Immunogenic compositions and uses thereof

Abstract

This disclosure provides compositions, pharmaceutical preparations, and uses of polyribonucleotides encoding Plasmodium falciparum antigens for use in treating and preventing Malaria.

Description

ATTORNEY DOCKET: 51719-011 WO2 PATENTIMMUNOGENIC COMPOSITIONS AND USES THEREOFSequence ListingThis application contains a Sequence Listing which has been filed electronically in Extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 31 , 2025, is named 51719-011 WO2_Sequence_Listing_7_31_25.XML and is 209,365 bytes in size.BackgroundVaccination has made an enormous contribution to both human and animal health. Since the invention of the first vaccine in 1796, vaccines have come to be considered the most successful method for preventing many infectious diseases by provoking an immune response in a subject. According to the World Health Organization, immunization currently prevents 2-3 million deaths every year across all age groups. Today, vaccines have been developed to prevent and control the spread of more than 20 infectious diseases, such as malaria. There remains a need to develop new and improved immunogenic compositions and uses thereof.SummaryThis disclosure provides compositions, pharmaceutical preparations, and uses of polyribonucleotides (e.g., circular or linear polyribonucleotides) encoding a plasmodium circumsporozoite polypeptide that can be used to treat or prevent malaria.In one aspect, featured is a linear polyribonucleotide including, from 5’-to-3’, a first circularization element, an open reading frame (ORF) encoding a plasmodium circumsporozoite polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 9, and a second circularization element.In another aspect, featured is a linear polyribonucleotide including, from 5’-to-3’, a first circularization element, an open reading frame (ORF) encoding a plasmodium circumsporozoite polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 65, and a second circularization element.In another aspect, featured is a linear polyribonucleotide including, from 5’-to-3’, a first circularization element, an open reading frame (ORF) encoding a plasmodium circumsporozoite polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 95, and a second circularization element.In some embodiments, the plasmodium circumsporozoite polypeptide has the sequence of SEQ ID NO: 9.In some embodiments, the plasmodium circumsporozoite polypeptide has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 65.In some embodiments, the plasmodium circumsporozoite polypeptide has the sequence of SEQ ID NO: 65.In some embodiments, the ORF includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the any one of SEQ ID NOs: 1 -8.In some embodiments, the ORF includes the sequence of any one of SEQ ID NOs: 1 -8.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, the ORF includes the sequence of SEQ ID NO: 1 .In some embodiments, the ORF includes the sequence of SEQ ID NO: 2.In some embodiments, the ORF includes the sequence of SEQ ID NO: 3.In some embodiments, the ORF includes the sequence of SEQ ID NO: 4.In some embodiments, the ORF includes the sequence of SEQ ID NO: 5.In some embodiments, the ORF includes the sequence of SEQ ID NO: 6.In some embodiments, the ORF includes the sequence of SEQ ID NO: 7.In some embodiments, the ORF includes the sequence of SEQ ID NO: 8.In some embodiments, the plasmodium circumsporozoite polypeptide is a full length PfCSP polypeptide.In some embodiments, the plasmodium circumsporozoite polypeptide has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 95.In some embodiments, the plasmodium circumsporozoite polypeptide has the sequence of SEQ ID NO: 95.In some embodiments, the ORF includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 74-79.In some embodiments, the ORF includes the sequence of any one of SEQ ID NOs: 74-79.In some embodiments, the ORF includes the sequence of SEQ ID NO: 74.In some embodiments, the ORF includes the sequence of SEQ ID NO: 75.In some embodiments, the ORF includes the sequence of SEQ ID NO: 76.In some embodiments, the ORF includes the sequence of SEQ ID NO: 77.In some embodiments, the ORF includes the sequence of SEQ ID NO: 78.In some embodiments, the ORF includes the sequence of SEQ ID NO: 79.In some embodiments, the first circularization element includes, from 5’-to-3’, a 3’ portion of a catalytic intron fragment (e.g., a group I catalytic intron fragment), a 3’ splice site, and a 3’ exon fragment.In some embodiments, the second circularization element includes, from 5’-to-3’, a 5’ portion of a catalytic intron fragment (e.g., a group I catalytic intron fragment), a 5’ splice site, and a 5’ exon fragment.In some embodiments, the first circularization element includes, from 5’-to-3’, a 3’ portion of a catalytic intron fragment (e.g., a group I catalytic intron fragment), a 3’ splice site, and a 3’ exon fragment, and the second circularization element includes, from 5’-to-3’, a 5’ portion of a catalytic intron fragment (e.g., a group I catalytic intron fragment), a 5’ splice site, and a 5’ exon fragment.In some embodiments, the first circularization element includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAG ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGA AAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA (SEQ ID NO: 10).In some embodiments, the first circularization element includes the sequence of AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAG ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGA AAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA (SEQ ID NO: 10).In some embodiments, the second circularization element includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity toATTORNEY DOCKET: 51719-011 WO2 PATENTCCCACACGACCGTTTAGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGA TGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTT (SEQ ID NO: 11 ).In some embodiments, the second circularization element includes the sequence of CCCACACGACCGTTTAGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGA TGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTT (SEQ ID NO: 11 ).In some embodiments, the linear polynucleotide further includes an internal ribosomal entry site (IRES) operatively linked to the ORF.In some embodiments, the linear polyribonucleotide further includes a spacer region positioned between the first circularization element and the ORF.In some embodiments, the linear polyribonucleotide further includes a spacer region positioned between the ORF and the second circularization element.In some embodiments, the linear polyribonucleotide includes a first spacer region positioned between the first circularization element and the ORF and a second spacer region positioned between the ORF and the second circularization element.In some embodiments, the linear polyribonucleotide includes a spacer region positioned between the circularization element and the IRES.In some embodiments, the linear polyribonucleotide includes a spacer region positioned between the circularization element and the IRES and a spacer region positioned between the ORF and the second circularization element.In some embodiments, the spacer region includes a polyA-C or a polyA-T sequence.In some embodiments, the spacer region is from 5 to 500 (e.g., 5 to 500, 10 to 400, 20 to 300, 30 to 250, 40 to 200, or 50 to 150, e.g., 5, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length.In some embodiments, the spacer region is from 50 to 150 ribonucleotides in length. 12. The linear polyribonucleotide of any one of claims 9-11 , wherein the IRES is a Coxsackievirus B3 (CVB3) IRES.In some embodiments, the CVB3 IRES has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA CTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG GCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAA TTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCC TTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACA GCAA (SEQ ID NO: 12).ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, the CVB3 IRES has a the sequence of TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA CTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG GCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAA TTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCC TTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACA GCAA (SEQ ID NO: 12).In some embodiments, the CVB3 IRES has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of CCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGAT CAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTC GTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCA CTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTG CGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTA TTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTC AAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTG TTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGAT TGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAG AGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAA (SEQ ID NO: 96).In some embodiments, the CVB3 IRES has the sequence of CCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGAT CAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTC GTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCA CTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTG CGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTA TTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTC AAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTG TTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGAT TGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAG AGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAA (SEQ ID NO: 96).In some embodiments, the linear polyribonucleotide is from 500 to 20,000 (e.g., 500, 600, 700, 800, 900, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000) ribonucleotides.In some embodiments, the linear polyribonucleotide is at least 1 ,000 ribonucleotides.In some embodiments, the ORF further encodes a signal sequence.In some embodiments, the signal sequence is an IL-2 signal sequence.In some embodiments, the signal sequence is an IgK signal sequence.ATTORNEY DOCKET: 51719-011 WO2PATENTIn another aspect, featured is a DNA vector that includes an RNA polymerase promoter operably linked to a DNA sequence that encodes the linear polyribonucleotide of any of the above embodiments.In another aspect, featured is a circular polyribonucleotide produced from the linear polyribonucleotide or DNA vector of any of the above embodiments.In another aspect, featured is a circular polyribonucleotide that includes a splice junction joining a 5’ exon fragment and a 3’ exon fragment and an ORF encoding a plasmodium circumsporozoite polypeptide of SEQ ID NO: 9.In another aspect, featured is a circular polyribonucleotide that includes a splice junction joining a 5’ exon fragment and a 3’ exon fragment and an ORF encoding a plasmodium circumsporozoite polypeptide of SEQ ID NO: 65.In another aspect, featured is a circular polyribonucleotide that includes a splice junction joining a 5’ exon fragment and a 3’ exon fragment and an ORF encoding a plasmodium circumsporozoite polypeptide of SEQ ID NO: 95.In some embodiments, the ORF includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the any one of SEQ ID NOs: 1 -8.In some embodiments, the ORF includes the sequence of any one of SEQ ID NOs: 1 -8.In some embodiments, the ORF includes the sequence of SEQ ID NO: 1 .In some embodiments, the ORF includes the sequence of SEQ ID NO: 2.In some embodiments, the ORF includes the sequence of SEQ ID NO: 3.In some embodiments, the ORF includes the sequence of SEQ ID NO: 4.In some embodiments, the ORF includes the sequence of SEQ ID NO: 5.In some embodiments, the ORF includes the sequence of SEQ ID NO: 6.In some embodiments, the ORF includes the sequence of SEQ ID NO: 7.In some embodiments, the ORF includes the sequence of SEQ ID NO: 8.In some embodiments, the ORF includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 74-79.In some embodiments, the ORF includes the sequence of any one of SEQ ID NOs: 74-79.In some embodiments, the ORF includes the sequence of SEQ ID NO: 74.In some embodiments, the ORF includes the sequence of SEQ ID NO: 75.In some embodiments, the ORF includes the sequence of SEQ ID NO: 76.In some embodiments, the ORF includes the sequence of SEQ ID NO: 77.In some embodiments, the ORF includes the sequence of SEQ ID NO: 78.In some embodiments, the ORF includes the sequence of SEQ ID NO: 79.In some embodiments, the circular polyribonucleotide further includes an IRES operably linked to the ORF.In some embodiments, the circular polyribonucleotide further includes a spacer region between the IRES and the ORF, the ORF and the splice junction, and / or the splice junction and the IRES.In some embodiments, the spacer region includes a polyA-C or a polyA-T sequence.In some embodiments, the spacer region is from 5 to 500 (e.g., 5 to 500, 10 to 400, 20 to 300, 30 to 250, 40 to 200, or 50 to 150, e.g., 5, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length.In some embodiments, the spacer region is from 50 to 150 ribonucleotides in length.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, the circular polyribonucleotide is from 500 to 20,000 (e.g., 500, 600, 700, 800, 900, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000) ribonucleotides.In some embodiments, the circular polyribonucleotide is at least 500 ribonucleotides.In another aspect, featured is a polynucleotide that includes an ORF including a sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 1 -8.In some embodiments, the ORF includes the sequence of SEQ ID NO: 1 .In some embodiments, the ORF includes the sequence of SEQ ID NO: 2.In some embodiments, the ORF includes the sequence of SEQ ID NO: 3.In some embodiments, the ORF includes the sequence of SEQ ID NO: 4.In some embodiments, the ORF includes the sequence of SEQ ID NO: 5.In some embodiments, the ORF includes the sequence of SEQ ID NO: 6.In some embodiments, the ORF includes the sequence of SEQ ID NO: 7.In some embodiments, the ORF includes the sequence of SEQ ID NO: 8.In another aspect, featured is a polynucleotide that includes an ORF including a sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 74-79.In some embodiments, the ORF includes the sequence of SEQ ID NO: 74.In some embodiments, the ORF includes the sequence of SEQ ID NO: 75.In some embodiments, the ORF includes the sequence of SEQ ID NO: 76.In some embodiments, the ORF includes the sequence of SEQ ID NO: 77.In some embodiments, the ORF includes the sequence of SEQ ID NO: 78.In some embodiments, the ORF includes the sequence of SEQ ID NO: 79.In some embodiments, the polynucleotide further includes an IRES operably linked to the ORF.In some embodiments, the polynucleotide is a polyribonucleotide.In some embodiments, the polyribonucleotide is a linear polyribonucleotide.In some embodiments, the polyribonucleotide is a circular polyribonucleotide.In another aspect, featured is a DNA vector that includes an RNA polymerase promoter operably linked to a DNA sequence that encodes the polynucleotide of any of the above embodiments.In another aspect, featured is an immunogenic composition that includes the circular polyribonucleotide or the polynucleotide of any of the above embodiments.In another aspect, featured is a pharmaceutical composition that includes the circular polyribonucleotide, the polynucleotide, or the immunogenic composition of any of the above embodiments and a pharmaceutically acceptable excipient.In some embodiments, the pharmaceutical composition further includes an adjuvant.In some embodiments, the adjuvant is an inorganic adjuvant, a small molecule adjuvant, and oil in water emulsion, a lipid or polymer, a peptide or peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a viral particle, a bacterial adjuvant, a hybrid molecule, a fungal or oocyte microbe-associated molecular pattern (MAMP), an inorganic nanoparticle, or a multi-component adjuvant.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn another aspect, featured is a method of treating or preventing malaria in a subject. The method includes administering to the subject the circular polyribonucleotide, polynucleotide, immunogenic composition, or pharmaceutical composition of any of the above embodiments.In another aspect, featured is method of inducing an immune response in a subject. The method includes administering to the subject the circular polyribonucleotide, polynucleotide, immunogenic composition, or pharmaceutical composition of any of the above embodiments.In some embodiments, the method further includes administering an adjuvant to the subject.In some embodiments, the adjuvant is an inorganic adjuvant, a small molecule adjuvant, and oil in water emulsion, a lipid or polymer, a peptide or peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a viral particle, a bacterial adjuvant, a hybrid molecule, a fungal or oocyte MAMP, an inorganic nanoparticle, or a multi-component adjuvant.In some embodiments, the circular polyribonucleotide is administered to the subject as a single dose.In some embodiments, the circular polyribonucleotide is administered to the subject two or more times, three or more times, four or more times, or five or more times.In some embodiments, administration of the circular polyribonucleotide occurs about weekly, about every two weeks, about every three weeks, about every month, about every two months, about every three months, about every four months, about every five months, about every six months, about every year, about every two years, about every three years, about every four years, about every five years, or about every ten years.In some embodiments, the method further includes administering to the subject a plasmodium circumsporozoite polypeptide.In some embodiments, the plasmodium circumsporozoite polypeptide is administered to the subject after administering the circular polyribonucleotide.In some embodiments, administration of the plasmodium circumsporozoite polypeptide maintains or enhances an immune response in the subject against the plasmodium circumsporozoite polypeptide.DefinitionsThe present disclosure will be described with respect to particular embodiments and with reference to certain figures, but the disclosure is not limited thereto but only by the claims. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.As used herein, the term “adaptive immune response” means either a humoral or cell-mediated immune response. For purposes of the present disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and / or other white blood cells.As used herein, the term “adjuvant” refers to a compound that augments or otherwise alters or modifies an immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain immunogen-specific immune responses.As used herein, the term “carrier” means a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., a linear or a circular polyribonucleotide) into aATTORNEY DOCKET: 51719-011 WO2 PATENT subject, a tissue, or a cell. Non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride-modified phytoglycogen or glycogen-type material), nanoparticles (e.g., a nanoparticle that encapsulates or is covalently linked binds to the circular or linear polyribonucleotide), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., a protein covalently linked to the polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent).As used herein, the term “cell-penetrating agent” means an agent that, when contacted to a cell, facilitates entry into the cell. In some cases, a cell-penetrating agent facilitates direct penetration of the cell membrane, for instance, via direct electrostatic interaction with negatively charged phospholipids of the cell membrane, or transient pore formation by inducing configurational changes in membrane proteins or the phospholipid bilayer. In some cases, a cell-penetrating agent facilitates endocytosis-mediated translocation into the cell. For example, under certain situations, the cell-penetrating agent can stimulate the cell to undergo the endocytosis process, by which the cell membrane can fold inward into the cell. In certain embodiments, a cell-penetrating agent helps form a transitory structure that transports across the cell membrane. Without wishing to be bound to a particular theory, a cell-penetrating agent as provided herein can increase the permeability of the cell membrane or increase internalization of a molecule into the cell, as a result of which, delivery into the cell can be more efficient when the cell is contacted with the cell-penetrating agent simultaneously as compared to otherwise identical delivery without the cellpenetrating agent.As used herein, the terms “circRNA,” “circular polyribonucleotide,” “circular RNA,” and “circular polyribonucleotide molecule” are used interchangeably and mean a polyribonucleotide molecule that has a structure having no free ends (i.e. , no free 3’ and / or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent or non-covalent bonds.As used herein, the term “circularization efficiency” is a measurement of resultant circular polyribonucleotide versus its non-circular starting material.As used herein, the terms “circRNA preparation,” “circular polyribonucleotide preparation,” and “circular RNA preparation” are used interchangeably and mean a composition including circRNA molecules and a diluent, carrier, first adjuvant, or a combination thereof.The wording “compound, composition, product, etc. for treating, modulating, etc.” is to be understood to refer a compound, composition, product, etc. per se which is suitable for the indicated purposes of treating, modulating, etc. The wording “compound, composition, product, etc. for treating, modulating, etc.” additionally discloses that, as a preferred embodiment, such compound, composition, product, etc. is for use in treating, modulating, etc.The wording “compound, composition, product, etc. for use in ...” or “use of a compound, composition, product, etc. in the manufacture of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for ...” indicates that such compounds, compositions, products, etc. are to be used in therapeutic methods which may be practiced on the human or animal body. They are considered as an equivalent disclosure of embodiments and claims pertaining to methods of treatment, etc. If an embodiment or a claim thus refers to “a compound for use in treating a human or animal being suspected to suffer from a disease”, this is considered to be also a disclosure of a “use of a compound in the manufacture of a medicament for treating a human or animal being suspected to suffer from a disease” or a “method of treatment by administering a compound to a human or animal being suspected to suffer from a disease.”ATTORNEY DOCKET: 51719-011 WO2 PATENTThe term “diluent” means a vehicle including an inactive solvent in which a composition described herein (e.g., a composition including a circular or linear polyribonucleotide) may be diluted or dissolved. A diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof. A diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3- butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and 1 ,3-butanediol. Nonlimiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, or powdered sugar.As used herein, the terms “disease,” “disorder,” and “condition” each refer to a state of sub- optimal health, for example, a state that is or would typically be diagnosed or treated by a medical professional.As used herein, the term “encryptogen” is a nucleic acid sequence or structure of the circular polyribonucleotide that aids in reducing, evading, and / or avoiding detection by an immune cell and / or reduces induction of an immune response against the circular or linear polyribonucleotide.As used herein, the term “expression sequence” is a nucleic acid sequence that encodes a product, e.g., a peptide or polypeptide, or a regulatory nucleic acid. An exemplary expression sequence that codes for a peptide or polypeptide can include a plurality of nucleotide triads, each of which can code for an amino acid and is termed as a “codon.”As used herein, the term “impurity” is an undesired substance present in a composition, e.g., a pharmaceutical composition as described herein. In some embodiments, an impurity is a process-related impurity. In some embodiments, an impurity is a product-related substance other than the desired product in the final composition, e.g., other than the active drug ingredient, e.g., circular or linear polyribonucleotide, as described herein. As used herein, the term “process-related impurity” is a substance used, present, or generated in the manufacturing of a composition, preparation, or product that is undesired in the final composition, preparation, or product other than the linear polyribonucleotides described herein. In some embodiments, the process-related impurity is an enzyme used in the synthesis or circularization of polyribonucleotides. As used herein, the term “product-related substance” is a substance or byproduct produced during the synthesis of a composition, preparation, or product, or any intermediate thereof. In some embodiments, the product-related substance is deoxyribonucleotide fragments. In some embodiments, the product-related substance is deoxyribonucleotide monomers. In some embodiments, the product-related substance is one or more of: derivatives or fragments of polyribonucleotides described herein, e.g., fragments of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acids, diribonucleic acids, or triribonucleic acids.As used herein, the term “immunogen” refers to an any molecule or molecular structure that includes one or more epitopes recognized, targeted, or bound by an antibody or a T cell receptor. In particular, an immunogen induces an immune response in a subject (e.g., is immunogenic as defined herein). An immunogen is capable of inducing an immune response in a subject, wherein the immune response refers to a series of molecular, cellular, and organismal events that are induced when anATTORNEY DOCKET: 51719-011 WO2 PATENT immunogen is encountered by the immune system. The immune response may be humoral and / or cellular immune response. These may include the production of antibodies and the expansion of B- and T-cells. To determine whether an immune response has occurred and to follow its course, the immunized subject can be monitored for the appearance of immune reactants directed at the specific immunogen. Immune responses to most immunogen induce the production of both specific antibodies and specific effector T cells. In some embodiments, the immunogen is foreign to a host. In some embodiments, the immunogen is not foreign to a host. An immunogen may include all or a portion of a polypeptide, a polysaccharide, a polynucleotide, or a lipid. An immunogen may also be a mixed polypeptide, polysaccharide, polynucleotide, and / or lipid. For example, an immunogen maybe a polypeptide that has been translationally modified. A “polypeptide immunogen” refers to an immunogen that includes a polypeptide. A polypeptide immunogen may also include one or more post-translational modifications, and / or may form a complex with one or more additional molecules, and / or may adopt a tertiary or quaternary structure, each of which may determine or affect the immunogenicity of the polypeptide.As used herein, the term “immunogenic” is a potential to induce a response to a substance in a particular immune response assay above a pre-determined threshold. The assay can be, e.g., expression of certain inflammatory markers, production of antibodies, or an assay for immunogenicity as described herein. In some embodiments, an immune response may be induced when an immune system of an organism or a certain type of immune cells are exposed to an immunogen.An immunogenic response may be assessed may evaluating the antibodies in the plasma or serum of a subject using a total antibody assay, a confirmatory test, titration and isotyping of the antibodies, and neutralizing antibody assessment. A total antibody assay measures all the antibodies generated as part of the immune response in the serum or plasma of a subject that has been administered the immunogen. The most commonly used test to detect antibodies is an ELISA (enzyme- linked immunosorbent assay), which detects antibodies in the tested serum that bind to the antibody of interest, including IgM, IgD, IgG, IgA, and IgE. An immunogenic response can be further assessed by a confirmatory assay. Following a total antibody assessment, a confirmatory assay may be used to confirm the results of the total antibody assay. A competition assay may be used to confirm that antibody is specifically binding to target and that the positive finding in the screening assay is not a result of nonspecific interactions of the test serum or detection reagent with other materials in the assay.An immunogenic response can be assessed by isotyping and titration. An isotyping assay may be used to assess only the relevant antibody isotypes. For example, the expected isotypes may be IgM and IgG which may be specifically detected and quantified by isotyping and titration and then compared to the total antibodies present.An immunogenic response can be assessed by a neutralizing antibody assay (nAb). A neutralizing antibody assay (nAb) may be used to determine if the antibodies produced in response to the immunogen neutralized the immunogen thereby inhibiting the immunogen from having an effect on the target and leading to abnormal pharmacokinetic behaviors. An nAb assay is often a cell-based assay where the target cells are incubated with the antibody. A variety of cell based nAb assays may be used including but not limited to Cell Proliferation, Viability, Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Cytopathic Effect Inhibition (CPE), Apoptosis, Ligand Stimulated Cell Signaling, Enzyme Activity, Reporter Gene Assays, Protein Secretion, Metabolic Activity, Stress and Mitochondrial Function. Detection readouts include Absorbance, Fluorescence,ATTORNEY DOCKET: 51719-011 WO2 PATENTLuminescence, Chemiluminescence, or Flow Cytometry. A ligand-binding assay may also be used to measure the binding affinity of an immunogen and an antibody in vitro to evaluate neutralization efficacy.Furthermore, induction of a cellular immune response may be assessed by measuring T cell activation in a subject using cellular markers on T cells obtained from the subject. A blood sample, lymph node biopsy, or tissue sample can be collected from a subject and T cells from the sample evaluated for one or more (e.g., 2, 3, 4 or more) activation markers: CD25, CD71 , CD26, CD27, CD28, CD30, CD154, CD40L, CD134, CD69, CD62L or CD44. T cell activation can also be assessed using the same methods in an in vivo animal model. This assay can also be performed by adding an immunogen to T cells in vitro (e.g., T cells obtained from a subject, animal model, repository, or commercial source) and measuring the aforementioned markers to evaluate T cell activation. Similar approaches can be used to assess the effect of an on activation of other immune cells, such as eosinophils (markers: CD35, CD11 b, CD66, CD69 and CD81 ), dendritic cells (makers: IL-8, MHC class II, CD40, CD80, CD83, and CD86), basophils (CD63, CD13, CD4, and CD203c), and neutrophils (CD11 b, CD35, CD66b and CD63). These markers can be assessed using flow cytometry, immunohistochemistry, in situ hybridization, and other assays that allow for measurement of cellular markers. Comparing results from before and after administration of an immunogen can be used to determine its effect.As used herein, the term “inducing an immune response” refers to initiating, amplifying, or sustaining an immune response by a subject. Inducing an immune response may refer to an adaptive immune response or an innate immune response. The induction of an immune response may be measured as discussed above.As used herein, the term “linear counterpart” is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence identity) as a circular polyribonucleotide and having two free ends (i.e. , the uncircularized version (and its fragments) of the circularized polyribonucleotide). In some embodiments, the linear counterpart (e.g., a pre-circularized version) is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence identity) and same or similar nucleic acid modifications as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide). In some embodiments, the linear counterpart is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence identity) and different or no nucleic acid modifications as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide). In some embodiments, a fragment of the polyribonucleotide molecule that is the linear counterpart is any portion of linear counterpart polyribonucleotide molecule that is shorter than the linear counterpart polyribonucleotide molecule. In some embodiments, the linear counterpart further includes a 5’ cap. In some embodiments, the linear counterpart further includes a poly adenosine tail. In some embodiments, the linear counterpart further includes a 3’ UTR. In some embodiments, the linear counterpart further includes a 5’ UTR.As used herein, the terms “linear RNA,” “linear polyribonucleotide,” and “linear polyribonucleotide molecule” are used interchangeably and mean polyribonucleotide molecule having a 5’ and 3’ end. One or both of the 5’ and 3’ ends may be free ends or joined to another moiety. Linear RNA includes RNA that has not undergone circularization (e.g., is pre-circularized) and can be used as a starting material forATTORNEY DOCKET: 51719-011 WO2 PATENT circularization through, for example, splint ligation, or chemical, enzymatic, ribozyme- or splicing- catalyzed circularization methods.As used herein, the term “mixture” means a material made of two or more different substances that are mixed. In some cases, a mixture described herein can be a homogenous mixture of the two or more different substances, e.g., the mixture can have the same proportions of its components (e.g., the two or more substances) throughout any given sample of the mixture. In some cases, a mixture as provided herein can be a heterogeneous mixture of the two or more different substances, e.g., the proportions of the components of the mixture (e.g., the two or more substances) can vary throughout the mixture. In some cases, a mixture is a liquid solution, e.g., the mixture is present in liquid phase. In some instances, a liquid solution can be regarded as comprising a liquid solvent and a solute. Mixing a solute in a liquid solvent can be termed as “dissolution” process. In some cases, a liquid solution is a liquid-in-liquid solution (e.g., a liquid solute dissolved in a liquid solvent), a solid-in-liquid solution (e.g., a solid solute dissolved in a liquid solvent), or a gas-in-liquid solution (e.g., a solid solute dissolved in a liquid solvent). In some cases, there is more than one solvent and / or more than one solute. In some cases, a mixture is a colloid, liquid suspension, or emulsion. In some cases, a mixture is a solid mixture, e.g., the mixture is present in solid phase.As used herein, the term “modified ribonucleotide” means a nucleotide with at least one modification to the sugar, the nucleobase, or the internucleoside linkage.As used herein, the term “naked delivery” means a formulation for delivery to a cell without the aid of a carrier and without covalent modification to a moiety that aids in delivery to a cell. A naked delivery formulation is free from any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, naked delivery formulation of a circular or linear polyribonucleotide is a formulation that includes a circular or linear polyribonucleotide without covalent modification and is free from a carrier.As used herein, the terms “nicked RNA,” “nicked linear polyribonucleotide, ’’and “nicked linear polyribonucleotide molecule” are used interchangeably and mean a polyribonucleotide molecule having a 5’ and 3’ end that results from nicking or degradation of a circular RNA.As used