., malaria) or a condition (e.g., a malaria associated condition), individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
  • polypeptide- refers to a polymeric chain of amino acids.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
  • a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
  • a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids.
  • a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
  • a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
  • a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids.
  • a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • a polypeptide is a Plasmodium polypeptide construct described herein.
  • a Plasmodium polypeptide construct is a polypeptide that includes one or more Plasmodium proteins, or one or more portions thereof.
  • a Plasmodium polypeptide construct described herein includes at least one region of Plasmodium QSP or a portion thereof.
  • a Plasmodium polypeptide construct additionally includes one or more additional amino acid sequences, such as a secretory signal (e.g., a heterologous secretory signal), a transmembrane region (e.g., a heterologous transmembrane region), a helper antigen, a multimerization region, and/or a linker, as described herein.
  • a secretory signal e.g., a heterologous secretory signal
  • a transmembrane region e.g., a heterologous transmembrane region
  • helper antigen e.g., a multimerization region, and/or a linker, as described herein.
  • Prevent refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time. In some embodiments, prevention refers to reducing the risk of developing clinical malaria.
  • Reference- As used herein, the term “reference” describes a standard or control relative to which a comparison is performed.
  • an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
  • a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
  • a reference or control is a historical reference or control, optionally embodied in a tangible medium.
  • a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
  • RNA Ribonucleic acid
  • RNA Polyribonucleotide-.
  • ribonucleic acid refers to a polymer of ribonucleotides.
  • an RNA is single stranded.
  • an RNA is double stranded.
  • an RNA comprises both single and double stranded portions.
  • an RNA can comprise a backbone structure as described in the definition of "Nucleic acid I Polynucleotide” above.
  • An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA).
  • an RNA is a mRNA. In some embodiments, where an RNA is a mRNA, a RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).
  • a polyribonucleotide encodes a polypeptide, which is preferably is a Plasmodium polypeptide construct.
  • Ribonucleotide- encompasses unmodified ribonucleotides and modified ribonucleotides.
  • unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U).
  • Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2 1 position or 4 1 position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.)
  • base modifications
  • Subject refers to an organism to be administered with a composition described herein, 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, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., malaria and/or a malaria-associated condition). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., malaria and/or a malaria-associated condition).
  • a disease, disorder, or condition e.g., malaria and/or a malaria-associated condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., malaria and/or a malaria-associated condition). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., malaria and/or a malaria-associated condition). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., malaria and/or a malaria-associated condition). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., malaria and/or a malaria -associated condition). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • a disease, disorder, and/or condition e.g., malaria and/or a malaria-associated condition
  • a disease, disorder, and/or condition e.g., malaria and/or a malaria-associated condition
  • an individual who is "susceptible to" a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition (e.g., malaria and/or a malaria-associated condition) than does a member of the general public.
  • an individual who is susceptible to a disease, disorder and/or condition e.g., malaria and/or a malaria-associated condition
  • an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition (e.g., malaria and/or a malaria-associated condition).
  • an individual who is susceptible to a disease, disorder, and/or condition e.g., malaria and/or a malaria-associated condition
  • may not exhibit symptoms of the disease, disorder, and/or condition e.g., malaria and/or a malaria-associated condition.
  • an individual who is susceptible to a disease, disorder, and/or condition e.g., malaria and/or a malaria-associated condition
  • a disease, disorder, and/or condition e.g., malaria and/or a malaria-associated condition
  • an individual who is susceptible to a disease, disorder, and/or condition e.g., malaria and/or a malaria-associated condition
  • will not develop the disease, disorder, and/or condition e.g., malaria and/or a malaria- associated condition.
  • a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., malaria and/or a malaria-associated condition).
  • a therapeutic agent or therapy is a medical intervention that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • treatment refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., malaria and/or a malaria-associated condition).
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition (e.g., malaria and/or a malaria-associated condition).
  • treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition (e.g., malaria and/or a malaria-associated condition), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition (e.g., malaria and/or a malaria-associated condition).
  • variant refers to a molecule that shows significant structural (e.g., primary or secondary) identity with a reference molecule but differs structurally from the reference molecule.
  • a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties ⁇ e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid ⁇ e.g., that are attached to the polypeptide or nucleic acid backbone).
  • a variant polyribonucleotide differs in nucleotide content, but encodes the same polypeptide as the reference molecule. In some embodiments, a variant polyribonucleotide is a codon optimized variant.
  • the present disclosure provides technologies (e.g., compositions, methods, etc.) for delivery of a Plasmodium circumsporozoite protein (CSP) antigen.
  • CSP Plasmodium circumsporozoite protein
  • the present disclosure provides polyribonucleotides encoding a full-length Plasmodium CSP polypeptide, where the portion encoding the N-terminal region of the Plasmodium QSP polypeptide has a defined nucleotide content.
  • the present disclosure identifies a problem with certain polyribonucleotides that encode a full-length Plasmodium CSP polypeptide, in that they are characterized by an irregular peak (e.g., having a shoulder or a double peak), for example, when analyzed by electrophoresis (e.g., capillary electrophoresis, e.g., using a fragment analyzer).
  • an irregular peak e.g., having a shoulder or a double peak
  • electrophoresis e.g., capillary electrophoresis, e.g., using a fragment analyzer.
  • the present disclosure provides optimized polyribonucleotides that encode a full-length Plasmodium CSP polypeptide whose integrity can be confirmed using capillary electrophoresis (e.g., a fragment analyzer).
  • capillary electrophoresis e.g., a fragment analyzer.
  • the present disclosure encompasses a recognition that such polyribonucleotides and compositions including the same may be useful for treatment and/or prevention of malaria.
  • Malaria is a mosquito-borne infectious disease caused by single-celled eukaryotic Plasmodium parasites that are transmitted by the bite of Anopheles spp. mosquitoes (Phillips, M., et ai. Malaria. Nat Rev Dis Primers!, 17050, 2017, which is incorporated herein by reference in its entirety).
  • Mosquitoes that transmit malaria must have been infected through a previous blood meal taken from an infected subject (e.g., a human). When a mosquito bites an infected subject a small amount of blood is taken in containing Plasmodium parasites. The infected mosquito can then subsequently bite a non-infected subject, infecting the subject.
  • Malaria remains one of the most serious infectious diseases, causing approximately 200 million clinical cases and 500,000-600,000 deaths annually. Although significant effort has been invested in developing therapeutic treatments for malaria, many Plasmodium parasites have developed resistance to available therapeutics. According to Malaria Eradication Research Agenda Initiative, malaria eradication will only be achievable through effective vaccination. An effective malaria vaccine remains an unmet medical need of critical importance for global health.
  • CSP Circumsporozoite protein
  • Circumsporozoite protein is a multifunctional protein that is involved in Plasmodium life cycle, as it is required for the formation of sporozoites in the mosquito midgut, the release of sporozoites from the oocyst, invasion of salivary glands, attachment of sporozoites to hepatocytes in the liver, and sporozoite invasion of hepatocytes (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607, which is incorporated herein by reference in its entirety).
  • CSP CSP is present in all Plasmodium species, and although variation exists in the amino acid sequence across species, the overall domain structure of a central repeat region and nonrepeat flanking regions is well conserved (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607; Wahl et al. (2022) J. Exp. Med. 219: e20201313, each of which is incorporated herein by reference in its entirety).
  • CSP sequences are known (see, e.g., UniProt accession numbers A0A2L1CF52, AOA2L,1CF88, C6FGZ3, C6FH2,7 C6FHG7, M1V060, M1V0A3, M1V0B0, M1V0C4, M1V0E0, M1V9I4, M1VFN9, M1VKZ2, P02893, Q5EIJ9, Q5EIK2, Q5EIK8, Q5EIL3, Q5EIL5, Q5EIL8, Q5R2L2, Q7K740, Q8I9G5, Q8I9J3, Q8I9J4), and Table 1 includes exemplary sequences for CSP P. falciparum isolates from Asia, South America and Africa.
  • SEQ ID NO: 1 An exemplary wild-type CSP polypeptide amino sequence from Piasmoidum falciparum isolate 3D7 is presented in Table 2 as SEQ ID NO: 1, and includes the following: a secretory signal (amino acids 1-18); an N- terminal domain (amino acids 19-104); a junction region (amino acids 93-104), a central domain (amino acids 105- 272); and a C-terminal domain (amino acids 273-397).
  • the N-terminal domain includes an N-terminal region (amino acids 19-80); an N-terminal end region (amino acids 81-92); and a junction region (amino acids 93-104).
  • the junction region includes an R1 region (amino acids 93-97) and a junction (SEQ ID NO: 22) at positions 98-104.
  • the central domain includes a minor repeat region (amino acids 105-128) and a major repeat region (amino acids 129-272).
  • the minor repeat region includes three repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 29).
  • the major repeat region includes 35 repeats of the amino acid sequence NANP (SEQ ID NO: 33), wherein 35 repeats of the amino acid sequence NANP (SEQ ID NO: 33) are separated into two contiguous stretches, and wherein one stretch includes 17 repeats of the amino acid sequence NANP (SEQ ID NO: 33) and one includes 18 repeats of the amino acid sequence NANP (SEQ ID NO: 33) which flank an amino acid sequence of NVDP (SEQ ID NO: 34).
  • the major repeat region includes the amino acid sequences NPNANP (SEQ ID NO: 35) and NANPNA (SEQ ID NO: 36).
  • the C-terminal domain includes a C-terminal region (amino acids 273-375), a serine-valine (amino acids 376-377), and a transmembrane domain (amino acids 378-397).
  • the C-terminal region includes a Th2R region (amino acids 314-327) and a Th3R region (amino acids 352-363).
  • Exemplary CSP amino acid sequence is provided in Table 2.
  • RNA technologies as a modality to express a full-length Plasmodium CSP polypeptide.
  • a full-length Plasmodium CSP polypeptide described herein includes one or more regions or portions of CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7.
  • a full-length Plasmodium CSP polypeptide described herein includes one or more of a N-terminal region, a N-terminal end region, a junction region, a minor repeat region, a major repeat region and a C-terminal region or corresponding portions thereof of CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7.
  • a full-length Plasmodium CSP polypeptide comprises (i) a secretory signal, (ii) a Plasmodium CSP N-terminal domain, where the Plasmodium CSP N-terminal domain comprises an N-terminal region, an N-terminal end region, and a junction region, (iii) a Plasmodium CSP central domain comprising a minor repeat region and a major repeat region, and (iv) a Plasmodium CSP C-terminal domain comprising a C-terminal region and a transmembrane region.
  • a full-length PiasmodiumCSP polypeptide described herein has the structure: N-terminal region - N-terminal end region - junction region - minor repeat region - major repeat region - C-terminal region, where the regions are from CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7.
  • such a full-length Plasmodium CSP polypeptide has immediately following the C-terminal region a serine or a serine and a valine.
  • an N-terminal region comprises an amino acid sequence of positions 19 to 80 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to the amino acid sequence of positions 19 to 80 of SEQ ID NO: 1.
  • an N-terminal region comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 18.
  • an N-terminal region comprises or consists of an amino acid sequence of SEQ ID NO: 18.
  • an N-terminal end region comprises an amino acid sequence of positions 81 to 92 of SEQ ID NO: 1, or an amino acid sequence of positions 81 to 92 of SEQ ID NO:1 having 1 or 2 amino acid substitutions. In some embodiments, an N-terminal end region comprises an amino acid sequence of SEQ ID NO: 19 having 1 or 2 amino acid substitutions. In some embodiments, an N-terminal end region comprises or consists of an amino acid sequence of SEQ ID NO: 19.
  • a junction region comprises an amino acid sequence of positions 93 to 104 of SEQ ID NO:1, or an amino acid sequence of positions 93 to 104 of SEQ ID NO: 1 having 1 or 2 amino acid substitutions. In some embodiments, a junction region comprises an amino acid sequence of SEQ ID NO: 20 having 1 or 2 amino acid substitutions. In some embodiments, a junction region comprises or consists of an amino acid sequence of SEQ ID NO: 20.
  • an N-terminal domain comprises an amino acid sequence of positions 19 to 104 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to the amino acid sequence of positions 19 to 104 of SEQ ID NO: 1.
  • an N-terminal domain comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 7.
  • an N-terminal domain comprises or consists of an amino acid sequence of SEQ ID NO: 7.
  • a minor repeat region comprises an amino acid sequence of positions 105 to 128 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to the amino acid sequence of positions 105 to 128 of SEQ ID NO: 1. In some embodiments, a minor repeat region comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 26. In some embodiments, a minor repeat region comprises or consists of an amino acid sequence of SEQ ID NO: 26.
  • a major repeat region comprises an amino acid sequence of positions 129 to 272 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to the amino acid sequence of positions 129 to 272 of SEQ ID NO:1. In some embodiments, a major repeat region comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 30. In some embodiments, a major repeat region comprises or consists of an amino acid sequence of SEQ ID NO: 30.
  • a central domain comprises an amino acid sequence of positions 105 to 272 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to the amino acid sequence of positions 105 to 272 of SEQ ID NO: 1. In some embodiments, a central domain comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 23. In some embodiments, a central domain comprises or consists of an amino acid sequence of SEQ ID NO: 23.
  • a C-terminal region comprises an amino acid sequence of positions 273 to 373 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273 to 373 of SEQ ID NO: 1.
  • a C-terminal region comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 40.
  • a C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO: 40.
  • a full-length PiasmodiumCsP polypeptide construct described herein includes a transmembrane region.
  • a transmembrane region comprises or consists of a Plasmodium transmembrane region.
  • a utilized transmembrane region is one that is normally associated with CSP in nature.
  • a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region.
  • a Plasmodium QSP GPI anchor region is from Plasmodium falciparum.
  • a Plasmodium CSP GPI anchor region is from Plasmodium falciparum isolate 3D7.
  • a Plasmodium QSP GPI anchor region comprises an amino acid sequence of amino acids 374 to 397 of SEQ ID NO: 1, or an amino acid sequence of positions 374 to 397 of SEQ ID NO:1 having 1 or 2 amino acid substitutions.
  • a Plasmodium QSP GPI anchor region comprises an amino acid sequence of SEQ ID NO: 43 having 1 or 2 amino acid substitutions.
  • a Plasmodium QSP GPI anchor region comprises or consists of an amino acid sequence: FNWNSSIGLIMVLSFLFLN (SEQ ID NO: 43).
  • a C-terminal domain comprises an amino acid sequence of positions 273 to 397 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273 to 397 of SEQ ID NO: 1.
  • a C-terminal domain comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 37.
  • a C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO: 37.
  • a full-length Plasmodium QSP polypeptide construct comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to the amino acid sequence of positions 19-375 of SEQ ID NO: 1.
  • a full-length Plasmodium CSP polypeptide construct comprises or consists of an amino acid sequence of positions 19-375 of SEQ ID NO: 1.
  • a full-length PiasmodiumCsP polypeptide construct described herein includes a Plasmodium secretory signal.
  • a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal.
  • a Plasmodium CSP secretory signal is from Plasmodium falciparum.
  • a Plasmodium CSP secretory signal is from Plasmodium falciparum isolate 3D7.
  • a Plasmodium QSP secretory signal comprises amino acids 1 to 18 of SEQ ID NO: 1, or an amino acid sequence of positions 1 to 18 of SEQ ID NO:1 having 1 or 2 amino acid substitutions.