herein, the term “non-circular RNA” means total nicked RNA and linear RNA.The terms “obtainable by,” “producible by,” and the like are used to indicate that a claim or embodiment refers to compound, composition, product, etc. per se, i.e. that the compound, composition, product, etc. can be obtained or produced by a method which is described for manufacture of the compound, composition, product, etc., but that the compound, composition, product, etc. may be obtained or produced by other methods than the described one as well. The terms “obtained by”, “produced by” or the like indicate that the compound, composition, product, is obtained or produced by a recited specific method. It is to be understood that the terms “obtainable by”, “producible by” and the like also disclose the terms “obtained by”, “produced by” and the like as a preferred embodiment of “obtainable by”, “producible by” and the like.As used herein, the term “pathogen” refers to an infectious agent, which causes disease or disease symptoms in a subject, for example, by directly infecting the subject, by producing agents that cause disease or disease symptoms in the subject, and / or by eliciting an immune response in the subject. As used herein, pathogens include, but are not limited to bacteria, protozoa, parasites, fungi, nematodes,ATTORNEY DOCKET: 51719-011 WO2 PATENT insects, viroids, and viruses, or any combination thereof, wherein each pathogen is capable, either by itself or in concert with another pathogen, of eliciting disease or symptoms a subject.The term “pharmaceutical composition” is intended to also disclose that the circular or linear polyribonucleotide included within a pharmaceutical composition can be used for the treatment of the human or animal body by therapy. It is thus meant to be equivalent to “a circular or linear polyribonucleotide for use in therapy”.The term “polynucleotide” as used herein means a molecule comprising one or more nucleic acid subunits, or nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide”. A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five- carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Polyribonucleotides or ribonucleic acids, or RNA, can refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.Polydeoxyribonucleotides or deoxyribonucleic acids, or DNA, means macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. A nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide means a deoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, that include detectable tags, such as luminescent tags or markers (e.g., fluorophores). A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e. , A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. In some cases, a polynucleotide is a short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some cases, a polynucleotide molecule is circular. A polynucleotide can have various lengths. A nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. A polynucleotide can be isolated from a cell or a tissue. As embodied herein, the polynucleotide sequences may include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.Polynucleotides, e.g., polyribonucleotides or polydeoxyribonucleotides, may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s) and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-ATTORNEY DOCKET: 51719-011 WO2 PATENT carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid(v), 5-methyl-2- thiouracil, 3-(3-amino- 3- N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates). Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and / or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone. Nucleic acid molecules may also contain amine -modified groups, such as amino ally 1 -dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide esters (NHS). Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photo-programmed polymerases, or lower secondary structure. Such alternative base pairs compatible with natural and mutant polymerases for de novo and / or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, which is herein incorporated by reference for all purposes.As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or may be a multi- molecular complex such as a dimer, trimer, or tetramer. They can also comprise single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.As used herein, the term “prevent” means to reduce the likelihood of developing a disease, disorder, or condition, or alternatively, to reduce the severity of a subsequently developed disease or disorder. A therapeutic agent can be administered to a subject who is at increased risk of developing a disease or disorder relative to a member of the general population in order to prevent the development of, or lessen the severity of, the disease or condition. A therapeutic agent can be administered as a prophylactic, e.g., before development of any symptom or manifestation of a disease or disorder.As used herein, the term “regulatory element” is a moiety, such as a nucleic acid sequence, that modifies expression of an expression sequence within the circular or linear polyribonucleotide.ATTORNEY DOCKET: 51719-011 WO2 PATENTAs used herein, the term “repetitive nucleotide sequence” is a repetitive nucleic acid sequence within a stretch of DNA or RNA or throughout a genome. In some embodiments, the repetitive nucleotide sequence includes poly CA or poly TG (UG) sequences. In some embodiments, the repetitive nucleotide sequence includes repeated sequences in the Alu family of introns.As used herein, the term “replication element” is a sequence and / or motif useful for replication or that initiate transcription of the circular polyribonucleotide.As used herein, the term “stagger element” is a moiety, such as a nucleotide sequence, that induces ribosomal pausing during translation. In some embodiments, the stagger element is a nonconserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V / l)ExNPGP (SEQ ID NO: 37), where x= any amino acid. In some embodiments, the stagger element may include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.As used herein, the terms “systemic delivery” and “systemic administration” means a route of administration of pharmaceutical compositions or other substances into the circulatory system (e.g., blood or lymphoid system). Systemic administration can include oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combinations thereof. As used herein, the term “non-systemic delivery” or “non-systemic administration” can refer to any other routes of administration than systemic delivery of pharmaceutical compositions or other substances, e.g., the delivered substances do not enter the circulation systems (e.g., blood and lymphoid system) of the subject body.As used herein, the term “sequence identity” is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) 18 (proteins) and gap extension penalty = 3 (nucleotides) 12 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively or additionally, percent identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.A “signal sequence” refers to a polypeptide sequence, e.g., between 10 and 30 amino acids in length, that is present at the N-terminus of a polypeptide sequence of a nascent protein which targets the polypeptide sequence to the secretory pathway.As used herein, the term “target” refers to any entity that includes one or more epitopes. For example, a target may be a chemical moiety, a portion of a molecule, a molecule (e.g., an allergen or a toxin), a macromolecule (e.g., a polypeptide, a nucleic acid, or carbohydrate), a post-translational modification state of a macromolecule (e.g., a macromolecule that is phosphorylated, glycosylated,ATTORNEY DOCKET: 51719-011 WO2 PATENT acylated, alkylated, and the like), a higher-order macromolecular structure (e.g., a complex of two or more polypeptides), a cell (e.g., a cancer cell), a portion of a cell (e.g., a tumor antigen), a receptor on the surface of a cell, a pathogen (e.g., a virus or a portion or a virus; a bacterium or a portion of a bacterium; a fungus or a portion of a fungus; or a parasite or a portion of a parasite), or a tissue-type.As used herein, the terms “treat” and “treating,” refer to a therapeutic treatment of a disease or disorder (e.g., an infectious disease, a cancer, a toxicity, or an allergic reaction) in a subject. The effect of treatment can include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e. , not worsening) the state of the disease or disorder, and / or preventing the spread of the disease or disorder as compared to the state and / or the condition of the disease or disorder in the absence of the therapeutic treatment.As used herein, the term “termination element” is a moiety, such as a nucleic acid sequence, that terminates translation of the expression sequence in the circular or linear polyribonucleotide.As used herein, the term “total ribonucleotide molecules” means the total amount of any ribonucleotide molecules, including linear polyribonucleotide molecules, circular polyribonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, fragments thereof, and modified variations thereof, as measured by total mass of the ribonucleotide molecules.As used herein, the term “translation efficiency” is a rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed as amount of protein or peptide produced per given amount of transcript that codes for the protein or peptide, e.g., in a given period of time, e.g., in a given translation system, e.g., an in vitro translation system like rabbit reticulocyte lysate, or an in vivo translation system like a eukaryotic cell or a prokaryotic cell.As used herein, the term “translation initiation sequence” is a nucleic acid sequence that initiates translation of an expression sequence in the circular or linear polyribonucleotide.Brief Description of the DrawingsFIG. 1 is a schematic drawing showing an exemplary linear polyribonucleotide that is used to produce a circular polyribonucleotide comprising an open reading frame for expressing a plasmodium circumsporozoite immunogen.FIG. 2 is a graph showing plasmodium circumsporozoite polypeptide expression from various codon optimized polyribonucleotide constructs (Seq1 -Seq8) as compared to a mock control and control constructs expressing human growth hormone (HGH), enhanced green fluorescent protein (eGFP), and Plasmodium falciparum circumsporozoite protein (PfCSP) as measured by ELISA.FIG. 3 is a graph showing antibody titer against full-length CSP from serum samples collected on Day 42 using different circular polyribonucleotide constructs encoding R21 or protein only.FIG. 4 is a graph showing binding antibody titers against full-length CSP measured using serum collected on Day 42 using different circular polyribonucleotide constructs encoding R21 , protein with CpG adjuvant, or standard vaccine comparator (RTS,S vaccine).FIG. 5 is a graph showing parasite liver burden measured on Day 44 from mice treated with different circular polyribonucleotide constructs encoding R21 , protein with CpG adjuvant, standard vaccine comparator, or mAb317 (positive control).ATTORNEY DOCKET: 51719-011 WO2 PATENTFIG. 6 is a graph showing post-challenge survival of mice treated with different circular polyribonucleotide constructs encoding R21 , protein with CpG adjuvant, standard vaccine comparator, or mAb317.FIG. 7 is a graph showing binding antibody titers against full-length CSP measured using serum collected on Day 42 using different circular polyribonucleotide constructs encoding R21 (Seq 2 and Seq 4), Full length construct 6 (Seq 79), and standard vaccine comparator (RTS,S vaccine).FIG. 8 is a graph showing parasite liver burden measured on Day 44 from mice treated with different circular polyribonucleotide constructs encoding R21 (Seq 2, Seq 4), Full length construct 6 (Seq 79), standard vaccine comparator, mAb317, and control.FIG. 9 is a graph showing survival post-challenge with circRNA encoding R21 (Seq 2 and Seq 4), full length (Seq 79), standard vaccine comparator (RTS,S vaccine), and mAb317.FIG. 10A and 10B are graphs showing binding antibody titers against full-length CSP (FIG. 10A) or against NANP5 (FIG. 10B) peptide measured using serum samples collected from mice on Day 42.FIGS. 11 A and 11B are graphs showing isotype binding antibody titers against full-length CSP were measured using serum samples collected on Day 42. FIG. 11 A shows anti-CSP isotypes (IgG, IgM, and IgA), and FIG. 11B shows IgG subclasses (IgG 1 , lgG2b, lgG2c, and lgG3).FIG. 12 is a graph showing IFNg response in vaccine treated mice following stimulation with circRNA encoding R21 (Seq 2) as compared to CSP protein.Detailed DescriptionThis disclosure provides compositions and pharmaceutical preparations of circular or linear polyribonucleotides encoding one or more polypeptide immunogens and uses thereof. In particular, the disclosure provides circular or linear polyribonucleotides encoding the malaria causing pathogen Plasmodium falciparum circumsporozoite protein (PfCSP) (e.g., full length CSP, a full length CSP with NANP and NVDP repeat regions, an R21 polypeptide that includes portions of the NANP repeat region and C-terminus followed by the Hepatitis B surface antigen (HBsAg)). The disclosure features immunogenic compositions including circular or linear polyribonucleotides. This disclosure further features pharmaceutical compositions and preparations including one or more circular or linear polyribonucleotides encoding the plasmodium circumsporozoite polypeptide. Compositions and pharmaceutical preparations of circular or linear polyribonucleotides described herein may induce an immune response in a subject upon administration. Compositions and pharmaceutical preparations of circular or linear polyribonucleotides described herein may be used to treat or prevent a disease, disorder, or condition in a subject, such as malaria.PolynucleotidesThe disclosure features circular polyribonucleotide compositions encoding a plasmodium circumsporozoite polypeptide, uses thereof, and methods of making circular polyribonucleotides encoding a plasmodium circumsporozoite polypeptide. In some embodiments, a circular polyribonucleotide is produced from a linear polyribonucleotide (e.g., by self-splicing compatible ends of the linear polyribonucleotide). In some embodiments, a linear polyribonucleotide is transcribed from a deoxyribonucleotide template (e.g., a vector, a linearized vector, or a cDNA). Accordingly, the disclosure features deoxyribonucleotides, linear polyribonucleotides, and circular polyribonucleotides andATTORNEY DOCKET: 51719-011 WO2 PATENT compositions thereof useful in the production of circular polyribonucleotides encoding a plasmodium circumsporozoite polypeptide.Template DeoxyribonucleotidesThe present invention features a template deoxyribonucleotide for making a circular RNA as described herein. In embodiments, the template deoxyribonucleotide includes the following, operably linked in a 5’-to-3’ orientation: a first circularization element, a polyribonucleotide cargo with an ORF that encodes a plasmodium circumsporozoite polypeptide, and a second circularization element. For example, in some embodiments, the template deoxyribonucleotide includes the following, operably linked in a 5’-to- 3’ direction: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo encoding a plasmodium circumsporozoite polypeptide; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment. In embodiments, the deoxyribonucleotide includes further elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E), (F), or (G). In embodiments, any of the elements (A), (B), (C), (D), (E), (F), or (G) is separated from each other by a spacer sequence, as described herein.In embodiments, the deoxyribonucleotide is, for example, a circular DNA vector, a linearized DNA vector, or a linear DNA (e.g., a cDNA, e.g., produced from a DNA vector).In some embodiments, the deoxyribonucleotide further includes an RNA polymerase promoter operably linked to a sequence encoding a linear RNA described herein. In embodiments, the RNA polymerase promoter is heterologous to the sequence encoding the linear RNA. In some embodiments, the RNA polymerase promoter is a T7 promoter, a T6 promoter, a T4 promoter, a T3 promoter, an SP6 virus promoter, or an SP3 promoter.In some embodiments, the deoxyribonucleotide includes a multiple-cloning site (MCS).In some embodiments, the deoxyribonucleotide is used to produce circular RNA with the size range of about 100 to about 20,000 nucleotides. In some embodiments, the circular RNA is at least 100, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000 nucleotides in size. In some embodiments, the circular RNA is no more than 20,000, 15,000 10,000, 9,000, 8,000, 7,000, 6,000, 5,000 or 4,000 nucleotides in size.Linear PolyribonucleotidesThe present invention also features linear polyribonucleotides encoding a plasmodium circumsporozoite polypeptide. The linear polyribonucleotide may be used to create a circular polyribonucleotide, e.g., by ligating or splicing (e.g., self-splicing) the linear polyribonucleotide to produce the circular polyribonucleotide. In embodiments, the line polyribonucleotide includes the following, operably linked in a 5’-to-3’ orientation: a first circularization element, a polyribonucleotide cargo with an ORF that encodes a plasmodium circumsporozoite polypeptide, and a second circularization element. In embodiments, the linear polyribonucleotide includes the following, operably linked in a 5’-to-3’ orientation: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo encoding a plasmodium circumsporozoite polypeptide; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment. In embodiments, the linear polyribonucleotide includes further elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E), (F), or (G). For example, anyATTORNEY DOCKET: 51719-01 1 WO2 PATENT of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence, as described herein.In certain embodiments, provided herein is a method of generating linear RNA encoding a plasmodium circumsporozoite polypeptide by performing transcription