  • a Plasmodium CSP secretory signal comprises an amino acid sequence of SEQ ID NO: 4 having 1 or 2 amino acid substitutions. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence of SEQ ID NO: 4.
  • a full-length Plasmodium QSP polypeptide construct comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, or 95% identity to the amino acid sequence of positions 1-397 of SEQ ID NO: 1.
  • a full-length Plasmodium CSP polypeptide construct comprises or consists of an amino acid sequence of positions 1-397 of SEQ ID NO: 1.
  • the present disclosure provides, among other things, polynucleotides encoding a full-length Plasmodium CSP polypeptide or portion thereof.
  • the present disclosure identifies a problem with certain polyribonucleotides that encode a full-length Plasmodium QSP polypeptide, in that they are characterized by an irregular peak (e.g., having a shoulder or a double peak), for example, when analyzed by electrophoresis (e.g., capillary electrophoresis, e.g., using a fragment analyzer).
  • electrophoresis e.g., capillary electrophoresis, e.g., using a fragment analyzer.
  • the present disclosure encompasses a recognition that such an irregular peak is inconsistent with standards of good manufacturing practices. For example, in some embodiments, RNA integrity cannot be confirmed if there is an irregular peak. In some embodiments, an irregular peak may disguise RNA degradation and/or a contaminant.
  • the present disclosure provides polyribon
  • the present disclosure provides the insight that the nucleotide content of a portion of a polyribonucleotide encoding, e.g., the N-terminal domain of the Plasmodium CSP polypeptide can affect peak shape.
  • the present disclosure provides polyribonucleotides that encode a full-length Plasmodium CSP polypeptide, where nucleotide content of the portion encoding the N-terminal domain of the Plasmodium CSP polypeptide is within defined ranges.
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N-terminal domain, and where the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N- terminal domain has an adenine content that is between 35% and 45%. In some embodiments, the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is between 36% and 42%. In some embodiments, the coding sequence has an adenine content that is between that is between 35% and 36.5%.
  • the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%. In some embodiments, the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium CSP N-terminal domain has a guanine content that is less than 27%.
  • the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a Plasmodium OS? N-terminal domain, and where the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N- terminal domain has a uracil content that is between 12% and 25%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is between 17% and 22%. In some embodiments, the coding sequence has a uracil content that is between that is between 15% and 16.5%.
  • the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%. In some embodiments, the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium CSP N-terminal domain has a guanine content that is less than 27%.
  • the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a PiasmodiumCSP N-terminal domain, and where the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a PiasmodiumCSP N-terminal domain has a guanine content that is less than 27%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N- terminal domain has a guanine content that is between 19% and 26%. In some embodiments, the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a guanine content that is between 19.5% and 24.5%. In some embodiments, the coding sequence has a guanine content that is between that is between 18.5% and 19.5%.
  • the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%.
  • the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • the present disclosure provides polyribonucleotides comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N-terminal domain, and where the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the PiasmodiumCSP N-terminal domain has a cytosine content that is less than 28%.
  • the portion of the coding sequence that encodes the Plasmodium CSP N- terminal domain has a cytosine content that is between 12% and 27% cytosine. In some embodiments, the portion of the coding sequence that encodes the PiasmodiumCSP N-terminal domain has a cytosine content that is between 15% and 22% cytosine. In some embodiments, the coding sequence has a cytosine content that is between that is between 28.5% and 30% cytosine.
  • the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium OS? N-terminal domain has an adenine content that is at least 35%.
  • the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%.
  • the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium QSP N-terminal domain has a guanine content that is less than 27%.
  • a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 11. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises or consists of a sequence of SEQ ID NO: 11.
  • a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 13. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises or consists of a sequence of SEQ ID NO: 13.
  • a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 15. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises or consists of a sequence of SEQ ID NO: 15.
  • a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 17. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP N-terminal domain comprises or consists of a sequence of SEQ ID NO: 17.
  • a polyribonucleotide encoding a Plasmodium CSP secretory signal comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 6. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP secretory signal comprises or consists of a sequence according to SEQ ID NO: 6
  • a polyribonucleotide encoding a Plasmodium CSP minor repeat region comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 28. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP minor repeat region comprises or consists of a sequence according to SEQ ID NO: 28.
  • a polyribonucleotide encoding a Plasmodium CSP major repeat region comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 32. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP major repeat region comprises or consists of a sequence according to SEQ ID NO: 32. [0140] In some embodiments, a polyribonucleotide encoding a Plasmodium CSP central domain comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 25. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP central domain comprises or consists of a sequence according to SEQ ID NO: 25.
  • a polyribonucleotide encoding a PiasmodiumCS? C-terminal region comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 42.
  • a polyribonucleotide encoding a Plasmodium CSP C-terminal region comprises or consists of a sequence according to SEQ ID NO: 42.
  • a polyribonucleotide encoding a Plasmodium CSP transmembrane region comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 45. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP transmembrane region comprises or consists of a sequence according to SEQ ID NO: 45.
  • a polyribonucleotide encoding a Plasmodium CSP C-terminal domain comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 39. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP C-terminal domain comprises or consists of a sequence according to SEQ ID NO: 39.
  • a polyribonucleotide encoding a Plasmodium CSP full-length polypeptide comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, and 62.
  • a polyribonucleotide encoding a Plasmodium CSP full-length polypeptide comprises or consists of a sequence according to any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, and 62.
  • a polyribonucleotide encoding a Plasmodium CSP full-length polypeptide comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 52. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP full-length polypeptide comprises or consists of a sequence according to SEQ ID NO: 52.
  • a polyribonucleotide encoding a Plasmodium CSP full-length polypeptide comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 54. In some embodiments, a polyribonucleotide encoding a Plasmodium CSP full-length polypeptide comprises or consists of a sequence according to SEQ ID NO: 54.
  • Polyribonucleotides described herein encode a full-length CSP polypeptide construct as described herein.
  • polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5'UTR of interest and/or a 3' UTR of interest.
  • polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail.
  • polyribonucleotides described herein may comprise a 5' cap, which may be incorporated during transcription, or joined to a polyribonucleotide post-transcription.
  • a structural feature of mRNAs is cap structure at five-prime end (5').
  • Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5 ' to 5 '-triphosphate bridge resulting in capO structure (m7GpppN).
  • capO structure m7GpppN
  • further modifications can occur at the 2'-hydroxy-group (2'-OH) ⁇ e.g., the 2'-hydroxyl group may be methylated to form 2'-0-Me) of the first and subsequent nucleotides producing "capl" and "cap2" five-prime ends, respectively).
  • RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference.
  • a 5'-cap structure which may be suitable in the context of the present invention is a capO (methylation of the first nudeobase, e.g., m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA ("anti-reverse cap analogue"), modified ARCA (e.g.
  • RNA e.g., mRNA
  • 5'-cap refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')).
  • a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions ⁇ e.g., at the 7-position) on a base (guanine), and/or by methylation at one or more positions of a ribose.
  • a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'OMeG).
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine (m7G).
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3' O methylation at a ribose (m7(3'OMeG)).
  • m7(3'OMeG) a 3' O methylation at a ribose
  • providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes.
  • co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator.
  • improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide.
  • alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.
  • a utilized 5' caps is a capO, a capl, or cap2 structure. See, e.g., Fig. 1 of Ramanathan A etai., and Fig. 1 of Decroly E etai., each of which is incorporated herein by reference in its entirety. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E eta/., each of which is incorporated herein by reference in its entirety.
  • an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.
  • an RNA described herein comprises a capO structure.
  • a capO structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G).
  • such a capO structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as (m 7 )Gppp.
  • a capO structure comprises a guanosine nucleoside methylated at the 2'- position of the ribose of guanosine.
  • a capO structure comprises a guanosine nucleoside methylated at the 3'-position of the ribose of guanosine.
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((m2 7 ' 2 ''°)G).
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((m2 7 ' 3 ''°)G).
  • a capl structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m 7 )G) and optionally methylated at the 2' or 3' position pf the ribose, and a 2'0 methylated first nucleotide in an RNA ((m 2 '‘°)Ni).
  • a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3' position of the ribose, and a 2'0 methylated first nucleotide in an RNA ((m 2 '-°)Ni).
  • a capl structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g., ((m 7 )Gppp( 2 ''°)Ni) or (m2 7 ' 3 '' 0 )Gppp( 2 ''°)Ni), where Ni is as defined and described herein.
  • a capl structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g., (m 7 )Gppp( 2 ''°)NipN2 or (m2 7 ' 3 '' 0 )Gppp( 2 ''°)NipN2 , where each of Ni and N2 is as defined and described herein.
  • a cap2 structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m 7 )G) and optionally methylated at the 2' or 3' position of the ribose, and a 2'0 methylated first and second nucleotides in an RNA ((m 2 '-°)Nip(m 2 '' 0 )N2).
  • a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3' position of the ribose, and a 2'0 methylated first and second nucleotide in an RNA.
  • a cap2 structure is connected to an RNA via a 5'- to 5'- triphosphate linkage and is also referred to herein as, e.g., ((m 7 )Gppp( 2 '' 0 )Nip( 2 ''°)N2) or (m2 7 ' 3 ''°)Gppp( 2 '' 0 )Nip( 2 ''°)N2), where each of Ni and N2 is as defined and described herein.
  • the 5' cap is a dinucleotide cap structure. In some embodiments, the 5' cap is a dinucleotide cap structure comprising Ni, where Ni is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G*Ni, where Ni is as defined above and herein, and G* comprises a structure of formula (I): or a salt thereof, where each R 2 and R 3 is -OH . [0157] In some embodiments, R 2 is -OH. In some embodiments, R 2 is -OCH3. In some embodiments, R 3 is -OH. In some embodiments, R 3 is -OCH3.
  • R 2 is -OH and R 3 is -OH. In some embodiments, R 2 is -OH and R 3 is -CH 3 . In some embodiments, R 2 is -CH 3 and R 3 is -OH. In some embodiments, R 2 is -CH 3 and R 3 is -CH 3 . [0158] In some embodiments, X is O. In some embodiments, X is S.
  • the 5’ cap is a dinucleotide cap0 structure (e.g., (m7)GpppN 1 , (m 2 7,2’- O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’-O )GppSpN 1 , or (m 2 7,3’-O )GppSpN 1 ), where N 1 is as defined and described herein.
  • m7GpppN 1 , (m 2 7,2’- O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’-O )GppSpN 1 , or (m 2 7,3’-O )GppSpN 1 where N 1 is as defined and described herein.
  • the 5’ cap is a dinucleotide cap0 structure (e.g., (m 7 )GpppN1, (m2 7,2’-O )GpppN1, (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’-O )GppSpN 1 , or (m 2 7,3’-O )GppSpN 1 ), where N 1 is G.
  • a dinucleotide cap0 structure e.g., (m 7 )GpppN1, (m2 7,2’-O )GpppN1, (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’-O )GppSpN 1 , or (m 2 7,3’-O )GppSpN 1
  • N 1 is G.
  • the 5’ cap is a dinucleotide cap0 structure (e.g., (m 7 )GpppN 1 , (m 2 7,2’-O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’- O )GppSpN1, or (m2 7,3’-O )GppSpN1), where N1 is A, U, or C.
  • a dinucleotide cap0 structure e.g., (m 7 )GpppN 1 , (m 2 7,2’-O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’- O )GppSpN1, or (m2 7,3’-O )GppSpN1
  • N1 is A, U, or C.
  • the 5’ cap is a dinucleotide cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N 1 , (m 2 7,2’-O )Gppp(m 2’-O )N 1 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 , (m 7 )GppSp(m 2’-O )N 1 , (m 2 7,2’- O )GppSp(m 2’-O )N 1 , or (m 2 7,3’-O )GppSp(m 2’-O )N 1 ), where N 1 is as defined and described herein.
  • N 1 is as defined and described herein.
  • the 5’ cap is selected from the group consisting of (m 7 )GpppG (“Ecap0”), (m 7 )Gppp(m 2’-O )G (“Ecap1”), (m2 7,3’-O )GpppG (“ARCA” or “D1”), and (m 2 7,2’-O )GppSpG (“beta-S-ARCA”).
  • the 5’ cap is (m 7 )GpppG (“Ecap0”), having a structure: or a salt thereof.
  • the 5’ cap is (m 7 )Gppp(m 2’-O )G (“Ecap1”), having a structure: or a salt thereof.
  • the 5’ cap is (m2 7,3 -O )GpppG ( ARCA” or D1”), having a structure: [0162] In some embodiments, the 5’ cap is (m 2 7,2’-O )GppSpG (“beta-S-ARCA”), having a structure: or a salt thereof [0163] In some embodiments, the 5’ cap is a trinucleotide cap structure. In some embodiments, the 5’ cap is a trinucleotide cap structure comprising N1pN2, where N1 and N2 are as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*N1pN2, where N1 and N2 are as defined above and herein, and G* comprises a structure of formula (I):
  • the 5 cap is a trinucleotide cap0 structure (e.g. (m 7 )GpppN 1 pN 2 , (m 2 7,2’- O )GpppN 1 pN 2 , or (m 2 7,3’-O )GpppN 1 pN 2 ), where N 1 and N 2 are as defined and described herein).
  • a trinucleotide cap0 structure e.g. (m 7 )GpppN 1 pN 2 , (m 2 7,2’- O )GpppN 1 pN 2 , or (m 2 7,3’-O )GpppN 1 pN 2 ), where N 1 and N 2 are as defined and described herein).
  • the 5’ cap is a trinucleotide cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N1pN2, (m2 7,2’-O )Gppp(m 2’-O )N1pN2, (m2 7,3’-O )Gppp(m 2’- O )N1pN2), where N1 and N2 are as defined and described herein.
  • a trinucleotide cap1 structure e.g., (m 7 )Gppp(m 2’-O )N1pN2, (m2 7,2’-O )Gppp(m 2’-O )N1pN2, (m2 7,3’-O )Gppp(m 2’- O )N1pN2
  • N1 and N2 are as defined and described herein.
  • the 5’ cap is a trinucleotide cap2 structure (e.g., (m 7 )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 , (m 2 7,2’-O )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 p(m 2’- O )N 2 ), where N 1 and N 2 are as defined and described herein.
  • the 5’ cap is selected from the group consisting of (m2 7,3’-O )Gppp(m 2’-O )ApG (“CleanCap AG”, “CC413”), (m2 7,3’-O )Gppp(m 2’-O )GpG (“CleanCap GG”), (m 7 )Gppp(m 2’-O )ApG, (m 7 )Gppp(m 2’-O )G, (m 2 7,3’-O )Gppp(m 2 6,2’-O )ApG, and (m 7 )Gppp(m 2’-O )ApU.
  • the 5’ cap is (m 2 7,3’-O)Gppp(m2’-O)ApG (“CleanCap AG”, “CC413”), having a structure: or a salt thereo [0166] In some embodiments, the 5’ cap is (m 2 7,3’-O)Gppp(m2’-O)GpG (“CleanCap GG”), having a structure:
  • the 5 cap is (m 7 )Gppp(m 2 -O )ApG, having a structure: or a salt thereof.
  • the 5’ cap is (m 7 )Gppp(m 2’-O )GpG, having a structure: or a salt thereof.