in a cell-free system (e.g., in vitro transcription) using a deoxyribonucleotide (e.g., a vector, linearized vector, or cDNA) encoding a plasmodium circumsporozoite polypeptide provided herein as a template (e.g., a vector, linearized vector, or cDNA provided herein with an RNA polymerase promoter positioned upstream of the region that codes for the linear RNA).In embodiments, a deoxyribonucleotide template is transcribed to a produce a linear RNA containing the components described herein. Upon expression, the linear polyribonucleotide produces a splicing-compatible polyribonucleotide, which may be self-spliced in order to produce a circular polyribonucleotide.In some embodiments, the linear polyribonucleotide is from 50 to 20,000, 100 to 20,000, 200 to 20,000, 300 to 20,000 (e.g., 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600, 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 1 1 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) ribonucleotides in length. In embodiments, the linear polyribonucleotide is, e.g., at least 500, at least 1 ,000, at least 2,000, at least 3,000, at least 4,000, or at least 5,000 ribonucleotides in length.Circular PolyribonucleotidesIn some embodiments, the invention features a circular polyribonucleotide including an expression sequence encoding a plasmodium circumsporozoite polypeptide. In embodiments, the polyribonucleotide includes an IRES operably linked to an expression sequence encoding a plasmodium circumsporozoite polypeptide. The circular polyribonucleotide may include a splice junction, e.g., joining a 5’ exon fragment and a 3’ exon fragment. The circular polyribonucleotide may include any one or more of the elements described herein. In some embodiments, the circular polyribonucleotide includes any feature or any combination of features as disclosed in International Patent Publication No. WO2019 / 1 18919, which is hereby incorporated by reference in its entirety.In embodiments, the circular polynucleotide further includes a polyribonucleotide cargo. In embodiments, the polyribonucleotide cargo includes an expression (or coding) sequence, a non-coding sequence, or a combination of an expression (coding) sequence and a non-coding sequence. In embodiments, the polyribonucleotide cargo includes an expression (coding) sequence encoding a polypeptide. In embodiments, the polyribonucleotide includes an IRES operably linked to an expression sequence encoding a polypeptide. In some embodiments, the IRES is located upstream of the expression sequence. In some embodiments, the IRES is located downstream of the expression sequence. In some embodiments, the circular polyribonucleotide further includes a spacer region between the IRES and the 3’ exon fragment or the 5’ exon fragment. The spacer region may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length ribonucleotides in length. The spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides. In some embodiments, the spacer region includes a polyA sequence. In some embodiments, the spacer region includes a polyA-C sequence. In some embodiments, the spacer region includes a polyA-G sequence. In some embodiments, the spacer region includes a polyA-T sequence. InATTORNEY DOCKET: 51719-011 WO2 PATENT some embodiments, the spacer region includes a random sequence. In some embodiments, the first annealing region and the second annealing region are joined, thereby forming a circular polyribonucleotide.In some embodiments, the circular RNA is produced by a deoxyribonucleotide template or a linear RNA described herein. In some embodiments, the circular RNA is produced by any of the methods described herein.In some embodiments, the circular polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.In some embodiments, the circular polyribonucleotide is between 500 nucleotides and 20,000 nucleotides, between 1 ,000 and 20,000 nucleotides, between 2,000 and 20,000 nucleotides, or between 5,000 and 20,000 nucleotides. In some embodiments, the circular polyribonucleotide is between 500 nucleotides and 10,000 nucleotides, between 1 ,000 and 10,000 nucleotides, between 2,000 and 10,000 nucleotides, or between 5,000 and 10,000 nucleotides.As a result of its circularization, the circular polyribonucleotide may include certain characteristics that distinguish it from linear RNA. For example, the circular polyribonucleotide is less susceptible to degradation by exonuclease as compared to linear RNA. As such, the circular polyribonucleotide is more stable than a linear RNA, especially when incubated in the presence of an exonuclease. The increased stability of the circular polyribonucleotide compared with linear RNA makes circular polyribonucleotide more useful as a cell transforming reagent to produce polypeptides and can be stored more easily and for longer than linear RNA. The stability of the circular polyribonucleotide treated with exonuclease can be tested using methods standard in art which determine whether RNA degradation has occurred (e.g., by gel electrophoresis). Moreover, unlike linear RNA, the circular polyribonucleotide is less susceptible to dephosphorylation when the circular polyribonucleotide is incubated with phosphatase, such as calf intestine phosphatase.The circular polyribonucleotides described herein and compositions or pharmaceutical compositions thereof may be used in therapeutic and veterinary methods of dosing to produce a level of circular polyribonucleotide, a level of binding to a target, or a level of protein in a plurality of cells after providing the plurality with at least two doses of circular polyribonucleotide. In some embodiments, the circular polyribonucleotide is capable of replicating or replicates in a cell from an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In someATTORNEY DOCKET: 51719-011 WO2 PATENT embodiments, the invention includes a cell that includes the circular polyribonucleotide described herein, wherein the cell is a cell from an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc.), a human cell, a cultured cell, a primary cell or a cell line, a stem cell, a progenitor cell, a differentiated cell, a germ cell, a cancer cell (e.g., tumorigenic, metastatic), a non-tumorigenic cell (normal cells), a fetal cell, an embryonic cell, an adult cell, a mitotic cell, a non-mitotic cell, or any combination thereof. In some embodiments, the cell is modified to include the circular polyribonucleotide.In some embodiments, the circular polyribonucleotide includes sequences for expression products. In some embodiments, the circular polyribonucleotide includes a binding site for binding to a target. In some embodiments, the circular polyribonucleotide is provided to a plurality of cells via any a dosing regimen described herein. In some embodiments, the circular polyribonucleotide as described herein induces a response or response level in a subject. In some embodiments, the expression products encoded by the sequences included in the circular polyribonucleotide are expressed in one or more of cells in the plurality of cells.In some embodiments, the circular polyribonucleotide has a half-life of at least that of a linear counterpart, e.g., linear expression sequence, or linear polyribonucleotide. In some embodiments, the circular polyribonucleotide has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell for at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell for from about 1 hour to about 60 days, e.g., about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell post division. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a dividing cell for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a dividing cell of from about 10 minutes to about 60 days, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 60 days.In some embodiments, the circular polyribonucleotide modulates a cellular function, e.g., transiently, or long term. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3ATTORNEY DOCKET: 51719-011 WO2 PATENT hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for from about 1 hour to about 60 days, e.g., from about 1 hour to about 30 days, e.g., for at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 60 days.Elements of PolynucleotidesThe polynucleotides (e.g., circular polyribonucleotides) described herein may include any one or more of the elements described herein and an expression sequence encoding a plasmodium circumsporozoite polypeptide.Plasmodium Circumsporozoite PolypeptidesThe disclosure provides polynucleotides (e.g., circular polyribonucleotides) that encode at least one expression sequence encoding a plasmodium circumsporozoite polypeptide.The polynucleotides described herein include an ORF that encodes a plasmodium circumsporozoite polypeptide.In some embodiments, the plasmodium circumsporozoite polypeptide is based on the full length Plasmodium falciparum circumsporozite polypeptide (PfCSP) (GenBank: AAN87606.1 ) MMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNS RSLGENDDGNNNNGDNGREGKDEDKRDGNNEDNEKLRKPKHKKLKQPGDGNPDPNANPNVDPNANP NVDPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN PNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNA NPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANNAVKNNNNEEPSDKHIEKYLK KIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYENDIEKKICKMEKCSSVFNVVNSSIGLIMVLSF LFLN (SEQ ID NO: 65).In some embodiments, the plasmodium circumsporozoite polypeptide has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 65.In some embodiments, the plasmodium circumsporozoite polypeptide has the sequence of SEQ ID NO: 65.In some embodiments, the plasmodium circumsporozoite polypeptide is a fragment that has at least 100 amino acids (e.g., at least 150 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids) of the sequence of SEQ ID NO: 65.In some embodiments, the plasmodium circumsporozoite polyeptide is the R21 polypeptide, which encompasses portions of the NANP repeat region and C-terminus of full length PfCSP followed by the Hepatitis B surface antigen (HBsAg).The R21 polypeptide is based on the amino acid sequence of SEQ ID NO: 9.MDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANP NANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSL STEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSVPVTNMENITSGFLGPLLVLQA GFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLATTORNEY DOCKET: 51719-011 WO2PATENTCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMEPSCCCTKPTDGNCTCIPIPSSWAFAK YLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI (SEQ ID NO: 9).In some embodiments, the plasmodium circumsporozoite polypeptide has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 9.In some embodiments, the plasmodium circumsporozoite polypeptide has the sequence of SEQ ID NO: 9.In some embodiments, the plasmodium circumsporozoite polypeptide is a fragment that has at least 100 amino acids (e.g., at least 150 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids) of the sequence of SEQ ID NO: 9.In some embodiments, the ORF includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the any one of SEQ ID NOs: 1 -8.In some embodiments, the ORF includes the sequence of any one of SEQ ID NOs: 1 -8.In some embodiments, the ORF includes the sequence of SEQ ID NO: 1 .In some embodiments, the ORF includes the sequence of SEQ ID NO: 2.In some embodiments, the ORF includes the sequence of SEQ ID NO: 3.In some embodiments, the ORF includes the sequence of SEQ ID NO: 4.In some embodiments, the ORF includes the sequence of SEQ ID NO: 5.In some embodiments, the ORF includes the sequence of SEQ ID NO: 6.In some embodiments, the ORF includes the sequence of SEQ ID NO: 7.In some embodiments, the ORF includes the sequence of SEQ ID NO: 8.In some embodiments, the plasmodium circumsporozoite polyeptide is a full length PfCSP polypeptide.In some embodiments, the plasmodium circumsporozoite polyeptide is a full length PfCSP polypeptide with 38 NANP repeats and 4 NVDP repeats.In some embodiments, the full length PfCSP polypeptide is based on the amino acid sequence of SEQ ID NO: 95: LFQEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGENDDGNNEDNEKL RKPKHKKLKQPADGNPDPNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNANPNANPN ANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPNANPNANPNANPNANPNANP NANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMP NDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPK DELDYANDIEKKICKMEKCSSVFNVVN (SEQ ID NO: 95)In some embodiments, the plasmodium circumsporozoite polypeptide is a fragment that has at least 100 amino acids (e.g., at least 100, 150 amino acids, 200 amino acids, 250, 300, or 350 amino acids) of the sequence of SEQ ID NO: 95.In some embodiments, the ORF includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 74-79.In some embodiments, the ORF includes the sequence of any one of SEQ ID NOs: 74-79.In some embodiments, the ORF includes the sequence of SEQ ID NO: 74.In some embodiments, the ORF includes the sequence of SEQ ID NO: 75.In some embodiments, the ORF includes the sequence of SEQ ID NO: 76.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, the ORF includes the sequence of SEQ ID NO: 77.In some embodiments, the ORF includes the sequence of SEQ ID NO: 78.In some embodiments, the ORF includes the sequence of SEQ ID NO: 79.Polyribonucleotide CargoA polyribonucleotide cargo described herein includes any sequence including at least one polyribonucleotide. In some embodiments, the polyribonucleotide cargo includes an expression sequence, a non-coding sequence, or an expression sequence and a non-coding sequence. In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a plasmodium circumsporozoite polypeptide. In some embodiments, the polyribonucleotide cargo includes an IRES operably linked to an expression sequence encoding a plasmodium circumsporozoite polypeptide. In some embodiments, the polyribonucleotide cargo includes an expression sequence that encodes a plasmodium circumsporozoite polypeptide that has a biological effect on a subject.A polyribonucleotide cargo may, for example, include at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the polyribonucleotide cargo includes from 1 -20,000 nucleotides, 1 -10,000 nucleotides, 1 -5,000 nucleotides, 100-20,000 nucleotide, 100-10,000 nucleotides, 100-5,000 nucleotides, 500-20,000 nucleotides, 500-10,000 nucleotides, 500- 5,000 nucleotides, 1 ,000-20,000 nucleotides, 1 ,000-10,000 nucleotides, or 1 ,000-5,000 nucleotides.In embodiments, the polyribonucleotide cargo includes one or multiple expression (or coding) sequences, wherein each expression (or coding) sequence encodes a polypeptide (e.g., a plasmodium circumsporozoite polypeptide). In embodiments, the polyribonucleotide cargo includes one or multiple noncoding sequences. In embodiments, the polyribonucleotide cargo consists entirely of non-coding sequence(s). In embodiments, the polyribonucleotide cargo includes a combination of expression (or coding) and noncoding sequences.In some embodiments, the polyribonucleotide includes any feature, or any combination of features as disclosed in International Patent Publication No. WQ2019 / 118919, which is hereby incorporated by reference in its entirety.Polypeptide Expression SequencesIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the circular polyribonucleotide) includes one or more expression (or coding) sequences, wherein each expression sequence encodes a plasmodium circumsporozoite polypeptide. In some embodiments, the circular polyribonucleotide includes two, three, four, five, six, seven, eight, nine, ten or more expression (or coding) sequences.ATTORNEY DOCKET: 51719-011 WO2 PATENTEach encoded polypeptide may be linear or branched. In various embodiments, the polypeptide has a length from about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1 ,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, the polypeptide has a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1 ,500 amino acids, less than about 1 ,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less may be useful.Polypeptides included herein may include naturally occurring polypeptides or non-naturally occurring polypeptides. In some embodiments, the polypeptide is or includes a functional fragment or variant of a reference polypeptide (e.g., a biologically active fragment or variant of a plasmodium circumsporozoite polypeptide). For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a polypeptide described herein or a naturally occurring polypeptide. In some instances, the polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to SEQ ID NO: 9.In embodiments, polypeptides include multiple polypeptides, e.g., multiple copies of one polypeptide sequence, or multiple different polypeptide sequences. In embodiments, multiple polypeptides are connected by linker amino acids or spacer amino acids.In embodiments, the polynucleotide cargo includes a sequence encoding a signal peptide. Many signal peptide sequences have been described, for example, the Tat (Twin-arginine translocation) signal sequence is typically an N-terminal peptide sequence containing a consensus SRRxFLK (SEQ ID NO: 36) “twin-arginine” motif, which serves to translocate a folded protein containing such a Tat signal peptide across a lipid bilayer. See also, e.g., the Signal Peptide Database publicly available at www[dot]signalpeptide[dot]de. Signal peptides are also useful for directing a protein to specific organelles; see, e.g., the experimentally determined and computationally predicted signal peptides disclosed in the Spdb signal peptide database, publicly available at proline. bic.nus.edu.sg / spdb.In some embodiments, the expression (or coding) sequence includes a poly-A sequence (e.g., at the 3’ end of an expression sequence). In some embodiments, the length of a poly-A sequence is greater than 10 nucleotides in length. In one embodiment, the poly-A sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600, 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-ATTORNEY DOCKET: 51719-011 WO2 PATENTA sequence is designed according to the descriptions of the poly-A sequence in