  • the 5’ cap is (m2 7,3’-O )Gppp(m2 6,2’-O )ApG, having a structure: Page 36 of 108
  • the 5’ cap is (m 7 )Gppp(m 2’-O )ApU, having a structure: or a salt thereof.
  • the 5’ cap is a tetranucleotide cap structure.
  • the 5’ cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1, N2, and N3 are as defined and described herein.
  • the 5’ cap is a tetranucleotide cap G*N1pN2pN3, where N1, N2, and N3 are as defined above and herein, and G* comprises a structure of formula (I):
  • the 5 cap is a tetranucleotide cap0 structure (e.g. (m7)GpppN 1 pN 2 pN 3 , (m 2 7,2’- O )GpppN1pN2pN3, or (m2 7,3’-O )GpppN1N2pN3), where N1, N2, and N3 are as defined and described herein).
  • a tetranucleotide cap0 structure e.g. (m7)GpppN 1 pN 2 pN 3 , (m 2 7,2’- O )GpppN1pN2pN3, or (m2 7,3’-O )GpppN1N2pN3
  • the 5’ cap is a tetranucleotide Cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N1pN2pN3, (m2 7,2’-O )Gppp(m 2’- O )N 1 pN 2 pN 3 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 pN 2 N 3 ), where N 1 , N 2 , and N 3 are as defined and described herein.
  • tetranucleotide Cap1 structure e.g., (m 7 )Gppp(m 2’-O )N1pN2pN3, (m2 7,2’-O )Gppp(m 2’- O )N 1 pN 2 pN 3 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 pN 2 N 3 ), where N 1 , N 2 , and N 3 are
  • the 5’ cap is a tetranucleotide Cap2 structure (e.g., (m 7 )Gppp(m 2’-O )N1p(m 2’-O )N2pN3, (m2 7,2’-O )Gppp(m 2’- O )N1p(m 2’-O )N2pN3, (m2 7,3’-O )Gppp(m 2’-O )N1p(m 2’-O )N2pN3), where N1, N2, and N3 are as defined and described herein.
  • tetranucleotide Cap2 structure e.g., (m 7 )Gppp(m 2’-O )N1p(m 2’-O )N2pN3, (m2 7,2’-O )Gppp(m 2’- O )N1p(m 2’-O )N2pN3, (m2 7,3’-O )Gppp(m 2’
  • the 5’ cap is selected from the group consisting of (m 2 7,3’-O )Gppp(m 2’-O )Ap(m 2’-O )GpG, (m 2 7,3’- O )Gppp(m 2’-O )Gp(m 2’-O )GpC, (m 7 )Gppp(m 2’-O )Ap(m 2’-O )UpA, and (m 7 )Gppp(m 2’-O )Ap(m 2’-O )GpG.
  • the 5’ cap is (m2 7,3’-O )Gppp(m 2’-O )Ap(m 2’-O )GpG, having a structure: or a salt thereof.
  • the 5’ cap is (m 2 7,3’-O )Gppp(m 2’-O )Gp(m 2’-O )GpC, having a structure:
  • the 5’ cap is (m7)Gppp(m2’-O)Ap(m2’-O)UpA, having a structure: or a salt thereo [0176] In some embodiments, the 5’ cap is (m 7 )Gppp(m 2’-O )Ap(m 2’-O )GpG, having a structure: Page 39 of 108
  • a 5’ UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein.
  • a cap proximal sequence comprises a sequence adjacent to a 5’ cap.
  • a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap structure comprises one or more polynucleotides of a cap proximal sequence.
  • a cap structure comprises an m 7 Guanosine cap and nucleotide +1 (N 1 ) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotides +1 and +2 (N 1 and N 2 ) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotides +1, +2, and +3 (N1, N2, and N3) of an RNA polynucleotide.
  • one or more residues of a cap proximal sequence may be included in an RNA by virtue of having been included in a cap entity (e.g., a cap1 or cap2 structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase).
  • the 5’ cap is a dinucleotide cap structure, where the cap proximal sequence comprises N1 of the 5’ cap, where N1 is any nucleotide, e.g., A, C, G or U.
  • the 5’ cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), where the cap proximal sequence comprises N1 and N2 of the 5’ cap, where N1 and N2 are independently any nucleotide, e.g., A, C, G or U.
  • the 5’ cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), where the cap proximal sequence comprises N 1 , N 2 , and N 3 of the 5’ cap, where N 1 , N 2 , and N 3 are any nucleotide, e.g., A, C, G or U.
  • a cap proximal sequence comprises N 1 of a the 5’ cap, and N 2 , N 3 , N 4 and N 5 , where N 1 to N 5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap proximal sequence comprises N 1 and N 2 of a the 5’ cap, and N 3 , N 4 and N 5 , where N 1 to N 5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap proximal sequence comprises N1, N2, and N3 of a the 5’ cap, and N4 and N5, where N1 to N5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • N 1 is A.
  • N 1 is C.
  • N 1 is G.
  • N1 is U.
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N 2 is U.
  • N 3 is A.
  • N 3 is C. In some embodiments, N 3 is G. In some embodiments, N 3 is U. In some embodiments, N 4 is A. In some embodiments, N 4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, N5 is A. In some embodiments, N 5 is C. In some embodiments, N 5 is G. In some embodiments, N 5 is U. It will be understood that, each of the embodiments described above and herein (e.g., for N 1 through N 5 ) may be taken singly or in combination and/or may be combined with other embodiments of variables described above and herein (e.g., 5’ caps). C.
  • a nucleic acid utilized in accordance with the present disclosure comprises a 5'-UTR.
  • 5’-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element).
  • a 5’ UTR comprises multiple different sequence elements.
  • untranslated region or “UTR” is commonly used in the art to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule.
  • An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR).
  • the terms “five prime untranslated region” or “5' UTR” refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide.
  • “5' UTR” refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context.
  • a 5' UTR comprises a Kozak sequence.
  • a 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap.
  • a 5’ UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein.
  • a cap proximal sequence comprises a sequence adjacent to a 5’ cap.
  • Exemplary 5' UTRs include a human alpha globin (hAg) 5'UTR or a fragment thereof, a TEV 5' UTR or a fragment thereof, a HSP70 5' UTR or a fragment thereof, or a c-Jun 5' UTR or a fragment thereof.
  • an RNA disclosed herein comprises a hAg 5' UTR or a fragment thereof.
  • an RNA disclosed herein comprises a 5' UTR having at least 80%, at least
  • an RNA disclosed herein comprises a 5' UTR having the sequence AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAACCCGCCACC (SEQ ID NO: 63).
  • a polynucleotide ⁇ e.g., DNA, RNA) disclosed herein comprises a polyadenylate (polyA) sequence, e.g., as described herein.
  • a polyA sequence is situated downstream of a 3‘- UTR, e.g., adjacent to a 3'-UTR.
  • poly(A) sequence or "poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide.
  • Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein.
  • An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical.
  • polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence.
  • polynucleotides disclosed herein comprise interrupted Poly(A) sequence.
  • RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.
  • a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly(A) sequence (Holtkamp eta/., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).
  • a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly(A) sequence typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • consists of means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides.
  • a nucleotide or “A” refers to adenylate.
  • a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al, which is incorporated herein by reference in its entirety, may be used in accordance with the present disclosure.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. co/iand is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed.
  • the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U).
  • Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length .
  • no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'- end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.
  • the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and u p to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.
  • a poly A tail comprises a specific number of Adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200.
  • a poly A tail of a string construct may comprise 200 A residues or less.
  • a poly A tail of a string construct may comprise about 200 A residues.
  • a poly A tail of a string construct may comprise 180 A residues or less.
  • a poly A tail of a string construct may comprise about 180 A residues.
  • a poly A tail may comprise 150 residues or less.
  • RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 66), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of
  • a poly(A) tail comprises a plurality of A residues interrupted by a linker.
  • a linker comprises the nucleotide sequence GCAUAUGAC.
  • a linker comprises the nucleotide sequence GCAUAUGACU (SEQ ID NO: 68).
  • an RNA utilized in accordance with the present disclosure comprises a 3'- UTR.
  • the terms "three prime untranslated region,” “3' untranslated region,” or “3' UTR” refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence.
  • the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context.
  • the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.
  • the term "3‘- UTR” does preferably not include the poly(A) sequence.
  • the 3'-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.
  • an RNA construct comprises an F element.
  • a F element sequence is a 3'-UTR of amino-terminal enhancer of split (AES).
  • an RNA disclosed herein comprises a 3' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3' UTR with the sequence of CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCA GGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 64).
  • an RNA disclosed herein comprises a 3' UTR with the sequence of CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCA GGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 64).
  • a 3'UTR is an FI element as described in W02017/060314, which is herein incorporated by reference in its entirety.
  • the present disclosure provides RNA constructs encoding a full-length Plasmodium CSP polypeptide having beneficial characteristics, e.g., for manufacturing.
  • the present disclosure recognizes a problem with certain RNA constructs encoding a full-length Plasmodium CSP polypeptide, in that their integrity cannot be confirmed using convention methods (e.g., capillary electrophoresis, e.g., a fragment analyzer).
  • RNA construct 23 having an RNA sequence of SEQ ID NO: 69, is associated with a double peak on a fragment analyzer.
  • the present disclosure provides RNA constructs that, among other things, overcome this problem.
  • an RNA construct encoding a Plasmodium CSP full-length polypeptide comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to any one of SEQ ID NOs: 70 to 76. In some embodiments, an RNA construct encoding a Plasmodium CSP full-length polypeptide comprises or consists of a sequence according to any one of SEQ ID NOs: 70 to 76.
  • an RNA construct encoding a Plasmodium CSP full-length polypeptide comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 71. In some embodiments, an RNA construct encoding a Plasmodium CSP full-length polypeptide comprises or consists of a sequence according to SEQ ID NO: 71.
  • an RNA construct encoding a Plasmodium CSP full-length polypeptide comprises a sequence having at least 99%, 98%, 97%, 96%, or 95% identity to SEQ ID NO: 72. In some embodiments, an RNA construct encoding a Plasmodium CSP full-length polypeptide comprises or consists of a sequence according to SEQ ID NO: 72.
  • RNA compositions e.g., pharmaceutical compositions
  • uRNA non-modified uridine containing mRNA
  • modRNA nudeoside-modified mRNA
  • saRNA self-amplifying mRNA
  • RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3' end.
  • ORFs open reading frames flanked by untranslated regions
  • An ORF of an uRNA and modRNA vectors encode an antibody agent or portion thereof.
  • An saRNA has multiple ORFs.
  • the RNA described herein may have modified nucleosides.
  • the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.
  • uracil describes one of the nudeobases that can occur in the nucleic acid of RNA.
  • the structure of uracil is:
  • uridine describes one of the nucleosides that can occur in RNA.
  • the structure of uridine is:
  • UTP uridine 5'-tri phosphate
  • Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:
  • Pseudouridine is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.
  • Nl-methyl-pseudouridine (m!4J), which has the structure:
  • Nl-methyl-pseudo-UTP has the following structure:
  • m5U 5-methyl-uridine
  • one or more uridine in the RNA described herein is replaced by a modified nucleoside.
  • the modified nucleoside is a modified uridine.
  • RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U).
  • the modified nucleoside comprises pseudouridine (ip).
  • the modified nucleoside comprises Nl-methyl-pseudouridine (mlip).
  • the modified nucleoside comprises 5-methyl-uridine (m5U).
  • RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U).
  • the modified nucleosides comprise pseudouridine (ip) and Nl-methyl-pseudouridine (mlip). In some embodiments, the modified nucleosides comprise pseudouridine (ip) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise Nl-methyl-pseudouridine (mlip) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U).
  • the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-th io-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-u rid ine, 5-halo-u ridi ne (e.g., 5-iodo-u rid ine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine
  • the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine.
  • modified cytidine in the RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine.
  • the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U).
  • the RNA comprises 5-methylcytidine and Nl-methyl-pseudouridine (mlip).
  • the RNA comprises 5-methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (mlip) in place of each uridine.
  • the RNA is "replicon RNA” or simply a “replicon,” in particular "self-replicating RNA” or “self-amplifying RNA.”
  • the replicon or self-replicating RNA is derived from or comprises elements derived from a single-stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus.
  • ss single-stranded
  • Alphaviruses are typical representatives of positive-stranded RNA viruses.
  • Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp.
  • the total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail.
  • the genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome.
  • the four non-structural proteins are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome.
  • first ORF is larger than the second ORF, the ratio being roughly 2:1.
  • RNA RNA molecule that resembles eukaryotic messenger RNA
  • mRNA messenger RNA
  • the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).
  • Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms.
  • a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA.
  • a second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding a Plasmodium polypeptide construct described herein.
  • Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system).
  • Trans- replication requires the presence of both these nucleic acid molecules in a given host cell.
  • the nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.
  • non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, as well as good tolerability and safety.
  • modified uridine ⁇ e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus good tolerability and safety.
  • self-amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.
  • RNA constructs optimized for example, for improved manufacturability, encapsulation, expression level (and/or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein.
  • polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs, or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid compositions, nanoparticles ⁇ e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vectors. See, e.g., Wadhwa et al.
  • one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery ⁇ e.g., administration).
  • lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and/or engineered for systemic delivery of the RNA to target cells (e.g., liver cells). In some embodiments, such lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intravenously or intramuscularly administered to a subject.
  • lipid and "lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, mu Itilamellar/u nilamellar liposomes, or membranes in an aqueous environment.
  • Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • the hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
  • an amphiphilic compound has a polar head attached to a long hydrophobic tail.
  • the polar portion is soluble in water, while the non-polar portion is insoluble in water.
  • the polar portion may have either a formal positive charge, or a formal negative charge.
  • the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt.
  • the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.
  • a "lipid-like material” is a substance that is structurally and/or functionally related to a lipid but may not be considered a lipid in a strict sense.
  • the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, mu Itilamellar/u nilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
  • the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
  • amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits).
  • lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides.
  • Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.
  • Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain.
  • Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
  • Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides.
  • triacylglycerol is sometimes used synonymously with "triglyceride”.
  • the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids.
  • Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage.
  • Examples of glycerophospholipids usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone.
  • the major sphingoid base in mammals is commonly referred to as sphingosine.
  • Ceramides N-acyl-sphingoid bases
  • the fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups.
  • the glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
  • Sterols such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.
  • Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E.
  • Kdo2-Lipid A a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.
  • Lipids and lipid-like materials may be cationic, anionic or neutral.
  • Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.
  • suitable lipids or lipid-like materials for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid.
  • cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • Cationic lipids or lipid-like materials are characterized in that they have a net positive charge (e.g., at a relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH.
  • This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.
  • a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.
  • cationic lipids include, but are not limited to l,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N— (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3-dimethylammonium propanes; 1,2- dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N
  • DODAC di
  • Suitable cationic lipids for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • Further suitable cationic lipids for use in the present disclosure include those described in W02010/053572 (including Cl 2-200 described at paragraph [00225]) and W02012/170930, both of which are incorporated herein by reference for the purposes described herein.
  • Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1, which is incorporated herein by reference in its entirety).
  • formulations that are useful for pharmaceutical compositions can comprise at least one cationic lipid.
  • Representative cationic lipids include, but are not limited to, 1 ,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin - DAC), 1 ,2-dilinoleyoxy-3morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2- DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA
  • amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH.
  • physiological pH e.g. pH 7.4
  • second pH preferably at or above physiological pH.
  • a protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.
  • a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure.
  • formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials).
  • non-cationic lipids or lipid-like materials including non-cationically ionizable lipids or lipid-like materials.
  • anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials.
  • optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.
  • a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles.
  • such lipid or lipid-like material is a non-cationic lipid or lipid- like material.
  • a non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • An "anionic lipid” is negatively charged (e.g., at a selected pH).
  • a "neutral lipid” exists either in an uncharged or neutral zwitterionic form (e.g., at a selected pH).
  • a formulation comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • Specific exemplary phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin.
  • Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), l-oleo
  • a formulation utilized in accordance with the present disclosure includes DSPC or DSPC and cholesterol.
  • formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.
  • formulations herein include a polymer conjugated lipid such as a pegylated lipid.
  • a polymer conjugated lipid such as a pegylated lipid.
  • Pegylated lipids comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
  • the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.
  • a non-cationic lipid in particular a neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in a formulation.
  • a neutral lipid e.g., one or more phospholipids and/or cholesterol
  • RNA described herein may be present in RNA lipoplex particles.
  • RNA lipoplex particle contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.
  • RNA lipoplex particles include both a cationic lipid and an additional lipid.
  • the cationic lipid is DOTMA and the additional lipid is DOPE.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1: 1.
  • the molar ratio may be about 3:1, about 2.75:1, about 2.5: 1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.
  • RNA lipoplex particles have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm.
  • the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm.
  • the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.
  • RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration.
  • the RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase.
  • the aqueous phase has an acidic pH.
  • the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM.
  • Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA.
  • the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.
  • the at least one cationic lipid comprises l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or l,2-dioleoyl-3- trimethylammonium-propane (DOTAP).
  • DOTMA l,2-di-O-octadecenyl-3-trimethylammonium propane
  • DOTAP l,2-dioleoyl-3- trimethylammonium-propane
  • the at least one additional lipid comprises l,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Choi) and/or l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC).
  • the at least one cationic lipid comprises l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA) and the at least one additional lipid comprises l,2-di-(9Z-octadecenoyl)-sn- glycero-3-phosphoethanolamine (DOPE).
  • the liposomes and RNA lipoplex particles comprise 1,2- di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE).
  • DOTMA 1,2- di-O-octadecenyl-3-trimethylammonium propane
  • DOPE 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine
  • RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells.
  • the antigen presenting cells are dendritic cells and/or macrophages.
  • LNPs Lipid Nanoparticles
  • nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs).
  • LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.
  • an LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.
  • an LNP comprises a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid; and an RNA, encapsulated within or associated with the lipid nanopartide.
  • a neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM.
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
  • the neutral lipid is DSPC.
  • a sterol is cholesterol
  • a polymer conjugated lipid is a pegylated lipid.
  • a pegylated lipid has the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, where:
  • R 12 and R 13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
  • R 12 and R 13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • w has a mean value ranging from 40 to 55.
  • the average w is about 45.
  • R 12 and R 13 are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.
  • a pegylated lipid is DMG-PEG 2000, e.g., having the following structure:
  • G 3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R a is H or C1-C12 alkyl
  • R 1 and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl
  • R 4 is C1-C12 alkyl
  • R 5 is H or Ci-Ce alkyl; and x is 0, 1 or 2.
  • the lipid has one of the following structures
  • A is a 3 to 8-membered cycloalkyl or cycloalkylene ring
  • R 6 is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
  • the lipid has one of the following structures (IIIC) or (HID):
  • the lipid has one of the following structures (HIE) or (IIIF):
  • the lipid has one of the following structures
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is 4.
  • n is 5.
  • n is 6.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H. In other of the foregoing embodiments, R 6 is C1-C24 alkyl. In other embodiments, R 6 is OH.
  • G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C1-C24 alkylene or linear C1-C24 alkenylene.
  • R 1 or R 2 is C6-C24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: where:
  • R 7a and R 7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, where R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • At least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is Ci-Cs alkyl.
  • Ci-Cs alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the following structures:
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has one of the structures set forth in in Table 3 below.
  • a cationic lipid has one of the structures set forth in Table 4 below.
  • an LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid).
  • lipidoid ionizable lipid-like material
  • a cationic lipid has the following structure:
  • lipid nanoparticles can have an average size ⁇ e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm.
  • lipid nanoparticles in accordance with the present disclosure can have an average size ⁇ e.g., mean diameter) of about 50 nm to about 100 nm.
  • lipid nanoparticles may have an average size ⁇ e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size ⁇ e.g., mean diameter) of about 60 nm to about 120 nm.
  • lipid nanoparticles in accordance with the present disclosure can have an average size ⁇ e.g., mean diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.
  • average size ⁇ e.g., mean diameter
  • average diameter refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference).
  • average diameter “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.
  • lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less.
  • lipid nanopartides can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.
  • the "polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanopartides (e.g., ribonucleic acid nanopartides).
  • Lipid nanopartides described herein can be characterized by an "N/P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N/P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N/P) in RNA.
  • N/P ratio is the molar ratio of cationic (nitrogen) groups (the "N” in N/P) in the cationic polymer to the anionic (phosphate) groups (the "P” in N/P) in RNA.
  • N/P ratio is the molar ratio of cationic (nitrogen) groups (the "N” in N/P) in the cationic polymer to the anionic (phosphate) groups (the "P” in N/P) in RNA.
  • a lipid nanopartide described herein has an N/P ratio greater than or equal to 5. In some embodiments, a lipid nanopartide described herein has an N/P ratio that is about 5, 6, 7, 8, 9, or 10. In some embodiments, an N/P ratio for a lipid nanopartide described herein is from about 10 to about 50. In some embodiments, an N/P ratio for a lipid nanopartide described herein is from about 10 to about 70. In some embodiments, an N/P ratio for a lipid nanopartide described herein is from about 10 to about 120.
  • Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos.
  • cationic lipids, neutral lipids ⁇ e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a pre-determined molar ratio ⁇ e.g., ones described herein).
  • lipid nanoparticles are prepared at a total lipid to polyribonucleotides weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg/mL in acetate buffer.
  • a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, neutral lipids, and polymer-conjugated lipids, is injected into an aqueous solution comprising polyribonucleotides ⁇ eg., ones described herein).
  • lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution at controlled flow rates into a mixing unit, for example, using piston pumps.
  • the flow rates of a lipid solution and a RNA solution into a mixing unit are maintained at a ratio of 1:3.
  • nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous polyribonucleotides. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged RNA.
  • a solution comprising RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.
  • RNA-encapsulated lipid nanoparticles can be processed through filtration.
  • particle size and/or internal structure of lipid nanoparticles (with or without
  • RNAs may be monitored by appropriate techniques such as, e.g., small-angle X-ray scattering (SAXS) and/or transmission electron cryomicroscopy (CryoTEM).
  • SAXS small-angle X-ray scattering
  • CasoTEM transmission electron cryomicroscopy
  • compositions e.g., pharmaceutical compositions comprising one or more polyribonucleotides described herein.
  • Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, 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.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, 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.
  • Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
  • an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • USP United States Pharmacopoeia
  • EP European Pharmacopoeia
  • British Pharmacopoeia the British Pharmacopoeia
  • International Pharmacopoeia International Pharmacopoeia
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
  • compositions described herein can be administered by appropriate methods known in the art.
  • the route and/or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and/or pharmacokinetics and/or pharmacodynamics of pharmaceutical compositions described herein.
  • compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intraarticular injection and infusion.
  • pharmaceutical compositions described herein are formulated for intravenous, intramuscular, or subcutaneous administration.
  • pharmaceutical compositions described herein are formulated for intramuscular administration.
  • pharmaceutical compositions described herein are formulated for intravenous administration.
  • pharmaceutically acceptable excipients that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants.
  • prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and/or microfiltration.
  • pharmaceutical compositions can be prepared as described herein and/or methods known in the art.
  • a pharmaceutical composition includes ALC- 0315; ALC-0159; DSPC; Cholesterol; Sucrose; NaCI; KCI; Na2HPO4; KH2PO4; Water for injection.
  • normal saline is used as diluent.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to
  • Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another 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.
  • a pharmaceutical composition in accordance with the present disclosure may 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 at least one RNA product produced using a system and/or method described herein.
  • Relative amounts of polyribonucleotides encapsulated in lipid nanoparticles, a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition can vary, depending upon the subject to be treated, target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.
  • compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start doses of active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • active ingredients e.g., polyribonucleotides encapsulated in lipid nanoparticles
  • a pharmaceutical composition is formulated (e.g., but not limited to, for intravenous, intramuscular, or subcutaneous administration) to deliver a dose of about 5 mg RNA/kg.
  • a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions.
  • additives may include but are not limited to salts, buffer substances, preservatives, and carriers.
  • a pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and/or an aqueous buffered solution, which may in some embodiments include one or more salts, including, e.g., alkali metal salts or alkaline earth metal salts such as, e.g., sodium salts, potassium salts, and/or calcium salts.
  • a pharmaceutical composition provided herein is a preservative-free, sterile RNA-lipid nanoparticle dispersion in an aqueous buffer for intravenous or intramuscular administration.
  • compositions suitable for administration to humans are principally directed to pharmaceutical compositions that 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 all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • technologies of the present disclosure are used for therapeutic and/or prophylactic purposes.
  • technologies of the present disclosure are used in the treatment and/or prophylactic of an infection with a Plasmodium parasite.
  • Prophylactic purposes of the present disclosure comprise pre-exposure prophylaxis and/or post-exposure prophylaxis.
  • a Plasmodium parasite is, for example, Plasmodium falciparum, Plasmodium knowiesi, Plasmodium ovale, Plasmodium simiovale, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, and/or Plasmodium berghei.
  • technologies of the present disclosure are used in the treatment and/or prophylaxis of a disorder related to such an infection.
  • a disordered related to such an infection comprises, for example, a typical symptom and/or a complication of a malaria infection.
  • compositions may be useful to detect and/or characterize one or more features of an anti-malarial immune response ⁇ eg., by detecting binding to a provided antigen by serum from an infected subject).
  • compositions are useful to raise antibodies to one or more epitopes included therein; such antibodies may themselves be useful, for example for detection or treatment of Plasmodium parasite(s) or infection thereby.
  • the present disclosure provides use of encoding nucleic acids ⁇ e.g., DNA or RNA) to produce encoded antigens and/or use of DNA constructs to produce RNA.
  • nucleic acids e.g., DNA or RNA
  • a subject population comprises an adult population.
  • an adult population comprises subjects between the ages of about 18 years and about 60 years of age (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, or 60 years of age).
  • a subject population comprises an adult population.
  • an adult population comprises subjects between the ages of about 19 years and about 60 years of age ⁇ eg., about 20, 25, 30, 35, 40, 45, 50, 55, or 60 years of age).
  • an adult population comprises subjects between the ages of about 18 years and about 55 years of age (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, or 60 years of age).
  • a subject population comprises an elderly population.
  • an elderly population comprises subjects of about 60 years of age, about 70 years of age, or older ⁇ e.g., about 65, 70, 75, 80, 85, 90, 95, or 100 years of age).
  • a subject population comprises a pediatric population.
  • a pediatric population comprises subjects approximately 18 years old or younger.
  • a pediatric population comprises subjects between the ages of about 1 year and about 18 years ⁇ e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age).
  • a subject population comprises a newborn population.
  • a newborn population comprises subjects about 12 months or younger ⁇ e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 months or younger).
  • subject populations to be treated with technologies described herein include infants ⁇ e.g., about 12 months or younger) whose mothers did not receive such technologies described herein during pregnancy.
  • subject populations to be treated with technologies described herein may include pregnant women; in some embodiments, infants whose mothers were treated with disclosed technologies during pregnancy ⁇ e.g., who received at least one dose, or alternatively only who received both doses), are not vaccinated during the first weeks, months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more) post-birth.
  • infants whose mothers were treated with disclosed technologies during pregnancy ⁇ e.g., who received at least one dose, or alternatively only who received both doses
  • are not vaccinated during the first weeks, months, or even years e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more post-birth.
  • infants whose mothers were treated with disclosed technologies during pregnancy ⁇ e.g., who received at least one dose, or alternatively only who received both doses
  • receive reduced treated with disclosed technologies ⁇ e.g., lower doses and/or smaller numbers of administrations - e.g., boosters - and/or lower total exposure over a given period of time
  • infants whose mothers were treated with disclosed technologies during pregnancy receive reduced treated with disclosed technologies ⁇ e.g., lower doses and/or smaller numbers of administrations - e.g., boosters - and/or lower total exposure over a given period of time) after birth, for example during the first weeks, months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more) post-birth or may need reduced vaccination (e.g., lower doses and/or smaller numbers of administrations - e.g.
  • a subject population is or comprises children aged 6 weeks to up to 17 months of age.
  • a subject has a body mass index over 15 kg/m 2 and under 40 kg/m 2 . In some embodiments, a subject has a body mass index over 18.5 kg/m 2 and under 35 kg/m. In some embodiments, a subject's body mass index is determined at an initial visit with a health professional. In some embodiments, a subject's body mass index is determined when a first dose of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein is administered.
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a subject weighs at least 40 kg. In some embodiments, a subject weighs at least 45 kg. In some embodiments, a subject's weight is determined at an initial visit with a health professional. In some embodiments, a subject's weight is determined when a first dose of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein is administered.
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a subject has a body mass index over 15 kg/m 2 and under 40 kg/m 2 and weighs at least 40kg. In some embodiments, a subject has a body mass index over 18.5 kg/m 2 and under 35 kg/m 2 and weighs at least 45kg. In some embodiments, a subject's body mass index and weight are determined at an initial visit with a health professional. In some embodiments, a subject's body mass index and weight are determined when a first dose of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein is administered.
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a subject population comprises a population with a high risk of infection (e.g., malaria).
  • a population may be deemed to have a high risk of infection due to a local epidemic or a global pandemic.
  • a population may be deemed to have a high risk of infection due to a subject population's geographic area.
  • a subject population comprises subjects that have been exposed to infection (e.g., malaria).
  • a subject is malaria naive, e.g., has not experienced a prior malarial infection.
  • a subject previously has been exposed to malaria e.g., Plasmodium falciparum). Previous exposure to malaria does not encompass a prior vaccination with a malarial parasite or a component thereof.
  • a subject previously has been infected with malaria (e.g., Plasmodium falciparum).
  • a subject has not traveled to a malaria-endemic region within the 3 months, 4 months, 5 months, 6 months or 1 year prior to receiving a first dose of a polyribonucleotide, RNA construct, or composition (e.g., pharmaceutical composition) described herein.
  • a subject has traveled to a malaria-endemic region within the 3 months, 4 months, 5 months, 6 months or 1 year prior to receiving a first dose.
  • a subject intends to travel to a malaria-endemic region within 3 months, 4 months, 5 months, 6 months or 1 year after receiving a first dose.
  • a subject resides in a malaria-endemic region.
  • a malaria-endemic region is defined by the Center for Disease Control and Prevention.