[0202] -

[0204] of International Patent Publication No. WO2019 / 118919A1 , which is incorporated herein by reference in its entirety. In some embodiments, the expression sequence lacks a poly-A sequence (e.g., at the 3’ end of an expression sequence).In some embodiments, a circular polyribonucleotide includes a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide lacking a polyA or having modified polyA improves one or more functional characteristics, e.g., immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response), half-life, and / or expression efficiency.Internal Ribosomal Entry SitesIn some embodiments, a circular polyribonucleotide described herein includes one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences. In embodiments, the IRES is located between a heterologous promoter and the 5’ end of a coding sequence.A suitable IRES element to include in a polyribonucleotide includes an RNA sequence capable of engaging a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.In some embodiments, the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, and Drosophila. Such viral DNA may be derived from, but is not limited to, picomavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.In some embodiments, the IRES sequence is an IRES sequence of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1 , Rhopalosiphum padi virus, Reticuloendotheliosis virus, fuman poliovirus 1 , Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus- 1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1 , Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus (AEV), Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1 , Human AML1 / RUNX1 , Drosophila antennapedia, Human AQP4, Human AT1 R, Human BAG-I, Human BCL2, Human BiP, Human c-IAPI , Human c-myc, Human elF4G, Mouse NDST4L, Human LEF1 , Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-I, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Salivirus, Cosavirus, Parechovirus, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1 , Human c-src, Human FGF-I, Simian picomavirus, Turnip crinkle virus, Aichivirus, Crohivirus,ATTORNEY DOCKET: 51719-011 WO2 PATENTEchovirus 11 , an aptamer to elF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2). In yet another embodiment, the IRES is an IRES sequence of Coxsackievirus B3 (CVB3). In a further embodiment, the IRES is an IRES sequence of Encephalomyocarditis virus. In a further embodiment, the IRES is an IRES sequence of Theiler's encephalomyelitis virus.The IRES sequence may have the sequence of wild-type CVB3 IRES sequence having the nucleic acid sequence of: TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA CTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG GCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAA TTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCC TTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACA GCAAA (SEQ ID NO: 22).The IRES sequence may be a CVB3 IRES sequence having the nucleic acid sequence of : TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA CTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG GCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAA TTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCC TTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACA GCAA (SEQ ID NO: 12).In some embodiments, the CVB3 IRES has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of CCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGAT CAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTC GTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCA CTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTG CGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTA TTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTC AAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTG TTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATATTORNEY DOCKET: 51719-011 WO2 PATENTTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAG AGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAA (SEQ ID NO: 96).In some embodiments, the CVB3 IRES has the sequence of CCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGAT CAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTC GTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCA CTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTG CGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTA TTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTC AAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTG TTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGAT TGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAG AGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAA (SEQ ID NO: 96).The IRES sequence may have a modified sequence in comparison to the wild-type IRES sequence. In some embodiments, when the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified such that it is a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence wherein the terminal adenosine residue is modified to cytosine residue. In some embodiments, the modified CVB3 IRES may have the nucleic acid sequence of: TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA CTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG GCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAA TTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCC TTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACA GCAAC (SEQ ID NO: 23).In some embodiments, the IRES sequence is an encephalomyocarditis virus (EMCV) IRES. In some embodiments, the ECMV IRES may have the nucleic acid sequence of: ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCAT ATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGG GGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGG AAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCG ACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGT GCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGG GCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTT TACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT GAAAAACACGATGATAATA (SEQ ID NO: 24).ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, the IRES sequence is an Enterovirus 71 (EV71 ) IRES. In some embodiments, the IRES sequence is a wild-type EV71 sequence having the nucleic acid sequence of: TTAAAACAGCTGTGGGTTGTCACCCACCCACAGGGTCCACTGGGCGCTAGTACACTGGTATCTCGG TACCTTTGTACGCCTGTTTTATACCCCCTCCCTGATTTGCAACTTAGAAGCAACGCAAACCAGATCAA TAGTAGGTGTGACATACCAGTCGCATCTTGATCAAGCACTTCTGTATCCCCGGACCGAGTATCAATA GACTGTGCACACGGTTGAAGGAGAAAACGTCCGTTACCCGGCTAACTACTTCGAGAAGCCTAGTAAC GCCATTGAAGTTGCAGAGTGTTTCGCTCAGCACTCCCCCCGTGTAGATCAGGTCGATGAGTCACCG CATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCTATGGGGTAACCCATAGGA CGCTCTAATACGGACATGGCGTGAAGAGTCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGC GGCTAATCCTAACTGCGGAGCACATACCCTTAATCCAAAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTGTATTGGCTGCTTATGGTGACAA TTAAAGAATTGTTACCATATAGCTATTGGATTGGCCATCCAGTGTCAAACAGAGCTATTGTATATCTCT TTGTTGGATTCACACCTCTCACTCTTGAAACGTTACACACCCTCAATTACATTATACTGCTGAACACG AAGCG (SEQ ID NO: 25).In some embodiments, the terminal guanosine residue of the EV71 IRES sequence is modified to a cytosine residue. In some embodiments, the modified EV71 IRES may have the nucleic acid sequence of: TTAAAACAGCTGTGGGTTGTCACCCACCCACAGGGTCCACTGGGCGCTAGTACACTGGTATCTCGG TACCTTTGTACGCCTGTTTTATACCCCCTCCCTGATTTGCAACTTAGAAGCAACGCAAACCAGATCAA TAGTAGGTGTGACATACCAGTCGCATCTTGATCAAGCACTTCTGTATCCCCGGACCGAGTATCAATA GACTGTGCACACGGTTGAAGGAGAAAACGTCCGTTACCCGGCTAACTACTTCGAGAAGCCTAGTAAC GCCATTGAAGTTGCAGAGTGTTTCGCTCAGCACTCCCCCCGTGTAGATCAGGTCGATGAGTCACCG CATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCTATGGGGTAACCCATAGGA CGCTCTAATACGGACATGGCGTGAAGAGTCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGC GGCTAATCCTAACTGCGGAGCACATACCCTTAATCCAAAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTGTATTGGCTGCTTATGGTGACAA TTAAAGAATTGTTACCATATAGCTATTGGATTGGCCATCCAGTGTCAAACAGAGCTATTGTATATCTCT TTGTTGGATTCACACCTCTCACTCTTGAAACGTTACACACCCTCAATTACATTATACTGCTGAACACG AAGCC (SEQ ID NO: 26).In some embodiments, the polyribonucleotide includes at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the IRES flanks both sides of at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, leading to separation of the resulting peptide(s) and or polypeptide(s). For example, a polyribonucleotide described herein may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.In some embodiments, a polyribonucleotide described herein includes an IRES (e.g., an IRES operably linked to a coding region). For example, the polyribonucleotide may include any IRES as described in Chen et al. MOL. CELL 81 (20):4300-18, 2021 ; Jopling et al. ONCOGENE 20:2664-70, 2001 ; Baranick et al. PNAS 105(12):4733-38, 2008; Lang et al. MOLECULAR BIOLOGY OF THE CELL 13(5):1792- 1801 , 2002; Dorokhov et al. PNAS 99(8):5301 -06, 2002; Wang et al. NUCLEIC ACIDS RESEARCH 33(7):2248-58, 2005; Petz et al. NUCLEIC ACIDS RESEARCH 35(8):2473-82, 2007; Chen et al. SCIENCEATTORNEY DOCKET: 51719-011 WO2 PATENT268:415-417, 1995; Fan et al. NATURE COMMUNICATION 13(1 ):3751 -3765, 2022, and International Publication No. WO2021 / 263124, each of which is hereby incorporated by reference in their entirety.Signal SequencesIn some embodiments, a plasmodium circumsporozoite polypeptide expressed from a circular polyribonucleotide disclosed herein includes a secreted protein, for example, a protein that naturally includes a signal sequence, or one that does not usually encode a signal sequence but is modified to contain one. In some embodiments, the plasmodium circumsporozoite polypeptide encoded by the circular polyribonucleotide includes a secretion signal. For example, the secretion signal may be the naturally encoded secretion signal for a secreted protein. In another example, the secretion signal may be a modified secretion signal for a secreted protein. In other embodiments, the plasmodium circumsporozoite polypeptide encoded by the circular polyribonucleotide does not include a secretion signal.In some embodiments, the signal sequence is selected from SecSP38 (MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO: 27); SecD4 (MWWLLLLLLLLWPMVWA; SEQ ID NO: 28), gLuc (MGVKVLFALICIAVAEAK; SEQ ID NO: 29); INHC1 (MASRLTLLTLLLLLLAG DRASS; SEQ ID NO: 30); Epo (MGVHECPAWLWLLLSLLSLPLGLPVLG; SEQ ID NO: 31 ); and IL-2 (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 32).In some embodiments, a circular polyribonucleotide encodes multiple copies of the same plasmodium circumsporozoite polypeptide (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more). In some embodiments, at least one copy of the plasmodium circumsporozoite polypeptide includes a signal sequence and at least one copy of the plasmodium circumsporozoite polypeptide does not include a signal sequence. In some embodiments, a circular polyribonucleotide encodes plurality of a plasmodium circumsporozoite polypeptides (e.g., a plurality of different plasmodium circumsporozoite polypeptides or a plurality of plasmodium circumsporozoite polypeptides having less than 100% sequence identity), where at least one of the plurality of plasmodium circumsporozoite polypeptides includes a signal sequence, and at least one copy of the plurality of plasmodium circumsporozoite polypeptides does not include a signal sequence.In some embodiments, the signal sequence is a wild-type signal sequence that is present on the N-terminus of the corresponding plasmodium circumsporozoite polypeptide. In some embodiments, the signal sequence is heterologous to the plasmodium circumsporozoite polypeptide, e.g., is not present when the wild-type plasmodium circumsporozoite polypeptide is expressed endogenously. A polyribonucleotide sequence encoding a plasmodium circumsporozoite polypeptide may be modified to remove the nucleotide sequence encoding a wild-type signal sequence and / or add a sequence encoding a heterologous signal sequence.A polypeptide encoded by a polyribonucleotide (e.g., a plasmodium circumsporozoite polypeptide) may include a signal sequence that directs the plasmodium circumsporozoite polypeptide to the secretory pathway. In some embodiments, the signal sequence may direct the plasmodium circumsporozoite polypeptide to reside in certain organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes). In some embodiments, the signal sequence directs the plasmodium circumsporozoite polypeptide to be secreted from the cell. For secreted proteins, the signal sequence may be cleaved after secretion, resulting in a mature protein. In other embodiments, the signal sequenceATTORNEY DOCKET: 51719-011 WO2 PATENT may become embedded in the membrane of the cell or certain organelles, creating a transmembrane segment that anchors the protein to the membrane of the cell, endoplasmic reticulum, or Golgi apparatus. In certain embodiments, the signal sequence of a transmembrane protein is a short sequence at the N- terminal of the polypeptide. In other embodiments, the first transmembrane domain acts as the first signal sequence, which targets the protein to the membrane.In some embodiments, the secretion signal is a human interleukin-2 (IL-2) secretion signal. In some embodiments, the IL-2 secretion signal has an amino acid sequence of at least 90% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 32). In some embodiments, the IL-2 secretion signal has an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 32. In some embodiments, the IL-2 secretion signal has an amino acid sequence of at least 99% sequence identity to SEQ ID NO: 32. In some embodiments, the IL-2 secretion signal has an amino acid sequence of 100% sequence identity to SEQ ID NO: 32.In some embodiments, the secretion signal is an IgK signal peptide. In some embodiments, the IgK secretion signal has an amino acid sequence of at least 90% sequence identify to DMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 132). In some embodiments, the IgK secretion signal has an amino acid sequence of at least 95% sequence identify to DMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 132). In some embodiments, the IgK secretion signal has an amino acid sequence of at least 99% sequence identify to DMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 132). In some embodiments, the IgK secretion signal has an amino acid sequence of 100% sequence identify to DMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 132).In some embodiments, the secretion signal is Gaussia luciferase secretion signal. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of at least 90% sequence identity of MGVKVLFALICIAVAEAK (SEQ ID NO: 33). In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 33. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 33. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 33.In some embodiments, the secretion signal is an EPO (e.g., a human EPO) secretion signal. In some embodiments, the EPO secretion signal has an amino acid sequence of at least 90% sequence identity of MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 34). In some embodiments, the EPO secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 34. In some embodiments, the EPO secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 34. In some embodiments, the EPO secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 34.In some embodiments, the secretion signal is a wildtype SARS-CoV-2 secretion signal. In some embodiments, the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of at least 90% sequence identity of MFVFLVLLPLVSS (SEQ ID NO: 35). In some embodiments, the wildtype SARS- CoV-2 secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 35. In some embodiments, the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 35. In some embodiments, the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 35.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, a plasmodium circumsporozoite polypeptide encoded by a polyribonucleotide includes either a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.Regulatory ElementsIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes one or more regulatory elements. In some embodiments, the polyribonucleotide includes a regulatory element, e.g., a sequence that modifies expression of an expression sequence within the polyribonucleotide.A regulatory element may include a sequence that is located adjacent to an expression sequence that encodes an expression product. A regulatory element may be linked operatively to the adjacent sequence. A regulatory element may increase an amount of product expressed as compared to an amount of the expressed product when no regulatory element exists. In addition, one regulatory element can increase an amount of products expressed for multiple expression sequences attached in tandem. Hence, one regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements are well-known to persons of ordinary skill in the art.In some embodiments, the regulatory element is a translation modulator. A translation modulator can modulate translation of the expression sequence in the polyribonucleotide. A translation modulator can be a translation enhancer or suppressor. In some embodiments, the polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the polyribonucleotide includes a translation modulator adjacent each expression sequence. In some embodiments, the translation modulator is present on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s).In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.Further examples of regulatory elements are described, e.g., in paragraphs