  • a malaria-endemic region is defined by the Center for Disease Control and Prevention at www.cdc.gov/malaria/travelers/country_table/a.html, as of July 24, 2023.
  • a malaria-endemic region includes all or a portion of Afghanistan, Angola, Bangladesh, Bhutan, Cambodia, Botswana, Brazil, Burma, Cameroon, Cambodia, Central African Republic, Chad, Colombia, Comoros, Congo, Costa Rica, Cote d'Irium, Democratic Republic of the Congo, Djibouti, Dominican Republic, Ecuador, Equatorial Guinea, Eritrea, Eswatini, Ethopia, French Guiana, Gabon, Gambia, Ghana, Guatemala, Guinea, Guinea-Bissau, Guyana, Haiti, Honduras, India, Indonesia, Kenya, Laos, Liberia, Madagascar, Malawi, Mali, Mauritania, Mexico, Mozambique, Indonesia, Sri, Niger, Nigeria, North Korea, Pakistan, Panama, Papua New Guinea, Peru, Philippines, Cambodia, Sao Tome and Principe, Saudia Arabia, Senegal, Sierra Leone, Solomon Islands, Somalia, South Africa, South Korea, South Sudan, Sudan, Suriname, Africa,
  • a subject population is or includes pregnant women
  • provided technologies offer a particular advantage of interrupting malaria's transmission cycle, including, for example, in some embodiments, by reducing or eliminating transmission from pregnant mothers to their fetuses.
  • a subject population is or comprises immunocompromised individuals. In some embodiments, a subject population does not include immunocompromised individuals.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • another pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • therapeutic intervention e.g., to treat or prevent malaria or another disease, disorder, or condition.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a protein vaccine e.g., a DNA vaccine, an RNA vaccine, a cellular vaccine, a conjugate vaccine, etc.
  • one or more doses of a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a provided pharmaceutical composition may be administered to subjects who have been exposed, or expect they have been exposed, to malaria.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • technologies of the present disclosure may be administered to subjects according to a particular dosing regimen.
  • a dosing regimen may involve a single administration; in some embodiments, a dosing regimen may comprise one or more "booster" administrations after the initial administration.
  • initial and boost doses are the same amount; in some embodiments they differ.
  • two or more booster doses are administered.
  • a plurality of doses are administered at regular intervals. In some embodiments, periods of time between doses become longer.
  • one or more subsequent doses is administered if a particular clinical ⁇ e.g., reduction in neutralizing antibody levels) or situational ⁇ e.g., local development of a new strain) even arises or is detected.
  • administered pharmaceutical compositions comprising RNA constructs that encode Plasmodium polypeptide constructs are administered in RNA doses of from about 0.1 pg to about 300 pg, about 0.5 pg to about 200 pg, or about 1 pg to about 100 pg, such as about 1 pg, about 3 pg, about 10 pg, about 30 pg, about 50 pg, about 70 pg, or about 100 pg.
  • administered pharmaceutical compositions comprising RNA constructs that encode Plasmodium polypeptide constructs are administered in RNA doses of 10 pg or less, 30 pg or less, 50 pg or less, 70 pg or less, or 100 pg or less.
  • an saRNA construct is administered at a lower dose ⁇ e.g., 2, 4, 5, 10 fold or more lower) than a modRNA or uRNA construct.
  • a first booster dose is administered within a about six months of the initial dose, and preferably within about 5, 4, 3, 2, or 1 months.
  • a first booster dose is administered in a time period that begins about 1, 2, 3, or 4 weeks after the first dose, and ends about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks of the first dose ⁇ e.g., between about 1 and about 12 weeks after the first dose, or between about 2 or 3 weeks and about 5 and 6 weeks after the first dose, or about 3 weeks or about 4 weeks after the first dose).
  • a plurality of booster doses ⁇ e.g., 2, 3, or 4) doses are administered within 6 months of the first dose, or within 12 months of the first dose.
  • RNA dose is about 60 pg or lower, 50 pg or lower, 40 pg or lower, 30 pg or lower, 20 pg or lower, 10 pg or lower, 5 pg or lower, 2.5 pg or lower, or 1 pg or lower.
  • an RNA dose is about 0.25 pg, at least 0.5 pg, at least 1 pg, at least 2 pg, at least 3 pg, at least 4 pg, at least 5 pg, at least 10 pg, at least 20 pg, at least 30 pg, or at least 40 pg.
  • an RNA dose is about 0.25 pg to 60 pg, 0.5 pg to 55 pg, 1 pg to 50 pg, 5 pg to 40 pg, or 10 pg to 30 pg may be administered per dose.
  • an RNA dose is about 30 pg. In some embodiments, at least two such doses are administered.
  • a second dose may be administered about 21 days following administration of the first dose.
  • a first booster dose is administered about one month after an initial dose.
  • at least one further booster is administered at one-month interval(s).
  • a longer interval is introduced and no further booster is administered for at least 6, 9, 12, 18, 24, or more months.
  • a single further booster is administered after about 18 months.
  • no further booster is required unless, for example, a material change in clinical or environmental situation is observed.
  • one or more outcomes may be assessed following administration of one or more doses of a pharmaceutical composition provided herein.
  • Exemplary outcomes include, but are not limited to: local reaction at the injection site (e.g., pain, erythema/redness, indu ration/swelling, etc.), frequency of solicited local reactions at the injection site (e.g., pain, erythema/redness, induration/swelling, etc.), systemic reactions (e.g., vomiting, diarrhea, headache, fatigue, musde/joint pain, fever, etc.), frequency of solicited systemic reactions (e.g., vomiting, diarrhea, headache, fatigue, musde/joint pain, fever, etc.), adverse events, medically attended adverse events, severe adverse events, frequency of subjects with at least one adverse event, frequency of subjects with at least one medically attended adverse events, frequency of subjects with at least one severe adverse events, number of subjects protected from blood stage parasitemia, proportion of subjects protected from blood stage parasitemia, and combinations thereof
  • blood stage parasitemia can be assessed by PCR, e.g., qPCR.
  • time points can include at the time of a first dose, the time of a second dose, the time of a third dose, following a known or suspected malaria exposure, or following a known or suspected malaria infection.
  • polyribonucleotides can be produced by methods known in the art.
  • polyribonucleotides can be produced by in vitro transcription, for example, using a DNA template.
  • a plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure.
  • a DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase ⁇ e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase) with ribonucleotide triphosphates ⁇ e.g., ATP, CTP, GTP, UTP).
  • an appropriate RNA polymerase e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase
  • ribonucleotide triphosphates e.g., ATP, CTP, GTP, UTP.
  • polyribonucleotides ⁇ e.g., ones described herein
  • pseudouridine (ip), Nl-methyl-pseudouridine (mlip), or 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).
  • pseudouridine (ip) can be used to replace uridine triphosphate (UTP).
  • Nl- methyl-pseudouridine (mlip) can be used to replace uridine triphosphate (UTP).
  • 5-methyl- uridine (m5U) can be used to replace uridine triphosphate (UTP).
  • an RNA polymerase typically traverses at least a portion of a single-stranded DNA template in the 3'-> 5' direction to produce a single-stranded complementary RNA in the 5'-> 3' direction.
  • a polyribonucleotide comprises a polyA tail
  • a polyA tail may be encoded in a DNA template, e.g., by using an appropriately tailed PCR primer, or it can be added to a polyribonucleotide after in vitro transcription, e.g., by enzymatic treatment ⁇ e.g., using a poly(A) polymerase such as an E. co// Poly(A) polymerase).
  • Suitable poly(A) tails are described herein above.
  • a poly(A) tail comprises a nucleotide sequence of: AAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 66).
  • a poly(A) tail comprises a plurality of A residues interrupted by a linker.
  • a linker comprises the nucleotide sequence GCATATGAC.
  • RNA e.g., mRNA
  • a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life.
  • capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system such as, e.g., capping enzymes of vaccinia virus).
  • a cap may be introduced during in vitro transcription, along with a plurality of ribonucleotide triphosphates such that a cap is incorporated into a polyribonucleotide during transcription (also known as co-transcriptional capping).
  • a GTP fed-batch procedure with multiple additions in the course of the reaction may be used to maintain a low concentration of GTP in order to effectively cap the RNA.
  • Suitable 5' cap are described herein above.
  • a 5' cap comprises m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.
  • RNA transcription Following RNA transcription, a DNA template is digested. In some embodiments, digestion can be achieved with the use of DNase I under appropriate conditions.
  • in-vitro transcribed polyribonucleotides may be provided in a buffered solution, for example, in a buffer such as HEPES, a phosphate buffer solution, a citrate buffer solution, an acetate buffer solution; in some embodiments, such solution may be buffered to a pH within a range of, for example, about 6.5 to about 7.5; in some embodiments approximately 7.0.
  • production of polyribonucleotides may further include one or more of the following steps: purification, mixing, filtration, and/or filling.
  • polyribonucleotides can be purified (e.g., in some embodiments after in vitro transcription reaction), for example, to remove components utilized or formed in the course of the production, like, e.g., proteins, DNA fragments, and/or or nucleotides.
  • Various nucleic acid purifications that are known in the art can be used in accordance with the present disclosure.
  • Certain purification steps may be or include, for example, one or more of precipitation, column chromatography (including, e.g., but not limited to anionic, cationic, hydrophobic interaction chromatography (HIC)), solid substrate- based purification (e.g., magnetic bead-based purification).
  • polyribonucleotides may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, polyribonucleotides may be purified using hydrophobic interaction chromatography (HIC) and/or diafiltration. In some embodiments, polyribonucleotides may be purified using HIC followed by diafiltration.
  • HIC hydrophobic interaction chromatography
  • dsRNA may be obtained as side product during in vitro transcri ption .
  • a second purification step may be performed to remove dsRNA contamination.
  • cellulose materials e.g., microcrystalline cellulose
  • cellulose materials can be pretreated to inactivate potential RNase contamination, for example in some embodiments by autoclaving followed by incubation with aqueous basic solution, e.g., NaOH.
  • cellulose materials may be used to purify polyribonucleotides according to methods described in WO 2017/182524, the entire content of which is incorporated herein by reference.
  • a batch of polyribonucleotides may be further processed by one or more steps of filtration and/or concentration.
  • polyribonudeotide(s) for example, after removal of dsRNA contamination, may be further subject to diafiltration ⁇ e.g., in some embodiments by tangential flow filtration), for example, to adjust the concentration of polyribonucleotides to a desirable RNA concentration and/or to exchange buffer to a drug substance buffer.
  • polyribonucleotides may be processed through 0.2 pm filtration before they are filled into appropriate containers.
  • polyribonucleotides and compositions thereof may be manufactured in accordance with a process as described herein, or as otherwise known in the art.
  • polyribonucleotides and compositions thereof may be manufactured at a large scale.
  • a batch of polyribonucleotides can be manufactured at a scale of greater than 1 g, greater than 2 g, greater than 3 g, greater than 4 g, greater than 5 g, greater than 6 g, greater than 7 g, greater than 8 g, greater than 9 g, greater than 10 g, greater than 15 g, greater than 20 g, or higher.
  • RNA quality control may be performed and/or monitored at any time during production process of polyribonucleotides and/or compositions comprising the same.
  • RNA quality control parameters including one or more of RNA identity ⁇ e.g., sequence, length, and/or RNA natures), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after each or certain steps of a polyribonucleotide manufacturing process, e.g., after in vitro transcription, and/or each purification step.
  • the stability of polyribonucleotides ⁇ eg., produced by in vitro transcription) and/or compositions comprising polyribonucleotides can be assessed under various test storage conditions, for example, at room temperatures vs. fridge or sub-zero temperatures over a period of time ⁇ e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer).
  • polyribonucleotides ⁇ e.g., ones described herein) and/or compositions thereof may be stored stable at a fridge temperature ⁇ e.g., about 4°C to about 10°C) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer.
  • polyribonucleotides ⁇ e.g., ones described herein) and/or compositions thereof may be stored stable at a sub-zero temperature ⁇ e.g., -20°C or below) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer.
  • polyribonucleotides ⁇ e.g., ones described herein) and/or compositions thereof may be stored stable at room temperature ⁇ e.g., at about 25°C) for at least 1 month or longer.
  • one or more assessments may be utilized during manufacture, or other preparation or use of polyribonucleotides ⁇ e.g., as a release test).
  • one or more quality control parameters may be assessed to determine whether polyribonucleotides described herein meet or exceed acceptance criteria ⁇ e.g., for subsequent formulation and/or release for distribution).
  • quality control parameters may include, but are not limited to RNA integrity, RNA concentration, residual DNA template and/or residual dsRNA.
  • Certain methods for assessing RNA quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests can be used for RNA quality assessment. Examples of such certain analytical tests may include but are not limited to gel electrophoresis, UV absorption, and/or PCR assay.
  • a batch of polyribonucleotides may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of polyribonucleotides can be designated for one or more further steps of manufacturing and/or formulation and/or distribution if RNA quality assessment indicates that such a batch of polyribonucleotides meet or exceed the relevant acceptance criteria. Otherwise, an alternative action can be taken ⁇ e.g., discarding the batch) if such a batch of polyribonucleotides does not meet or exceed the acceptance criteria.
  • a batch of polyribonucleotides that satisfy assessment results can be utilized for one or more further steps of manufacturing and/or formulation and/or distribution.
  • DNA constructs for example that may encode one or more antibody agents as described herein, or components thereof.
  • DNA constructs provided by and/or utilized in accordance with the present disclosure are comprised in a vector.
  • Non-limiting examples of a vector include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC).
  • a vector is an expression vector.
  • a vector is a cloning vector.
  • a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a payload, or that imparts a particular functionality, etc.).
  • Expression vectors which may be plasmid or viral or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest.
  • a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).
  • Cloning vectors are generally used to modify, engineer, and/or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process).
  • a cloning vector may lack expression signals.
  • a vector may include replication elements such as primer binding site(s) and/or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as restriction endonuclease recognition sites and/or guide RNA binding sites, etc. [0377] In some embodiments, a vector is a viral vector ⁇ e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.
  • polynudeotide(s) of the present disclosure are included in a DNA construct ⁇ e.g., a vector) amenable to transcription and/or translation.
  • an expression vector comprises a polynucleotide that encodes proteins and/or polypeptides of the present disclosure operatively linked to a sequence or sequences that control expression ⁇ e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.).
  • a sequence or sequences that control expression are selected to achieve a desired level of expression.
  • more than one sequence that controls expression ⁇ e.g., promoters) are utilized.
  • more than one sequence that controls expression ⁇ e.g., promoters
  • a plurality of polynucleotides that encode a plurality of proteins and/or polypeptides are utilized to achieve a desired level of expression of a plurality of polynucleotides that encode a plurality of proteins and/or polypeptides.
  • a plurality of recombinant proteins and/or polypeptides are expressed from the same vector ⁇ e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic).
  • a plurality of polypeptides are expressed, each of which is expressed from a separate vector.
  • an expression vector comprising a polynucleotide of the present disclosure is used to produce a RNA and/or protein and/or polypeptide in a host cell.
  • a host cell may be in vitro ⁇ e.g., a cell line) - for example a cell or cell line ⁇ e.g., Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc.) suitable for producing polynucleotides of the present disclosure and proteins and/or polypeptides encoded by said polynucleotides.
  • HEK cells Human Embryonic Kidney
  • Chinese Hamster Ovary cells etc.
  • an expression vector is an RNA expression vector.
  • an RNA expression vector comprises a polynucleotide template used to produce a RNA in cell-free enzymatic mix.
  • an RNA expression vector comprising a polynucleotide template is enzymatically linearized prior to in vitro transcription.
  • a polynucleotide template is generated through PCR as a linear polynucleotide template.
  • a linearized polynucleotide is mixed with enzymes su itable for RNA synthesis, RNA capping and/or purification.
  • the resulting RNA is suitable for producing proteins encoded by the RNA.
  • a vector may be introduced into host cells using transfection.
  • transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection.
  • a vector may be introduced into a host cell using transduction.
  • transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides.
  • a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer.
  • Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected.
  • growth conditions e.g., temperature, carbon-dioxide levels, growth medium
  • Embodiment 1 A polyribonucleotide comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N- terminal domain, and where the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • Embodiment 2 The polyribonucleotide of embodiment 1, where the portion of the coding sequence that encodes the Plasmodium QSP N-terminal domain has an adenine content that is between 35% and 45%.
  • Embodiment 3 The polyribonucleotide of embodiment 1 or 2, where the portion of the coding sequence that encodes the PiasmodiumCS? N-terminal domain has an adenine content that is between 36% and 42%.
  • Embodiment 4 The polyribonucleotide of any one of embodiments 1-3, where the coding sequence has an adenine content that is between that is between 35% and 36.5%.
  • Embodiment 5 The polyribonucleotide of any one of embodiments 1-4, where the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the PiasmodiumCS? N-terminal domain has a uracil content that is at least 10%.
  • Embodiment 6 The polyribonucleotide of any one of embodiments 1-5, where the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium CSP N-terminal domain has a guanine content that is less than 27%.
  • Embodiment 7 The polyribonucleotide of any one of embodiments 1-6, where the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • Embodiment 8 A polyribonucleotide comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N- terminal domain, and where the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the PiasmodiumCSP N-terminal domain has a uracil content that is at least 10%.
  • Embodiment 9 The polyribonucleotide of embodiment 8, where the portion of the coding sequence that encodes the Plasmodium QSP N-terminal domain has a uracil content that is between 12% and 25%.
  • Embodiment 10 The polyribonucleotide of embodiment 8 or 9, where the portion of the coding sequence that encodes the Plasmodium QSP N-terminal domain has a uracil content that is between 17% and 22%.
  • Embodiment 11 The polyribonucleotide of any one of embodiments 8-10, where the coding sequence has a uracil content that is between that is between 15% and 16.5%.
  • Embodiment 12 The polyribonucleotide of any one of embodiments 8-11, where the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • Embodiment 13 The polyribonucleotide of any one of embodiments 8-12, where the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a PiasmodiumCS? N-terminal domain has a guanine content that is less than 27%.
  • Embodiment 14 The polyribonucleotide of any one of embodiments 8-13, where the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium QSP N-terminal domain has a cytosine content that is less than 28%.
  • Embodiment 15 A polyribonucleotide comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N- terminal domain, and where the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a Plasmodium QSP N-terminal domain has a guanine content that is less than 27%.
  • Embodiment 16 The polyribonucleotide of embodiment 15, where the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a guanine content that is between 19% and 26%.
  • Embodiment 17 The polyribonucleotide of embodiment 15 or 16, where the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a guanine content that is between 19.5% and 24.5%.
  • Embodiment 18 The polyribonucleotide of any one of embodiments 15-17, where the coding sequence has a guanine content that is between that is between 18.5% and 19.5%.
  • Embodiment 19 The polyribonucleotide of any one of embodiments 15-18, where the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • Embodiment 20 The polyribonucleotide of any one of embodiments 15-19, where the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%.
  • Embodiment 21 The polyribonucleotide of any one of embodiments 15-20, where the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • Embodiment 22 A polyribonucleotide comprising a coding sequence that encodes a full-length Plasmodium CSP polypeptide, where the full-length Plasmodium CSP polypeptide comprises a Plasmodium CSP N- terminal domain, and where the coding sequence has a cytosine content that is between 22% and 31%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is less than 28%.
  • Embodiment 23 The polyribonucleotide of embodiment 22, where the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is between 12% and 27% cytosine.
  • Embodiment 24 The polyribonucleotide of embodiment 22 or 23, where the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a cytosine content that is between 15% and 22% cytosine.
  • Embodiment 25 The polyribonucleotide of any one of embodiments 22-24, where the coding sequence has a cytosine content that is between that is between 28.5% and 30% cytosine.
  • Embodiment 26 The polyribonucleotide of any one of embodiments 22-25, where the coding sequence has an adenine content that is between 35% and 42%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has an adenine content that is at least 35%.
  • Embodiment 27 The polyribonucleotide of any one of embodiments 22-26, where the coding sequence has a uracil content that is between 14% and 20%, and the portion of the coding sequence that encodes the Plasmodium CSP N-terminal domain has a uracil content that is at least 10%.
  • Embodiment 28 The polyribonucleotide of any one of embodiments 22-27, where the coding sequence has a guanine content that is between 15% and 19.5%, and the portion of the coding sequence encoding a PiasmodiumCS? N-terminal domain has a guanine content that is less than 27%.
  • Embodiment 29 The polyribonucleotide of any one of embodiments 1-28, where the full-length Plasmodium QSP polypeptide comprises: (i) a secretory signal, (ii) the Plasmodium QSP N-terminal domain, where the Plasmodium CSP N-terminal domain comprises an N-terminal region, an N-terminal end region, and a junction region, (iii) a Plasmodium CSP central domain comprising a minor repeat region and a major repeat region, and (iv) a PiasmodiumCS? C-terminal domain comprising a C-terminal region and a transmembrane region.
  • Embodiment 30 The polyribonucleotide of embodiment 29, where the secretory signal comprises or consists of a Plasmodium secretory signal.
  • Embodiment 31 The polyribonucleotide of embodiment 29 or 30, where the Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal.
  • Embodiment 32 The polyribonucleotide of embodiment 31, where the Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 4.
  • Embodiment 33 The polyribonucleotide of any one of embodiments 29-32, where (i) the N-terminal region comprises or consists of an amino acid sequence according to SEQ ID NO: 18; (ii) the N-terminal end region comprises or consists of an amino acid sequence according to SEQ ID NO: 19, and/or (iii) the junction region comprises or consists of an amino acid sequence according to SEQ ID NO: 20.
  • Embodiment 34 The polyribonucleotide of embodiment 33, where the Plasmodium CSP N-terminal domain comprises or consists of an amino acid sequence according to SEQ ID NO: 7.
  • Embodiment 35 The polyribonucleotide of any one of embodiments 29-34, where the minor repeat region comprises exactly three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 29).
  • Embodiment 36 The polyribonucleotide of any one of embodiments 29-35, where the minor repeat region comprises or consists of an amino acid sequence according to SEQ ID NO: 26.
  • Embodiment 37 The polyribonucleotide of any one of embodiments 29-36, where the major repeat region comprises or consists of an amino acids sequence according to SEQ ID NO: 30.
  • Embodiment 38 The polyribonucleotide of any one of embodiments 29-37, where the Plasmodium CSP C-terminal domain comprises or consists of an amino acid sequence according to SEQ ID NO: 37.
  • Embodiment 39 The polyribonucleotide of any one of embodiments 1-38, where the full-length Plasmodium QSP polypeptide comprises an amino acid sequence of SEQ ID NO: 1.
  • Embodiment 40 The polyribonucleotide of any one of embodiments 1-39, where Plasmodium is Plasmodium falciparum.
  • Embodiment 41 The polyribonucleotide of embodiment 40, where Plasmodium falciparum is Plasmodium falciparum isolate 3D7.
  • Embodiment 42 The polyribonucleotide of any one of embodiments 29-41, where the portion of the coding sequence encoding the Plasmodium QSP secretory signal comprises or consists of a sequence according to SEQ ID NO: 6.
  • Embodiment 43 The polyribonucleotide of any one of embodiments 29-42, where the portion of the coding sequence encoding the Plasmodium CSP N-terminal domain comprises or consists of a sequence according to any one of SEQ ID NOs: 11, 13, 15, and 17.
  • Embodiment 44 The polyribonucleotide of any one of embodiments 29-43, where the portion of the coding sequence encoding the Plasmodium CSP N-terminal domain comprises or consists of a sequence according to SEQ ID NO: 13.
  • Embodiment 45 The polyribonucleotide of any one of embodiments 29-43, where the portion of the coding sequence encoding the Plasmodium CSP N-terminal domain comprises or consists of a sequence according to SEQ ID NO: 15.
  • Embodiment 46 The polyribonucleotide of any one of embodiments 29-45, where the portion of the coding sequence encoding the minor repeat region comprises or consists of a sequence according to SEQ ID NO: 28.
  • Embodiment 47 The polyribonucleotide of any one of embodiments 29-46, where the portion of the coding sequence encoding the major repeat region comprises or consists of a sequence according to SEQ ID NO: 32.
  • Embodiment 48 The polyribonucleotide of any one of embodiments 29-47, where the portion of the coding sequence encoding the central domain comprises or consists of a sequence according to SEQ ID NO: 25.
  • Embodiment 49 The polyribonucleotide of any one of embodiments 29-48, where the portion of the coding sequence encoding the C-terminal region comprises or consists of a sequence according to SEQ ID NO: 42.
  • Embodiment 50 The polyribonucleotide of any one of embodiments 29-49, where the portion of the coding sequence encoding the transmembrane region comprises or consists of a sequence according to SEQ ID NO: 45.
  • Embodiment 51 The polyribonucleotide of any one of embodiments 29-50, where the portion of the coding sequence encoding the Plasmodium CSP C-terminal domain comprises or consists of a sequence according to SEQ ID NO: 39.
  • Embodiment 52 The polyribonucleotide of any one of embodiments 1-51, where the coding sequence comprises or consists of a sequence according to any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, and 62.
  • Embodiment 53 The polyribonucleotide of any one of embodiments 1-52, where the coding sequence comprises or consists of a sequence according to SEQ ID NO: 52.
  • Embodiment 54 The polyribonucleotide of any one of embodiments 1-52, where the polyribonucleotide comprises or consists of a sequence according to SEQ ID NO: 54.
  • Embodiment 55 An RNA construct comprising in 5' to 3' order: (i) a 5' UTR that comprises or consists of a modified human alpha-globin 5'-UTR; (ii) a polyribonucleotide of any one of embodiments 1-54; (iii) a 3' UTR that comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA; and (iv) a polyA tail sequence.
  • AES amino terminal enhancer of split
  • Embodiment 56 The RNA construct of embodiment 55, where the 5' UTR comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 63.
  • Embodiment 57 The RNA construct of embodiment 55 or 56, where the 3' UTR comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 65.
  • Embodiment 58 The RNA construct of any one of embodiments 55-57, where the polyA tail sequence is a split polyA tail sequence.
  • Embodiment 59 The RNA construct of embodiment 58, where the split polyA tail sequence comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 66.
  • Embodiment 60 The RNA construct of any one of embodiments 55-59, further comprises a 5' cap.
  • Embodiment 61 The RNA construct of any one of embodiments 55-60, further comprises a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the polyribonucleotide.
  • Embodiment 62 The RNA construct of embodiment 60 or 61, where the 5' cap comprises or consists of m7(3'OMeG)(5')ppp(5')(2'OMeAi)pG2, where Ai is position +1 of the polyribonucleotide, and G2 is position +2 of the polyribonucleotide.
  • Embodiment 63 The RNA construct of embodiment 61 or 62, where the cap proximal sequence comprises Ai and G2 of the Capl structure, and a sequence comprising: A3A4U5 at positions +3, +4 and +5 respectively of the polyribonucleotide.
  • Embodiment 64 The RNA construct of any one of embodiments 55-63, where the RNA construct comprises a sequence of any one of SEQ ID NOs: 70 to 76.
  • Embodiment 65 The RNA construct of any one of embodiments 55-64, where the RNA construct comprises a sequence of SEQ ID NO: 71.
  • Embodiment 66 The RNA construct of any one of embodiments 55-63, where the RNA construct comprises a sequence of SEQ ID NO: 72.
  • Embodiment 67 A composition comprising one or more polyribonucleotides of any one of embodiments 1-54.
  • Embodiment 68 A composition comprising one or more RNA constructs of any one of embodiments 55-66.
  • Embodiment 69 The composition of embodiment 67 or 68, where the composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, where the one or more polyribonucleotides are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.
  • PLX polyplexes
  • LPLX lipidated polyplexes
  • liposomes where the one or more polyribonucleotides are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.
  • Embodiment 70 The composition of any one of embodiments 67-69, where the composition further comprises lipid nanoparticles, where the one or more polyribonucleotides are encapsulated within the lipid nanoparticles.
  • Embodiment 71 The composition of embodiment 69 or 70, where the lipid nanoparticles target liver cells.
  • Embodiment 72 The composition of embodiment 69 or 70, where the lipid nanoparticles target secondary lymphoid organ cells.
  • Embodiment 73 The composition of embodiment any one of embodiments 69-72, where the lipid nanoparticles are cationic lipid nanoparticles.
  • Embodiment 74 The composition of any one of embodiments 69-73, where the lipid nanopartides each comprise: (a) a polymer-conjugated lipid; (b) a cationic lipid; and (c) one or more neutral lipids.
  • Embodiment 75 The composition of embodiment 74, where the polymer-conjugated lipid comprises a PEG-conjugated lipid.
  • Embodiment 76 The composition of embodiment 74 or 75, where the polymer-conjugated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.
  • Embodiment 77 The composition of any one of embodiments 74-76, where the one or more neutral lipids comprise l,2-Distearoyl-sn-glycero-3-phosphocholine (DPSC).
  • DPSC l,2-Distearoyl-sn-glycero-3-phosphocholine
  • Embodiment 78 The composition of any one of embodiments 74-77, where the one or more neutral lipids comprise cholesterol.
  • Embodiment 79 The composition of any one of embodiments 74-78, where the cationic lipid comprises [(4-Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate).
  • Embodiment 80 The composition of any one of embodiments 74-79, where the lipid nanopartides have an average diameter of about 50-150 nm.
  • Embodiment 81 A pharmaceutical composition comprising the composition of any one of embodiments 67-80 and at least one pharmaceutically acceptable excipient.
  • Embodiment 82 The pharmaceutical composition of embodiment 81, where the pharmaceutical comprises a cryoprotectant, optionally where the cryoprotectant is sucrose.
  • Embodiment 83 The pharmaceutical composition of embodiment 81 or 82, where the pharmaceutical comprises an aqueous buffered solution, optionally where the aqueous buffered solution comprises one or more of Tris base, Tris HCI, NaCI, KCI, Na2HPO4, and KH2PO4.
  • Embodiment 84 A method comprising administering a polyribonucleotide according to any one of embodiments 1-54 to a subject.
  • Embodiment 85 A method comprising administering an RNA construct according to any one of embodiments 55-66 to a subject.
  • Embodiment 86 A method comprising administering a composition according to any one of embodiments 67-80 to a subject.
  • Embodiment 87 A method comprising administering one or more doses of the pharmaceutical composition of any one of embodiments 81-83 to a subject.