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[0161] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.Cleavage DomainsA circular polyribonucleotide of the disclosure can include a cleavage domain (e.g., a stagger element or a cleavage sequence).The term “stagger element” refers to a moiety, such as a nucleotide sequence, that induces ribosomal pausing during translation. In some embodiments, the stagger element is a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence - D(V / I)EXNPGP, where x= any amino acid (SEQ ID NO: 37). In some embodiments, the stagger element may include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.In some embodiments, the circular polyribonucleotide includes at least one stagger element adjacent to an expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s). In some embodiments, the stagger element is a portionATTORNEY DOCKET: 51719-011 WO2 PATENT of the one or more expression sequences. In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and each of the one or more expression sequences is separated from a succeeding expression sequence by a stagger element on the circular polyribonucleotide. In some embodiments, the stagger element prevents generation of a single polypeptide (a) from two rounds of translation of a single expression sequence or (b) from one or more rounds of translation of two or more expression sequences. In some embodiments, the stagger element is a sequence separate from the one or more expression sequences. In some embodiments, the stagger element includes a portion of an expression sequence of the one or more expression sequences.In some embodiments, the circular polyribonucleotide includes a stagger element. To avoid production of a continuous expression product, e.g., peptide or polypeptide, while maintaining rolling circle translation, a stagger element may be included to induce ribosomal pausing during translation. In some embodiments, the stagger element is at 3’ end of at least one of the one or more expression sequences. The stagger element can be configured to stall a ribosome during rolling circle translation of the circular polyribonucleotide. The stagger element may include, but is not limited to a 2A-like, or CHYSEL (SEQ ID NO: 38) (cis-acting hydrolase element) sequence. In some embodiments, the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP (SEQ ID NO: 39), where Xi is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid. In some embodiments, this sequence includes a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V / I)EXNPGP (SEQ ID NO: 40), where x= any amino acid. Some nonlimiting examples of stagger elements includes GDVESNPGP (SEQ ID NO: 41 ), GDIEENPGP (SEQ ID NO: 42), VEPNPGP (SEQ ID NO: 43), IETNPGP (SEQ ID NO: 44), GDIESNPGP (SEQ ID NO: 45), GDVELNPGP (SEQ ID NO: 46), GDIETNPGP (SEQ ID NO: 47), GDVENPGP (SEQ ID NO: 48), GDVEENPGP (SEQ ID NO: 49), GDVEQNPGP (SEQ ID NO: 50), IESNPGP (SEQ ID NO: 51 ), GDIELNPGP (SEQ ID NO: 52), HDIETNPGP (SEQ ID NO: 53), HDVETNPGP (SEQ ID NO: 54), HDVEMNPGP (SEQ ID NO: 55), GDMESNPGP (SEQ ID NO: 56), GDVETNPGP (SEQ ID NO: 57), GDIEQNPGP (SEQ ID NO: 58), and DSEFNPGP (SEQ ID NO: 59).In some embodiments, the stagger element described herein cleaves an expression product, such as between G and P of the consensus sequence described herein. As one non-limiting example, the circular polyribonucleotide includes at least one stagger element to cleave the expression product. In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element after each expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element is present on one or both sides of each expression sequence, leading to translation of individual peptide(s) and or polypeptide(s) from each expression sequence.In some embodiments, a stagger element includes one or more modified nucleotides or unnatural nucleotides that induce ribosomal pausing during translation. Unnatural nucleotides may include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Examples such as these are distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone), and any combination thereof that can induceATTORNEY DOCKET: 51719-011 WO2 PATENT ribosomal pausing during translation. Some of the exemplary modifications provided herein are described elsewhere herein.In some embodiments, the stagger element is present in the circular polyribonucleotide in other forms. For example, in some exemplary circular polyribonucleotides, a stagger element includes a termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a first translation initiation sequence of an expression succeeding the first expression sequence. In some examples, the first stagger element of the first expression sequence is upstream of (5’ to) a first translation initiation sequence of the expression succeeding the first expression sequence in the circular polyribonucleotide. In some cases, the first expression sequence and the expression sequence succeeding the first expression sequence are two separate expression sequences in the circular polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence can enable continuous translation of the first expression sequence and its succeeding expression sequence.In some embodiments, the first stagger element includes a termination element and separates an expression product of the first expression sequence from an expression product of its succeeding expression sequences, thereby creating discrete expression products. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the succeeding sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element of a second expression sequence that is upstream of a second translation initiation sequence of an expression sequence succeeding the second expression sequence is not continuously translated. In some cases, there is only one expression sequence in the circular polyribonucleotide, and the first expression sequence and its succeeding expression sequence are the same expression sequence. In some exemplary circular polyribonucleotides, a stagger element includes a first termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence. In some such examples, the first stagger element is upstream of (5’ to) a first translation initiation sequence of the first expression sequence in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence enables continuous translation of the first expression sequence and any succeeding expression sequences.In some embodiments, the first stagger element separates one round expression product of the first expression sequence from the next round expression product of the first expression sequences, thereby creating discrete expression products. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the first expression sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular polyribonucleotide is not continuously translated. In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x greater in the corresponding circular polyribonucleotide than a distance between the first stagger element and the first translation initiation in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15ATTORNEY DOCKET: 51719-011 WO2 PATENT nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater. In some embodiments, the distance between the second stagger element and the second translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance between the first stagger element and the first translation initiation. In some embodiments, the circular polyribonucleotide includes more than one expression sequence.Examples of stagger elements are described in paragraphs

[0172] -

[0175] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, a plurality of a plasmodium circumsporozoite polypeptides encoded by a circular ribonucleotide may be separated by an IRES between each plasmodium circumsporozoite polypeptide (e.g., each plasmodium circumsporozoite polypeptide is operably linked to a separate IRES). For example, a circular polyribonucleotide may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence. The IRES may be the same IRES between all plasmodium circumsporozoite polypeptides. The IRES may be different between different plasmodium circumsporozoite polypeptides.In some embodiments, the plurality of a plasmodium circumsporozoite polypeptides may be separated by a 2A self-cleaving peptide. For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first plasmodium circumsporozoite polypeptide, a 2A, and a second plasmodium circumsporozoite polypeptide.In some embodiments, the plurality of a plasmodium circumsporozoite polypeptides may be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first plasmodium circumsporozoite polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second plasmodium circumsporozoite polypeptide.In some embodiments, the plurality of a plasmodium circumsporozoite polypeptides may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first plasmodium circumsporozoite polypeptide, a 2A, a protease cleavage site (e.g., a furin cleavage site), and a second plasmodium circumsporozoite polypeptide. A circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first plasmodium circumsporozoite polypeptide, a protease cleavage site (e.g., a furin cleavage site), a 2A, and a second plasmodium circumsporozoite polypeptide. A tandem 2A and furin cleavage site may be referred to as a furin-2A (which includes furin-2A or 2A-furin, arranged in either orientation).Furthermore, the plurality of a plasmodium circumsporozoite polypeptides encoded by the circular ribonucleotide may be separated by both IRES and 2A sequences. For example, an IRES may be between one plasmodium circumsporozoite polypeptide and a second plasmodium circumsporozoite polypeptide while a 2A peptide may be between the second plasmodium circumsporozoite polypeptide and the third plasmodium circumsporozoite polypeptide. The selection of a particular IRES or 2A selfcleaving peptide may be used to control the expression level of a plasmodium circumsporozoite polypeptide under control of the IRES or 2A sequence. For example, depending on the IRES and or 2A peptide selected, expression on the polypeptide may be higher or lower.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, a circular polyribonucleotide includes at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to an expression sequence. In some embodiments, the cleavage sequence is between two expression sequences. In some embodiments, cleavage sequence is included in an expression sequence. In some embodiments, the circular polyribonucleotide includes between 2 and 10 cleavage sequences. In some embodiments, the circular polyribonucleotide includes between 2 and 5 cleavage sequences. In some embodiments, the multiple cleavage sequences are between multiple expression sequences; for example, a circular polyribonucleotide may include three expression sequences two cleavage sequences such that there is a cleavage sequence in between each expression sequence. In some embodiments, the circular polyribonucleotide includes a cleavage sequence, such as in an immolating circRNA or cleavable circRNA or self-cleaving circRNA. In some embodiments, the circular polyribonucleotide includes two or more cleavage sequences, leading to separation of the circular polyribonucleotide into multiple products, e.g., miRNAs, linear RNAs, smaller circular polyribonucleotide, etc.In some embodiments, a cleavage sequence includes a ribozyme RNA sequence. A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNA, but they have also been found to catalyze the aminotransferase activity of the ribosome. Catalytic RNA can be “evolved” by in vitro methods. Similar to riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In some embodiments, a catalytic RNA or ribozyme can be placed within a larger non-coding RNA such that the ribozyme is present at many copies within the cell for the purposes of chemical transformation of a molecule from a bulk volume. In some embodiments, aptamers and ribozymes can both be encoded in the same non-coding RNA.In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, a polyribonucleotide may encode two or more plasmodium circumsporozoite polypeptides, e.g., where the two or more plasmodium circumsporozoite polypeptides are encoded by a single open-reading frame (ORF). For example, two or more plasmodium circumsporozoite polypeptides may be encoded by a single open-reading frame, the expression of which is controlled by an IRES. In some embodiments, the ORF further encodes a polypeptide linker, e.g., such that the expression product of the ORF encodes two or more plasmodium circumsporozoite polypeptides each separated by a sequence encoding a polypeptide linker (e.g., a linker of 5-200, 5 to 100, 5 to 50, 5 to 20, 50 to 100, or 50 to 200 amino acids). The polypeptide linker may include a cleavage site, for example, a cleavage site recognized and cleaved by a protease (e.g., an endogenous protease in a subject following administration of the polyribonucleotide to that subject). In such embodiments, a single expression product including the amino acid sequence of two or more plasmodium circumsporozoite polypeptides is cleaved upon expression, such that the two or more plasmodium circumsporozoite polypeptides are separated following expression. Exemplary protease cleavage sites are known to those of skill in the art, for example, amino acid sequences that act as protease cleavage sites recognized by a metalloproteinase (e.g., a matrix metalloproteinase (MMP), such as any one or more of MMPs 1 -28), a disintegrin and metalloproteinase (ADAM, such as any one or more of ADAMs 2, 7-12, 15, 17-23, 28-30 and 33), a serine protease (e.g., furin), urokinase-type plasminogen activator, matriptase, a cysteine protease, an aspartic protease, or aATTORNEY DOCKET: 51719-011 WO2 PATENT cathepsin protease. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is matriptase.In some embodiments, a circular polyribonucleotide described herein is an immolating circular polyribonucleotide, a cleavable circular polyribonucleotide, or a self-cleaving circular polyribonucleotide. A circular polyribonucleotide can deliver cellular components including, for example, RNA, IncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some embodiments, a circular polyribonucleotide includes miRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites; (iii) degradable linkers; (iv) chemical linkers; and / or (v) spacer sequences. In some embodiments, circRNA includes siRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites (e.g., ADAR); (iii) degradable linkers (e.g., glycerol); (iv) chemical linkers; and / or (v) spacer sequences. Nonlimiting examples of self-cleavable elements include hammerhead, splicing element, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes.Translation Initiation SequencesIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes at least one translation initiation sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence operably linked to an expression sequence.In some embodiments, the polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide includes the translation initiation sequence, e.g., Kozak sequence, adjacent to an expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, the translation initiation sequence, e.g., Kozak sequence, is present on one or both sides of each expression sequence, leading to separation of the expression products. In some embodiments, the polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] -

[0165] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.The polyribonucleotide may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon.In some embodiments, the polyribonucleotide may initiate at a codon which is not the first start codon, e.g., AUG. Translation of the polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG. In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the polyribonucleotide may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiationATTORNEY DOCKET: 51719-011 WO2 PATENT sequence, CTG / CUG. As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiation sequence, GTG / GUG. As another non-limiting example, the polyribonucleotide may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC, CAG, CTG.Termination ElementsIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination element.In some embodiments, the polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product.In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the circular polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product, e.g., peptides or polypeptides, due to lack of ribosome stalling or fall-off. In such an embodiment, rolling circle translation expresses a continuous expression product through each expression sequence. In some other embodiments, a termination element of an expression sequence can be part of a stagger element. In some embodiments, one or more expression sequences in the circular polyribonucleotide includes a termination element. However, rolling circle translation or expression of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide is performed. In such instances, the expression product may fall off the ribosome when the ribosome encounters the termination element, e.g., a stop codon, and terminates translation. In some embodiments, translation is terminated while the ribosome, e.g., at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.In some embodiments, the circular polyribonucleotide includes a termination element at the end of one or more expression sequences. In some embodiments, one or more expression sequences includes two or more termination elements in succession. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome completely disengages with the circular polyribonucleotide. In some such embodiments, production of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide may require the ribosome to reengage with the circular polyribonucleotide prior to initiation of translation. Generally, termination elements include an in-frame nucleotide triplet that signals termination of translation, e.g., UAA, UGA, UAG. In some embodiments, one or more termination elements in the circular polyribonucleotide are frame-shifted termination elements, such as but not limited to, off-frame or -1 and + 1 shifted reading frames (e.g., hidden stop) that may terminate translation. Frame-shifted terminationATTORNEY DOCKET: 51719-011 WO2PATENT elements include nucleotide triples, TAA, TAG, and TGA that appear in the second and third reading frames of an expression sequence. Frame-shifted termination elements may be important in preventing misreads of mRNA, which is often detrimental to the cell. In some embodiments, the termination element is a stop codon.Further examples of termination elements are described in paragraphs

[0169] -

[0170] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.Untranslated RegionsIn some embodiments, a circular polyribonucleotide includes untranslated regions (UTRs). UTRs of a genomic region including a gene may be transcribed but not translated. In some embodiments, a UTR may be included upstream of the translation initiation sequence of an expression sequence described herein. In some embodiments, a UTR may be included downstream of an expression sequence described herein. In some instances, one UTR for a first expression sequence is the same as or continuous with or overlapping with another UTR for a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, e.g., ZKSCAN1.Exemplary untranslated regions are described in paragraphs

[0197] -

[0201] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, a circular polyribonucleotide includes a polyA sequence. Exemplary polyA sequences are described in paragraphs

[0202] -

[0205] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety. In some embodiments, a circular polyribonucleotide lacks a polyA sequence.In some embodiments, a circular polyribonucleotide includes a UTR with one or more stretches of Adenosines and Uridines embedded within. These AU rich signatures may increase turnover rates of the expression product.Introduction, removal, or modification of UTR AU rich elements (AREs) may be useful to modulate the stability, or immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide. When engineering specific circular polyribonucleotides, one or more copies of an ARE may be introduced to the circular polyribonucleotide and the copies of an ARE may modulate translation and / or production of an expression product. Likewise, AREs may be identified and removed or engineered into the circular polyribonucleotide to modulate the intracellular stability and thus affect translation and production of the resultant protein.It should be understood that any UTR from any gene may be incorporated into the respective flanking regions of the circular polyribonucleotide.In some embodiments, a circular polyribonucleotide lacks a 5’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 3’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA sequence and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination element and is competent for protein expression from its one or more expression sequences. In some embodiments, the circularATTORNEY DOCKET: 51719-011 WO2 PATENT polyribonucleotide lacks an internal ribosomal entry site and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a cap and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5’-UTR, a 3’-UTR, and an IRES, and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide includes one or more of the following sequences: a sequence that encodes one or more miRNAs, a sequence that encodes one or more replication proteins, a sequence that encodes an exogenous gene, a sequence that encodes a therapeutic, a regulatory element (e.g., translation modulator, e.g., translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids that targets endogenous genes (e.g., siRNA, IncRNAs, shRNA), and a sequence that encodes a therapeutic mRNA or protein.In some embodiments, a circular polyribonucleotide lacks a 5’-UTR. In some embodiments, the circular polyribonucleotide lacks a 3’-UTR. In some embodiments, the circular polyribonucleotide lacks a polyA sequence. In some embodiments, the circular polyribonucleotide lacks a termination element. In some embodiments, the circular polyribonucleotide lacks an internal ribosomal entry site. In some embodiments, the circular polyribonucleotide lacks degradation susceptibility by exonucleases. In some embodiments, the fact that the circular polyribonucleotide lacks degradation susceptibility can mean that the circular polyribonucleotide is not degraded by an exonuclease, or only degraded in the presence of an exonuclease to a limited extent, e.g., that is comparable to or similar to in the absence of exonuclease. In some embodiments, the circular polyribonucleotide is not degraded by exonucleases. In some embodiments, the circular polyribonucleotide has reduced degradation when exposed to exonuclease. In some embodiments, the circular polyribonucleotide lacks binding to a cap-binding protein. In some embodiments, the circular polyribonucleotide lacks a 5’ cap.Protein-Binding SequencesIn some embodiments, a circular polyribonucleotide includes one or more protein binding sites that allow a protein, e.g., a ribosome, to bind to an internal site in the RNA sequence. By engineering protein binding sites, e.g., ribosome binding sites, into the circular polyribonucleotide, the circular polyribonucleotide may evade or have reduced detection by the host’s immune system, have modulated degradation, or modulated translation, by masking the circular polyribonucleotide from components of the host’s immune system.In some embodiments, a circular polyribonucleotide includes at least one immunoprotein binding site, for example to evade immune responses, e.g., CTL (cytotoxic T lymphocyte) responses. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and aids in masking the circular polyribonucleotide as exogenous. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and aids in hiding the circular polyribonucleotide as exogenous or foreign.Traditional mechanisms of ribosome engagement to linear RNA involve ribosome binding to the capped 5' end of an RNA. From the 5' end, the ribosome migrates to an initiation codon, whereupon the first peptide bond is formed. According to the present disclosure, internal initiation (i.e., cap-independent) of translation of the circular polyribonucleotide does not require a free end or a capped end. Rather, a ribosome binds to a non-capped internal site, whereby the ribosome begins polypeptide elongation at anATTORNEY DOCKET: 51719-011 WO2 PATENT initiation codon. In some embodiments, the circular polyribonucleotide includes one or more RNA sequences including a ribosome binding site, e.g., an initiation codon.Natural 5' UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCRCCAUGG (SEQ ID NO: 60), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5’ UTRs also have been known to form secondary structures which are involved in elongation factor binding.In some embodiments, a circular polyribonucleotide encodes a protein binding sequence that binds to a protein. In some embodiments, the protein binding sequence targets or localizes the circular polyribonucleotide to a specific target. In some embodiments, the protein binding sequence specifically binds an arginine-rich region of a protein.In some embodiments, the protein binding site includes, but is not limited to, a binding site to the protein such as ACIN1 , AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1 , CELF2, CPSF1 , CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21 , DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, ELAVL1 , ELAVL3, FAM120A, FBL, FIP1 L1 , FKBP4, FMR1 , FUS, FXR1 , FXR2, GNL3, GTF2F1 , HNRNPA1 , HNRNPA2B1 , HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1 , IGF2BP1 , IGF2BP2, IGF2BP3, ILF3, KHDRBS1 , LARP7, LIN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1 , MSI2, NONO, NONO-, NOP58, NPM1 , NUDT21 , PCBP2, POLR2A, PRPF8, PTBP1 , RBFOX2, RBM10, RBM22, RBM27, RBM47, RNPS1 , SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1 , SND1 , SRRM4, SRSF1 , SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1 , TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1 , U2AF2, UNK, UPF1 , WDR33, XRN2, YBX1 , YTHDC1 , YTHDF1 , YTHDF2, YWHAG, ZC3H7B, PDK1 , AKT1 , and any other protein that binds RNA.Spacer SequencesIn some embodiments, the polyribonucleotide described herein includes one or more spacer sequences. A spacer refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacers may be present in between any of the nucleic acid elements described herein. Spacer may also be present within a nucleic acid element described herein.For example, wherein a nucleic acid includes any two or more of the following elements: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment; a spacer region may be present between any one or more of the elements. Any of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence, as described herein. For example, there may be a spacer between (A) and (B), between (B) and (C), between (C) and (D), between (D) and (E), between (E) and (F), or between (F) and (G).In some embodiments, the polyribonucleotide further includes a first spacer region between the 5’ exon fragment of (C) and the polyribonucleotide cargo of (D). The spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, the polyribonucleotide further includes a second spacer region between the polyribonucleotide cargo of (D) and the 5’ exon fragment of (E).ATTORNEY DOCKET: 51719-011 WO2 PATENTA spacer sequences may be used to separate an IRES from adjacent structural elements to martini the structure and function of the IRES or the adjacent element. A spacer can be specifically engineered depending on the IRES. In some embodiments, an RNA folding computer software, such as RNAFold, can be utilized to guide designs of the various elements of the vector, including the spacers.The spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, each spacer region is at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. Each spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA sequence. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA-C sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-G sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-T sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a random sequence.Spacers may also be present within a nucleic acid region described herein. For example, a polynucleotide cargo region may include one or multiple spacers. Spacers may separate regions within the polynucleotide cargo.In some embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the spacer sequence is from 20 to 50 nucleotides in length. In certain embodiments, the spacer sequence is 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, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.The spacer sequences can be polyA sequences, polyA-C sequences, polyC sequences, or poly- U sequences.In some embodiments, the spacer sequences can be polyA-T, polyA-C, polyA-G, or a random sequence.Exemplary spacer sequences are described in paragraphs

[0293] -

[0302] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, the polyribonucleotide includes a 5’ spacer sequence (e.g., between the 5’ annealing region and the polyribonucleotide cargo). In some embodiments, the 5’ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 5’ spacer sequence is at least 15 nucleotides in length. In a further embodiment, the 5’ spacer sequence is at least 30 nucleotides in length. In some embodiments, the 5’ spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5’ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5’ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 5’ spacer sequence is 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, 3637, 38, 39, 40,ATTORNEY DOCKET: 51719-011 WO2 PATENT41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 5’ spacer sequence is a polyA sequence. In another embodiment, the 5’ spacer sequence is a polyA-C sequence. In some embodiments, the 5’ spacer sequence includes a polyA-G sequence. In some embodiments, the 5’ spacer sequence includes a polyA-T sequence. In some embodiments, the 5’ spacer sequence includes a random sequence.In some embodiments, the polyribonucleotide includes a 3’ spacer sequence (e.g., between the 3’ annealing region and the polyribonucleotide cargo). In some embodiments, the 3’ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 3’ spacer sequence is at least 15 nucleotides in length. In a further embodiment, the 3’ spacer sequence is at least 30 nucleotides in length. In some embodiments, the 3’ spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 3’ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 3’ spacer sequence is from 20 to 50 nucleotides in length. In certain embodiments, the 3’ spacer sequence is 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, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 3’ spacer sequence is a polyA sequence. In another embodiment, the 5’ spacer sequence is a polyA-C sequence. In some embodiments, the 5’ spacer sequence includes a polyA-G sequence. In some embodiments, the 5’ spacer sequence includes a polyA-T sequence. In some embodiments, the 5’ spacer sequence includes a random sequence.In one embodiment, the polyribonucleotide includes a 5’ spacer sequence, but not a 3’ spacer sequence. In another embodiment, the polyribonucleotide includes a 3’ spacer sequence, but not a 5’ spacer sequence. In another embodiment, the polyribonucleotide includes neither a 5’ spacer sequence, nor a 3’ spacer sequence. In another embodiment, the polyribonucleotide does not include an IRES sequence. In a further embodiment, the polyribonucleotide does not include an IRES sequence, a 5’ spacer sequence or a 3’ spacer sequence.In some embodiments, the spacer sequence includes at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least about 200 ribonucleotides, at least about 250 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 600 ribonucleotides, at least about 700 ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, or at least about100 ribonucleotides.ModificationsA polyribonucleotide (e.g., circular polyribonucleotide) as described herein may include one or more substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, in particular, the parent polyribonucleotide, are included within the scope of this disclosure.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, a circular polyribonucleotide includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post- transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). In some embodiments, the first isolated nucleic acid includes messenger RNA (mRNA). In some embodiments, the polyribonucleotide includes at least one nucleoside selected from the group such as those described in