  • Embodiment 88 The pharmaceutical composition of any one of embodiments 81-83 for use in the treatment of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.
  • Embodiment 89 The pharmaceutical composition of any one of embodiments 81-83 for use in the prevention of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.
  • Embodiment 90 The method of embodiment 87 or the pharmaceutical composition for use of embodiment 88 or 89, comprising administering two or more doses of the pharmaceutical composition to a subject.
  • Embodiment 91 The method of embodiment 87 or 90, or the pharmaceutical composition for use of any one of embodiments 88-90, comprising administering three or more doses of the pharmaceutical composition to a subject.
  • Embodiment 92 The method or the pharmaceutical composition for use of embodiment 91, where the second of the three or more doses is administered to the subject at least 4 weeks after the first of the three or more doses is administered to the subject.
  • Embodiment 93 The method or the pharmaceutical composition for use of embodiment 91 or 92, where the third of the three or more doses is administered to the subject at least 4 weeks after the second of the three or more doses is administered to the subject.
  • Embodiment 94 The method of any one of embodiments 90-93, or the pharmaceutical composition for use of any one of embodiments 88-93, comprising administering a fourth dose of the pharmaceutical composition to a subject.
  • Embodiment 95 The method or the pharmaceutical composition for use of embodiment 94, where the fourth dose is administered to the subject at least one year after the third of the three or more doses is administered to the subject.
  • Embodiment 96 A combination comprising: (i) a first pharmaceutical composition comprising a first polyribonucleotide, where the first polyribonucleotide is a polyribonucleotide according to any one of embodiments 1- 54; and (ii) a second pharmaceutical composition comprising a second polyribonucleotide, where the second polyribonucleotide encodes a second polypeptide, and the second polypeptide comprises one or more Plasmodium ⁇ [- cell antigens.
  • Embodiment 97 A method comprising administering a combination of embodiment 96 to a subject.
  • Embodiment 98 The method of embodiment 97, where the first pharmaceutical composition and the second pharmaceutical composition are administered on the same day.
  • Embodiment 99 The method of embodiment 97, where the first pharmaceutical composition and the second pharmaceutical composition are administered on different days.
  • Embodiment 100 The method of any one of embodiments 96-99, where the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject at different locations on the subject's body.
  • Embodiment 101 The method of any one of embodiments 96-100, where the method is a method of treating a malaria infection.
  • Embodiment 102 The method of any one of embodiments 96-100, where the method is a method of preventing a malaria infection.
  • Embodiment 103 The method of any one of embodiments 96-102, where the subject has or is at risk of developing a malaria infection.
  • Embodiment 104 The method of any one of embodiments 96-103, where the subject is a human.
  • Embodiment 105 The method of any one of embodiments 96-104, where administration induces an anti-malaria immune response in the subject.
  • Embodiment 106 The method of embodiment 105, where the anti-malaria immune response in the subject comprises an adaptive immune response.
  • Embodiment 107 The method of embodiment 105 or 106, where the anti-malaria immune response in the subject comprises a T-cell response.
  • Embodiment 108 The method of embodiment 107, where the T-cell response is or comprises a CD4+ T cell response.
  • Embodiment 109 The method of embodiment 107 or 108, where the T-cell response is or comprises a CD8+ T cell response.
  • Embodiment 110 The method of any one of embodiments 105-109, where the anti-malaria immune system response comprises a B-cell response.
  • Embodiment 111 The method of any one of embodiments 105-110, where the anti-malaria immune system response comprises the production of antibodies directed against CSP.
  • Embodiment 112. Use of the pharmaceutical composition of any one of embodiments 81-83 in the treatment of a malaria infection.
  • Embodiment 113 Use of the pharmaceutical composition of any one of embodiments 81-83 in the prevention of a malaria infection.
  • Embodiment 114 Use of the pharmaceutical composition of any one of embodiments 81-83 in inducing an anti-malaria immune response in a subject.
  • Embodiment 115 A host cell comprising a polyribonucleotide of any one of embodiments 1-54.
  • Embodiment 116 A host cell comprising an RNA construct of any one of embodiments 55-66.
  • the disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.
  • EXEMPLIFICATION Example 1 A codon optimized polyribonucleotide encoding full-length CSP exhibits an irregular peak [0502]
  • This example describes synthesis and characterization of a polyribonucleotide encoding full-length PfCSP construct. Specifically, the present example describes in vitro transcription of a particular exemplary construct, RNA construct 23, and characterization of RNA integrity by capillary electrophoresis. The present example identifies a problem with this construct, in that it is exhibits an irregular peak when analyzed by capillary electrophoresis.
  • CleanCap m7(3'OMeG)(5')ppp(5')(2'OMeA)pG capped RNA corresponding to RNA construct 23 was produced following by in vitro transcription, under the following conditions: 3 mM CleanCap, 12.2 mM ATP/CTP, G/m1Y fed batch, 180 min, at pH 7.0 and pH 8.35.
  • RNA construct 23 has a sequence of: AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAUGCGGAAGCUGGCCAUCCUGAGCGU GUCCAGCUUCCUGUUUGUGGAAGCCCUGUUCCAAGAGUACCAGUGCUACGGCAGCAGCAGCAACACCAGAGUGCUGAACGAG CUGAACUACGACAACGCCGGCACCAACCUGUACAACGAGCUGGAAAUGAACUACUACGGCAAGCAAGAGAACUGGUACAGCCU GAAGAAGAACAGCAGAAGCCUGGGCGAGAACGACGACGGCAACAACGAGGACAACGAGAAGCUGCGGAAGCCCAAGCACAAGA AGCUGAAGCAGCCCGCCGACGGCAAUCCCGAUCCUAACGUGGACCCCAACGCUAACCCCAAUGUGGACCCA AAUGCCAAUCCAAAUGUCGAUCCAAACCCAAACGCAACCCAAACGCAACCCAAACGCUAAACCCCAACGCUAAUCCGCAACCCAAACGCAAA
  • RNA integrity was assessed by microfluidic-based electrophoresis, specifically using a Fragment Analyzer (FA; Agilent Technologies), Standard Sensitivity Kit (Agilent Technologies, article no. DNF-471-0500) and ProSize software (Agilent Technologies). Results are shown in FIG. 1 and FIG. 2.
  • RNA construct 23 was observed to have an irregular peak having a shoulder or a double peak when in vitro transcribed at pH 7 and pH 8.35, respectively.
  • In vitro transcription at pH 7.0 was performed and resulting RNA was purified using different methods: Tangential Flow Filtration (TFF), magnetic bead purification, or oligo-dT affinity chromatography.
  • RNA integrity was assessed using a Fragment Analyzer (FA; Agilent Technologies). Results are shown in FIG. 3. No significant difference in the peak shape was observed when the RNA was purified using these different methods. These results demonstrate the presence of an irregular peak and confirm that the irregular peak is independent of the means of RNA purification.
  • F Fragment Analyzer
  • the present example identifies an irregular peak issue with a particular RNA construct encoding a full-length CSP having an RNA sequence of SEQ ID NO: 48.
  • This irregular peak issue is consistent between batches and when purified by different purification methods.
  • the present disclosure encompasses a recognition that an irregular peak is inconsistent with GMP practices, which require a single peak to ensure RNA integrity.
  • This example describes a series of characterizations of the irregular peak observed with an exemplary full-length /7CSP construct, RNA construct 23. Specifically, this example describes several different analyses that were conducted to assess whether the irregular peak is the result of an impurity or degradation product.
  • a forced degradation study at 90°C was performed and degradation analyzed by a Fragment Analyzer (FA, Agilent Technologies) and PA 800 Plus (Sciex), both of which are capable of detecting degradation products. Exemplary fragment analyzer results are depicted in FIGS. 4A-4E. It was found that the shoulder of the peak degrades in a similar way as main peak, whether detected by FA (FIGS. 4A-4E) or PA 800 Plus (data not shown).
  • RNA construct 23 An exemplary full-length CSP construct, RNA construct 23, was purified by TFF or TFF + magnetic beads and protein expression after transfection into cells was analyzed by western blot. Specifically, HEK293T cells were transfected with 250 ng of RNA construct 23, using RiboJuiceTM as a transfection reagent. Eighteen hours post transfection, cells were harvested, lysed with a lysis buffer, and processed for western blot analysis in a 4-15% gel. For both methods of RNA purification, a single band was detected and this band was observed at the expected size of about 42 kDa (data not shown). This size is consistent with published data, see Lopaticki et al. (2017) Nat. Commun. 8(1):561.
  • RNA construct 23 was also analyzed by a translatability assay. RNA construct 23 was purified using magnetic beads and in vitro translated with biotinylated lysine. Biotinylated lysine is incorporated in the protein, allowing for subsequent visualization. A single translation product was detected, having the expected size (data not shown).
  • HPLC Fractionation Analysis An exemplary full-length CSP construct, RNA construct 23, was further analyzed by HPLC fractionation.
  • IP-RP ion-pair reversed phase
  • RNA construct 23 was analyzed by reverse transcription and DNA sequencing.
  • RNA construct 23 was analyzed by denaturing agarose gel electrophoresis and only a single band was visible.
  • RNA was then converted to cDNA by reverse transcriptions, which was performed at 55°C and 65°C using SuperScript IV, and the cDNA amplified by PCR using primers in the 5’-UTR and 3’-UTR, 29 cycles, followed by gel electrophoresis.
  • Sanger sequencing was performed on cDNA synthesized at 65°C.
  • Nanopore sequencing [0517] The polynucleotide identity of the irregular peak was analyzed by Nanopore. RNA construct 23 was analyzed by direct RNA sequencing. The sample was converted into a library by attaching a 3’-adapter to the RNA and sequenced with the help of a Nanopore flow cell (Oxford Nanopore Technologies Limited), followed by basecalling translating the electric signals into a nucleotide sequence. Nanopore sequencing confirmed identity and found no indication for second species. [0518] Taken together, these characterization studies all support that the irregular peak corresponds to a single species.
  • RNA construct 23 may be the result of different secondary structures of a single RNA sequence.
  • Example 3 Optimizing the Nucleotide Content of the CSP N-terminal Domain Resolves the Irregular Peak [0519]
  • the present disclosure provides the unexpected discovery that selectively optimizing the nucleotide content of the N-terminal domain region of an exemplary full-length PfCSP construct, RNA construct 23, resolves the irregular peak associated with this construct.
  • this example describes several RNA construct variants where the nucleotide content of the portion that encodes the Plasmodium CSP N-terminal domain has been optimized.
  • RNA construct 23 encodes the same polypeptide as RNA construct 23, namely, a polypeptide comprising amino acids 1-397 of SEQ ID NO:1. It was observed that those variants that specifically optimize the nucleotide content of the portion encoding the N-terminal region of CSP had an improved peak when analyzed by a Fragment Analyzer. Specifically, variants 6, 7, 8, 9, 10, 13, and 14 all showed an improved peak shape. Notably, variant 7 and variant 8 consistently exhibited single peak. See FIGS. 9 and 10 and Table 5 below.
  • this data supports that the nucleotide content of the portion of CSP encoding the N- terminal region specifically has an effect on the RNA integrity analysis. Without wishing to be bound by theory, it is suggested that optimizing the N terminal region will affect the secondary structure of the RNA construct.
  • the present Example demonstrates that exemplary optimized polyribonucleotides as described herein, exhibit in-vitro expression (e.g., intracellular, surface) in mammalian cells (HEK293T cells).
  • HEK293T cells were transfected with (i) 200 ng of RNA constructs or (ii) 50 ng of RNA constructs with MessengerMax as transfection reagent (similar to LNPs as it forms particles).
  • HEK293T cells transfected with 12.5 ng (see FIG. 13), 100 ng (see FIG. 12), and 200 ng (see FIG. 11) RNA constructs and assessed for protein expression by antibody staining and flow cytometry. Transfection rate was determined by measuring percentage of positive cells, and total expression was determined by measuring median fluorescence of the total HEK population.
  • RNA constructs had an overall high transfection rate. Variants 6 to 9 all show similar or higher expression than RNA Construct 23, particularly when looking at total expression, which measures expression in the overall live HEK population.
  • HEK293T cells were transfected with 12.5 ng of either RNA formulated with the transfection reagent MessengerMax. 18 hours (h) later, they were harvested and stained with a viability dye and, after permeabilization, with an anti-PfCSP monoclonal antibody. Flow cytometry was used to quantify the percentage of transfected cells (designated transfection rate), total protein expression and cell viability. Results are shown in FIG. 13.
  • RNA Construct 23 and variant 7 were evaluated in vivo in immunogenicity studies in mice.
  • splenocytes were harvested and analyzed by fluorospot.
  • Serum samples obtained from each group of immunized animals were analyzed by one or more of the following method(s): (1) Enzyme-linked Immunosorbent Assay (ELISA), (2) multiplex assay, (3) sporozoite ELISA, (4) traversal Assay, (5) inhibition of Liver Stage Development Assay (ILSDA), (6) Surface Plasmon Resonance (SPR), and/or (7) fluorospot assay.
  • ELISA Enzyme-linked Immunosorbent Assay
  • ILSDA inhibition of Liver Stage Development Assay
  • SPR Surface Plasmon Resonance
  • Enzyme-linked Immunosorbent Assay [0530] Endpoint antibodies titers against full-length PfCSP (PfCSP-FL) were assessed by ELISA for serum samples collected on Day 21 (pre-boost) and Day 35 (post-boost). Briefly, the RNA constructs were assessed for their ability to induce production of antibodies that may bind to Plasmodium falciparum (Pf) CSP full-length protein (“PfCSP- FL”), using an ELISA assay (see Table 7).
  • Pf Plasmodium falciparum
  • Table 7 List of PfCSP recombinant proteins and peptides assessed by ELISA analyses in the present
  • HRP horseradish peroxidase
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • immunization with both RNA Construct 23 and variant 7 elicited similar antibody responses against PfCSP on both days analyzed.
  • the second immunization increased the antibody titers against PfCSP by more than 10-fold for both variants, when compared to the titer before the boost (at Day 21). No titers have been detected for control mice (injected with the vehicle).
  • PfCSP peptides used in the multiplex analysis SEQ ID Designation AS sequence Sequence NO : [0535]
  • a Construct 23 or with variant 7 were able to bind the central region of the PfCSP protein, including three well-described neutralizing epitopes: 21C that corresponds to the CIS43-binding epitope, 22C that is the binding sequence of L9, and 29C, which is the main epitope for Ab317 (Jel ⁇ nková et al. 2022; Langowski et al. 2022; Francica et al. 2021).
  • RNA constructs were assessed for their ability to induce production of antibodies that bind to native CSP antigen from Plasmodium falciparum (Pf) sporozoite lysates, using a sporozoite ELISA assay. Specifically, 384-well plates were coated with non-denatured total protein lysate from P.
  • RNA construct 23-7 effectively induced the production of antibodies that bind native PfCSP above the background level, obtained with the serum from vehicle-injected mice.
  • FIG. 20B shows the binding of murine anti-CSP mAb3SP2, used as positive control in the assay.
  • RNA constructs are assessed for their ability to induce production of antibodies that have an inhibitory effect on Plasmodium falciparum (Pf) sporozoites traversal. Briefly, HC-04 cells, a human hepatocyte cell line, are seeded into the plates and incubated for 24 h at 5% CO2 and 37°C. Freshly isolated P. falciparum salivary gland sporozoites are pre-incubated with serially diluted (1:20, 1:80 and 1:320) serum samples from mice immunized with the formulated RNAs encoding different CSP-based constructs.
  • Pf Plasmodium falciparum
  • sporozoites are added to the HC-04 cells in a multiplicity of infection (MOI) of 1:1, in the presence of the impermeable dye dextran-rhodamine.
  • MOI multiplicity of infection
  • sporozoites are pre-treated with mAb317, an antibody that targets the NANP repeats and has a well- established impact on sporozoite traversal and infectivity.
  • Non-treated sporozoites are used as a negative control for inhibition.
  • the ability of P. falciparum sporozoites to traverse cells is quantified by determining the % of cells that incorporate dextran-rhodamine, by fluorescence microscopy.
  • parasite cytoplasm is stained with anti- PfHsp70 and DNA (from hepatocytes and from parasites) is stained with DAPI.
  • Sporozoites incubated with serum from mice injected with vehicle are used as a negative control for inhibition.
  • the ability of P. falciparum sporozoites to infect hepatocytes is quantified by determining the % of cells with parasites inside, by fluorescence microscopy. Parasite development is also assessed by measuring the area of the parasites (stained with anti-PfHsp70).
  • Serum samples are diluted in analysis buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, 0.22 ⁇ m filtered) and run using a flow rate of 10 ⁇ L/min for interaction analysis of the analyte (association and dissociation). Association of the antibodies in the serum is measured for 5 min, while dissociation is measured for 15 min. Analyzes are performed in triplicates with individually prepared dilutions. Buffer blanks are implemented regularly and used for referencing.
  • Evaluation of serum sample binding data is performed with regard to two parameters: a) height of binding signals, as relative comparison of the titer, and b) assessment of antibody:antigen complex stability based on dissociation, by calculating the binding signal after 5 min and 15 min, which is determined by calculating the dissociation relative to the maximal signal (% residual response). The higher the % residual response value the higher the complex stability.
  • Table 9 Polypeptides for SPR measurements Designa As SEQ ID tion seque Tag Sequence NO.: (7) FluoroSpot Assay [0541] Provided polyribonucleotides can be assessed for their ability to induce T-cell responses upon stimulation with peptides from Pf antigens.
  • a polyribonucleotide is determined to induce a useful immune response if splenocytes from a subject (e.g., a mouse) immunized with such construct, following incubation with peptide(s) as described herein, exhibit T-cell secretion of one or more pro-inflammatory cytokines (e.g., IFN- ⁇ , TNF- ⁇ , or IL-2) in a FluoroSpot Assay, using the IFN- ⁇ /IL-2/TNF- ⁇ FluoroSpotPLUS kit according to the manufacturer’s instructions (Mabtech).
  • pro-inflammatory cytokines e.g., IFN- ⁇ , TNF- ⁇ , or IL-2
  • Splenocytes were freshly isolated from the spleens of mice immunized with RNA constructs and resuspended in culture medium (RPMI1640 + 10% heat-inactivated fetal calf serum (FCS) + 1% non-essential amino acids (NEAA) + 1% Sodium Pyruvate + 1% HEPES + 0.5% Penicillin/Streptomycin, + 0.1% ⁇ -Mercaptoethanol).
  • a total of 5 ⁇ 10 5 splenocytes were added to each well and restimulated ex vivo overnight at 37°C with the appropriate target peptide pool, e.g., as indicated in Table 10 below or with controls (negative control: Trp1, 2 ⁇ g/mL; positive control: concanavalin A, 2 ⁇ g/mL).
  • controls negative control: Trp1, 2 ⁇ g/mL; positive control: concanavalin A, 2 ⁇ g/mL.
  • anti-IFN- ⁇ , anti-IL-2 and anti-TNF- ⁇ antibodies were added to the wells to detect production of these cytokines and then secondary antibodies conjugated with different fluorophores were added. Fluorescent spots were counted using a Mabtech IRIS FluoroSpot plate reader.
  • CD4 and CD8 isolation kits (Mylteni) were employed to specifically isolate these cells from all the cells isolated from the spleens, according to the manufacturer’s instructions. Shortly, splenocytes were resuspended in MACS buffer, the biotin-labelled antibody cocktail was added to the cells, mixed and incubated for 5 minutes at 2-8°C. Then, MACS buffer was added again, followed by anti-biotin microbeads and an incubation for 10 minutes at 2-8°C. Columns were conditioned by adding MACS buffer to them and allowing the MACS buffer to go through the columns completely.
  • anti-IFN- ⁇ , anti-IL-2 and anti-TNF- ⁇ antibodies were added to the wells to detect production of these cytokines and then secondary antibodies conjugated with different fluorophores were added. Fluorescent spots were counted using a Mabtech IRIS FluoroSpot plate reader.
  • Example 6 In vivo Immunogenicity of an Exemplary Polyribonucleotide Encoding Full-length Plasmodium CSP Polypeptide Construct Variant 7
  • Endpoint antibodies titers against PfCSP-FL were assessed by ELISA as described above for serum samples collected on Day 21 (pre-boost) and Day 35 (post-boost). As shown in FIG.17A and FIG.17B, immunization with variant 7 elicited an antibody response against PfCSP that was detectable on both days analyzed, with the titers at Day 35 being >10x higher than those at Day 21. [0547] The multiplex assay as described above was employed to quantify the binding of the antibodies present in the serum samples of the immunized HLA-A02 mice from Day 35. The results are shown in FIG. 18.
  • Example 7 In vivo Immunogenicity in Humans following Administration of an Exemplary Polyribonucleotides Encoding Plasmodium CSP Polypeptide Construct
  • the present Example demonstrates that exemplary polyribonucleotides encoding different malarial polypeptide, as described herein, can be immunogenic in vivo in humans.
  • the present Example provides a randomized, dose-escalation trial for the evaluation of safety, tolerability, and immunogenicity of a formulated RNA construct as provided herein.
  • the exemplary formulated RNA constructs to be assessed in this Example are RNA Construct 23 variant 7 (referred to as “variant 7”).
  • Trial Inclusion Criteria Subjects to be included in a trial as described in this Example: i.
  • ii. Have a body mass index over 18.5 kg/m 2 and under 35 kg/m 2 and weigh at least 45 kg at an initial visit (“Visit 0”).
  • iii. Are healthy, in the clinical judgment of a health practitioner (e.g., a trial investigator) based on reported medical history data, physical examination, 12-lead electrocardiogram (ECG), vital signs, and clinical laboratory test outcomes. Healthy subjects with pre-existing stable disease (e.g., obesity, hypertension, etc.), defined as disease but not requiring significant change in therapy or hospitalization for worsening disease during the three months (e.g., 90 days) before Visit 0, can be included.
  • HBV Hepatitis B surface antigen
  • anti-HCV Hepatitis C virus
  • negative HCV PCR test result if the anti-HCV is positive at Visit 0.
  • a subject has childbearing potential, the subject has a negative serum beta-human chorionic gonadotropin ( ⁇ -HCG) pregnancy test result at Visit 0 and negative urine pregnancy test results before each administration of a formulated RNA construct or a combination of formulated RNA constructs. Subjects born female who are postmenopausal or permanently sterilized are not be considered to have childbearing potential.
  • ⁇ -HCG negative serum beta-human chorionic gonadotropin
  • a subject has childbearing potential, the subject agree to practice a highly effective form of contraception starting at Visit 0 and continuously until 90 days after receiving a third dose of a formulated RNA construct or a combination of formulated RNA constructs.
  • a subject has childbearing potential, the subject agree not to donate or cryopreserve eggs (ova, oocytes) for the purposes of assisted reproduction during trial, starting at Visit 0 and continuously until 90 days after receiving a third dose of a formulated RNA construct or a combination of formulated RNA constructs.
  • xii the subject agree to practice a highly effective form of contraception starting at Visit 0 and continuously until 90 days after receiving a third dose of a formulated RNA construct or a combination of formulated RNA constructs.
  • a subject is male, does not have had a vasectomy and are sexually active with partners of childbearing potential agree to use condoms and to practice a highly effective form of contraception with their sexual partners of childbearing potential during the trial, starting at Visit 0 and continuously until 90 days after receiving a third dose of a formulated RNA construct or a combination of formulated RNA constructs. xiii. If a subject is male, will refrain from sperm donation, starting at Visit 0 and continuously until 90 days after receiving a third dose of a formulated RNA construct or a combination of formulated RNA constructs.
  • Trial Exclusion Criteria Subjects with the following are excluded from a trial described in the present Example: i.
  • Plasmodium parasitemia any species
  • a short-acting rescue inhaler typically a beta 2 agonist
  • ii Uses high dose inhaled corticosteroids (per American Academy of Allergy Asthma & Immunology), or iii.
  • a short-acting rescue inhaler typically a beta 2 agonist
  • high dose inhaled corticosteroids per American Academy of Allergy Asthma & Immunology
  • iii Uses a short-acting rescue inhaler daily, or ii.
  • high dose inhaled corticosteroids per American Academy of Allergy Asthma & Immunology
  • a person has a history of hypertension, or elevated blood pressure detected during screening, exclude for blood pressure that is not well controlled.
  • Well controlled blood pressure is defined as consistently ⁇ 140 mm Hg systolic and ⁇ 90 mm Hg diastolic, with or without medication, with only isolated, brief instances of higher readings, which must be ⁇ 150 mm Hg systolic and ⁇ 100 mm Hg diastolic at screening and enrollment; d. Malignancy within 5 years of screening, excluding localized basal or squamous cell skin cancer; e.
  • cardiovascular diseases e.g., myocarditis, pericarditis, myocardial infarction, congestive heart failure, cardiomyopathy or clinically significant arrhythmias
  • a health practitioner e.g., investigator’s
  • An abnormal screening ECG i.e., showing the corrected QT interval by Fridericia (QTcF) >150 ms; significant ST-T wave changes suggestive of myocardial ischemia or of an acute or indeterminate- age myocardial infarction; left ventricular hypertrophy; any non-sinus rhythm including isolated premature ventricular contractions; complete right or left bundle branch block [QRS >120 ms]; second-or third-degree atrioventricular [AV] block); or other clinically significant abnormalities on the ECG at the investigator’s discretion; g. Bleeding disorder diagnosed by a doctor (e.g., factor deficiency, coagulopathy, or platelet disorder requiring special precautions); or h.
  • QTcF Fridericia
  • Seizure disorder History of seizure(s) within past 3 years. Also exclude if volunteer has used medications in order to prevent or treat seizure(s) at any time within the past 3 years.
  • Documented major psychiatric illness including bipolar disorder, major depressive disorder, schizophrenia, autism, and attention deficit-hyperactivity disorder that at the discretion of the investigator could interfere with participation and follow-up as outlined by the trial.
  • autoimmune disease including and not limited to thyroid autoimmune disease, multiple sclerosis, psoriasis, etc.
  • viii Previous vaccination with an approved or investigational malaria vaccine at any time or having taken part in a human malaria challenge study.
  • ix Receipt of any investigational product within 28 days before Visit 0.
  • Seasonal influenza and COVID-19 vaccines are allowed; however, they should be administered at least 14 days before or 28 days after any IMP administration.
  • Emergency vaccinations, such as tetanus are allowed to be administered when medically indicated.
  • xii Received allergy treatment with antigen injections within 28 days before and after each IMP administration.
  • xiii Current or planned treatment with immunosuppressive therapy, including systemic corticosteroids (if systemic corticosteroids are administered for ⁇ 14 days at a dose of ⁇ 20 mg/d of prednisone or equivalent) starting at Visit 0 and continuously until a third dose of a formulated RNA construct or a combination of formulated RNA constructs.
  • Intraarticular, intrabursal, or topical (skin or eyes) corticosteroids are permitted.
  • xiv. Have a history of alcohol abuse or drug addiction within 1 year before Visit 0 or have a history (within the past 5 years) of substance abuse, which in the opinion of a health practitioner (e.g., an investigator), could compromise their wellbeing if they participate as a subject in a trial, or that could prevent, limit, or confound the protocol specified assessments.
  • ICH International Council for Harmonisation
  • ICH International Council for Harmonisation
  • Subjects with abnormal but not clinically significant parameters not included in the toxicity guidance may be considered eligible at discretion of a health practitioner (e.g., an investigator).
  • RNA Construct 23-7 is evaluated in different dose combinations in a 3 dose schedule to select a safe, tolerable, and immunogenic dose combination, and to assess the impact of third dose on immunogenicity.
  • subjects are divided into cohorts. Cohorts are randomized 5:1 active:placebo. Evaluation uses a staggered dose-escalation schema with sentinel subjects in all cohorts. Different cohorts receive different doses of RNA Construct 23-7. Table 11 provides an overview of the dose combinations used.
  • RNA Sample size Cohort Construct 23-7 (active:placebo) [0554] A u - u y . pproximately eight weeks later, a second dose of RNA Construct 23-7 is administered to the subject. A third dose of RNA Construct 23-7 is administered approximately 18 weeks after the second dose. Post-Trial Assessments [0555] Subjects are assessed for the following primary outcome measures after each dose of a formulated RNA Construct 23-7: i.
  • Frequency of solicited local reactions at the injection site e.g., pain, erythema/redness and/or induration/swelling
  • ii Frequency of solicited systemic reactions (vomiting, diarrhea, headache, fatigue, muscle/joint pain, and fever) recorded up to 7 days after each dose
  • iii Frequency of subjects with at least one AE occurring until 28 days after each dose
  • iv Frequency of subjects with at least one medically attended adverse event (MAAE) occurring until 28 days after each dose.
  • Subjects are assessed to determine a frequency of subjects in each cohort with at least one SAE occurring until 24 weeks after a third dose.
  • Descriptive statistics on antibody levels (e.g., anti-CSP antibodies) in a subject can be assessed at various time points. For example, levels of antigen-specific serum and/or plasma antibodies can be assessed using ELISA or similar assays. Additionally, the functionality and/or avidity of serum and/or plasma antibodies are evaluated. [0558] Other immune responses of subjects following administration of one or more doses are also assessed. For instance, CD4+ and CD8+ T-cell responses to antigens in a formulated RNA construct can be measured using polychromatic flow cytometry. Vaccine-induced plasmablasts and/or memory B cells can be measured using flow cytometry, and levels of inflammatory markers, chemokines, and/or cytokines can be measured.
  • RNA expression patterns in subjects following administration of one or more doses can be assessed using, e.g., RNASeq technology.

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Abstract

La présente divulgation concerne des compositions (par exemple, des compositions pharmaceutiques) pour l'administration d'antigènes de protéine circumsporozoïte (CSP) du plasmodium, ainsi que des technologies associées (par exemple, des composants de celles-ci et/ou des méthodes associées). Entre autres, la présente divulgation concerne des polyribonucléotides comprenant une séquence de codage qui code un polypeptide CSP de plasmodium pleine longueur. Dans certains modes de réalisation, un polypeptide CSP de plasmodium pleine longueur comprend un domaine N-terminal de CSP de plasmodium et la séquence de codage d'un polyribonucléotide codant le polypeptide CSP de plasmodium pleine longueur a une teneur en nucléotides particulière telle que présentement décrite.
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