[0311] of International Patent Publication No. WO2019 / 118919A1 , which is incorporated herein by reference in its entirety.A polyribonucleotide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.In some embodiments, a polyribonucleotide includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the m6A modification can reduce immunogenicity (e.g., reduce the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide.In some embodiments, a modification may include a chemical or cellular induced modification. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.In some embodiments, chemical modifications to the ribonucleotides of a circular polyribonucleotide may enhance immune evasion. The circular polyribonucleotide may be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), base modifications (e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases. The modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the circular polyribonucleotide. In some embodiments, the modification includes a bi-orthogonal nucleotide, e.g., an unnatural base. See for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039 / c7cc06661 a, which is hereby incorporated by reference.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar one or more ribonucleotides of the circular polyribonucleotide may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of circular polyribonucleotide include, but are not limited to, circular polyribonucleotide including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Circular polyribonucleotides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the circular polyribonucleotide will include ribonucleotides with a phosphorus atom in its internucleoside backbone.Modified polyribonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. In some embodiments, the circular polyribonucleotide may be negatively or positively charged.The modified nucleotides, which may be incorporated into the polyribonucleotide, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylenephosphonates).The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked to the circular polyribonucleotide is expected to reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-0-(l- thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-0-(l-thiophosphate)- guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(1 -thiophosphate)-pseudouridine).Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.ATTORNEY DOCKET: 51719-01 1 WO2 PATENTIn some embodiments, a circular polyribonucleotide may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into circular polyribonucleotide, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5- fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5- fluoro-l-(tetrahydrofuran-2- yl)pyrimidine-2,4(IH,3H)-dione), troxacitabine, tezacitabine, 2'- deoxy-2'- methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4-octadecyl-1 -beta-D-arabinofuranosylcytosine, N4- palmitoyl-l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5'- elaidic acid ester).A polyribonucleotide may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the circular polyribonucleotide, or in a given predetermined sequence region thereof. In some embodiments, the circular polyribonucleotide includes a pseudouridine. In some embodiments, the circular polyribonucleotide includes an inosine, which may aid in the immune system characterizing the circular polyribonucleotide as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability / reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.In some embodiments, all nucleotides in a polyribonucleotide (or in a given sequence region thereof) are modified. In some embodiments, the modification may include an m6A, which may augment expression; an inosine, which may attenuate an immune response; pseudouridine, which may increase RNA stability, or translational readthrough (stagger element), an m5C, which may increase stability; and a 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization).Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in a circular polyribonucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the circular polyribonucleotide, such that the function of the circular polyribonucleotide is not substantially decreased. A modification may also be a non-coding region modification. The circular polyribonucleotide may include from about 1 % to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1 % to 20%>, from 1 % to 25%, from 1 % to 50%, from 1 % to 60%, from 1 % to 70%, from 1 % to 80%, from 1 % to 90%, from 1 % to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).ATTORNEY DOCKET: 51719-01 1 WO2 PATENTMethods of CircularizationThe disclosure provides methods for producing circular polyribonucleotides encoding a plasmodium circumsporozoite polypeptide (e.g., a polypeptide of SEQ ID NO: 9), including, e.g., recombinant technology or chemical synthesis. For example, a DNA molecule used to produce an RNA circle can include a DNA sequence of a naturally occurring original nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide not normally found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant techniques, such as site- directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.In some embodiments, a linear polyribonucleotide for circularization may be cyclized, or concatemerized. In some embodiments, the linear polyribonucleotide for circularization may be cyclized in vitro prior to formulation and / or delivery. In some embodiments, the circular polyribonucleotide may be in a mixture with linear polyribonucleotides. In some embodiments, the linear polyribonucleotides have the same nucleic acid sequence as the circular polyribonucleotides.In some embodiments, a linear polyribonucleotide for circularization is cyclized, or concatemerized using a chemical method to form a circular polyribonucleotide. In some chemical methods, the 5'-end and the 3'-end of the nucleic acid (e.g., a linear polyribonucleotide for circularization) includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5'-end and the 3'-end of the molecule. The 5'-end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino- terminated nucleotide on the 3'-end of a linear RNA molecule will undergo a nucleophilic attack on the 5'- NHS-ester moiety forming a new 5'73'-amide bond.In some embodiments, a DNA or RNA ligase is used to enzymatically link a 5'-phosphorylated nucleic acid molecule (e.g., a linear polyribonucleotide for circularization) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) forming a new phosphodiester linkage. In an example reaction, a linear polyribonucleotide for circularization is incubated at 37°C for 1 hour with 1 -10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The ligation reaction may occur in the presence of a linear nucleic acid capable of base-pairing with both the 5'- and 3'- region in juxtaposition to assist the enzymatic ligation reaction. In some embodiments, the ligation is splint ligation. For example, a splint ligase, like SplintR® ligase, can be used for splint ligation, RNA ligase II, T4 RNA ligase, or T4 DNA ligase. For splint ligation, a single stranded polynucleotide (splint), like a single stranded RNA, can be designed to hybridize with both termini of a linear polyribonucleotide, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint. Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear polyribonucleotide, generating a circular polyribonucleotide.In some embodiments, a DNA or RNA ligase is used in the synthesis of the circular polynucleotides. In some embodiments, either the 5'-or 3'-end of the linear polyribonucleotide for circularization can encode a ligase ribozyme sequence such that during in vitro transcription, the resultantATTORNEY DOCKET: 51719-011 WO2 PATENT linear polyribonucleotide for circularization includes an active ribozyme sequence capable of ligating the 5'-end of the linear polyribonucleotide for circularization to the 3'-end of the linear polyribonucleotide for circularization. The ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.In some embodiments, a linear polyribonucleotide for circularization is cyclized or concatemerized by using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus and / or near the 3' terminus of the linear polyribonucleotide for circularization in order to cyclize or concatemerized the linear polyribonucleotide for circularization. In another aspect, the at least one non-nucleic acid moiety may be located in or linked to or near the 5' terminus and / or the 3' terminus of the linear polyribonucleotide for circularization. The non-nucleic acid moieties contemplated may be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage, and / or a cleavable linkage. As another non-limiting example, the non- nucleic acid moiety is a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.In some embodiments, the linear polyribonucleotide for circularization is synthesized using IVT and an RNA polymerase, where the nucleotide mixture used for IVT may contain an excess of guanosine monophosphate relative to guanosine triphosphate to preferentially produce RNA with a 5’ monophosphate; the purified IVT product may be circularized using a splint DNA.In some embodiments, a linear polyribonucleotide for circularization is cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near or linked to the 5' and 3' ends of the linear polyribonucleotide for circularization. As a non-limiting example, one or more linear polyribonucleotides for circularization may be cyclized or concatemerized by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibonding.In some embodiments, a linear polyribonucleotide for circularization may include a ribozyme RNA sequence near the 5' terminus and near the 3' terminus. The ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, the peptides covalently linked to the ribozyme RNA sequence near the 5' terminus and the 3 'terminus may associate with each other causing a linear polyribonucleotide for circularization to cyclize or concatemerized. In another aspect, the peptides covalently linked to the ribozyme RNA near the 5' terminus and the 3' terminus may cause the linear primary construct or linear mRNA to cyclize or concatemerized after being subjected to ligated using various methods known in the art such as, but not limited to, protein ligation. Non-limiting examples of ribozymes for use in the linear primary constructs or linear RNA of the present invention or a non-exhaustive listing of methods to incorporate and / or covalently link peptides are described in US patent application No. US20030082768, the contents of which is here in incorporated by reference in its entirety.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, a linear polyribonucleotide for circularization may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase). In some embodiments, the 5’ end of at least a portion of the linear polyribonucleotides includes a monophosphate moiety. In some embodiments, the population of polyribonucleotides including circular and linear polyribonucleotides is contacted with RppH prior to digesting at least a portion of the linear polyribonucleotides with a 5’ exonuclease and / or a 3’ exonuclease. Alternately, converting the 5' triphosphate of the linear polyribonucleotide for circularization into a 5' monophosphate may occur by a two-step reaction including: (a) contacting the 5' nucleotide of the linear polyribonucleotide for circularization with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.In some embodiments, circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%. In some embodiments, the circularization method provided has a circularization efficiency of between about 10% and about 100%; for example, the circularization efficiency may be about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 99%. In some embodiments, the circularization efficiency is between about 20% and about 80%. In some embodiments, the circularization efficiency is between about 30% and about 60%. In some embodiments the circularization efficiency is about 40%.In some embodiments, the circular polyribonucleotide includes an internal splicing element that when replicated the spliced ends are joined together. Some examples may include miniature introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in (suptable4 enriched motifs) c / s-sequence elements proximal to back splice events such as sequences in the 200 bp preceding (upstream of) or following (downstream from) a back splice site with flanking exons. In some embodiments, the linear polyribonucleotide includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repetitive nucleotide sequence may include repeated sequences from the Alu family of introns. In some embodiments, a splicing-related ribosome binding protein can regulate circular polyribonucleotide biogenesis (e.g., the Muscle blind and Quaking (QKI) splicing factors).In some embodiments, the linear polyribonucleotide may include canonical splice sites that flank head-to-tail junctions of the circular polyribonucleotide.In some embodiments, the linear polyribonucleotide may include a bulge-helix-bulge motif, including a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl group and 2', 3'-cyclic phosphate. Circularization proceeds by nucleophilic attack of the 5'-OH group onto the 2', 3'-cyclic phosphate of the same molecule forming a 3', 5'-phosphodiester bridge.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, the linear polyribonucleotide may include a multimeric repeating RNA sequence that harbors a HPR element. The HPR includes a 2',3'-cyclic phosphate and 5'-OH termini. The HPR element self-processes the 5'- and 3'-ends of the linear linear polyribonucleotide, thereby ligating the ends together.In some embodiments, the linear polyribonucleotide may include a sequence that mediates selfligation. In one embodiment, the linear polyribonucleotide may include a HDV sequence, e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUG CUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (Beeharry et al 2004) (SEQ ID NO: 61 ) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGC CGCCCGAGCC (SEQ ID NO: 62), to self-ligate. In one embodiment, the linear polyribonucleotide may include loop E sequence (e.g., in PSTVd) to self-ligate. In another embodiment, the linear polyribonucleotide may include a self-circularizing intron, e.g., a 5' and 3’ slice junction, or a selfcircularizing catalytic intron such as a Group I, Group II or Group III Introns. Nonlimiting examples of group I intron self-splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena.In some embodiments, linear polyribonucleotides for circularization may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. Repetitive nucleic acid sequence are sequences that occur within a segment of the linear polyribonucleotide. In some embodiments, the linear polyribonucleotide includes a repetitive nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence includes poly CA or poly UG sequences. In some embodiments, the linear polyribonucleotide includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, the linear polyribonucleotide includes between 1 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, and 10) repetitive nucleic acid sequences that hybridize to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, the linear polyribonucleotide includes 2 repetitive nucleic acid sequences that hybridize to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming internal double strands. In some embodiments, the complementary sequences are found at the 5’ and 3’ ends of the linear polyribonucleotides for circularization. In some embodiments, the complementary sequences include about 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.In some embodiments, chemical methods of circularization may be used to generate the circular polyribonucleotide. Such methods may include, but are not limited to click chemistry (e.g., alkyne and azide-based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, enzymatic methods of circularization may be used to generate the circular polyribonucleotide. In some embodiments, a ligation enzyme, e.g., DNA or RNA ligase, may be used to generate a template of the circular polyribonucleotide or complement, a complementary strand of the circular polyribonucleotide, or the circular polyribonucleotide.Circularization of the linear polyribonucleotide may be accomplished by methods known in the art, for example, those described in “RNA circularization strategies in vivo and in vitro” by Petkovic and Muller from Nucleic Acids Res, 2015, 43(4): 2454-2465, and “In vitro circularization of RNA” by Muller and Appel, from RNA Biol, 2017, 14(8):1018-1027.The circular polyribonucleotide may encode a sequence and / or motif useful for replication. Exemplary replication elements are described in paragraphs

[0280] -

[0286] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, linear polyribonucleotides may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. Repetitive nucleic acid sequence are sequences that occur within a segment of the circular polyribonucleotide. In some embodiments, the linear polyribonucleotide includes a repetitive nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence includes poly CA or poly UG sequences. In some embodiments, the linear polyribonucleotide includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming internal double strands. In some embodiments, the complementary sequences are found at the 5’ and 3’ ends of the linear polyribonucleotides. In some embodiments, the complementary sequences include about 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.Methods of making the circular polyribonucleotides described herein are described in, for example, Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); Muller and Appel, from RNA Biol, 2017, 14(8) :1018-1027; and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012). Other methods of making circular polyribonucleotides are described, for example, in International Publication No. WO2022 / 247943, US Patent No. US11000547, International Publication No. 2018 / 191722, International Publication No. WO2019 / 236673, International Publication No. W02020 / 023595, International Publication No. W02022 / 204460, International Publication No. WO2022 / 204464, and International Publication No. WO2022 / 204466.Various methods of synthesizing circular polyribonucleotides are also described elsewhere (see, e.g., US Patent No. US6210931 , US Patent No. US5773244, US Patent No. US5766903, US Patent No. US5712128, US Patent No. US5426180, US Publication No. US20100137407, International Publication No. WO1992001813, International Publication No. WO2010084371 , and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015); the contents of each of which are herein incorporated by reference in their entirety).ATTORNEY DOCKET: 51719-01 1 WO2 PATENTIn some embodiments, the circular polyribonucleotide is purified, e.g., free ribonucleic acids, linear or nicked RNA, DNA, proteins, etc. are removed. In some embodiments, the circular polyribonucleotides may be purified by any known method commonly used in the art. Examples of nonlimiting purification methods include, column chromatography, gel excision, size exclusion, etc.Methods of ProductionMethods of production in a cell-free systemThe disclosure also provides methods of producing a circular RNA. For example, a deoxyribonucleotide template may be transcribed in a cell-free system (e.g., by in vitro transcription) to produce a linear RNA. The linear polyribonucleotide produces a splicing-compatible polyribonucleotide, which may be self-spliced to produce a circular polyribonucleotide.In some embodiments, the disclosure provides a method of producing a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; and self-splicing linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide; thereby producing a circular polyribonucleotide.In some embodiments, the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding the linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.In some embodiments, the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide, wherein the transcribing occurs in a solution under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a 5’ split-intron and a 3’ split-intron (e.g., a self-splicing construct for producing a circular polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5’ annealing region and a 3’ annealing region.Suitable conditions for in vitro transcriptions and or self-splicing may include any conditions (e.g., a solution or a buffer, such as an aqueous buffer or solution) that mimic physiological conditions in one or more respects. In some embodiments, suitable conditions include between 0.1 -100 mM Mg2+ ions or a salt thereof (e.g., 1 -100 mM, 1 -50 mM, 1 -20 mM, 5- 50 mM, 5-20 mM, or 5-15 mM). In some embodiments, suitable conditions include between 1 -1000 mM K+ ions or a salt thereof such as KCI (e.g., 1 -1000 mM, 1 -500 mM, 1 -200 mM, 50- 500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include between 1 -1000 mM Cl- ions or a salt thereof such as KCI (e.g., 1 -1000 mM, 1 -500 mM, 1 -200 mM, 50- 500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include between 0.1 -100 mM Mn2+ ions or a salt thereof such as MnCI2 (e.g., 0.1 -100 mM, 0.1 -50 mM, 0.1 -20 mM, 0.1 -10 mM, 0.1 -5 mM, 0.1 -2 mM, 0.5-50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5 mM, 0.5-2 mM, or 0.1 -10 mM). In some embodiments, suitable conditions include dithiothreitol (DTT) (e.g., 1 - 1000 pM, 1 -500 pM, 1 -200 pM, 50- 500 pM, 100-500 pM, 100-300 pM, 0.1 -100 mM, 0.1 -50 mM, 0.1 -20 mM, 0.1 -10 mM, 0.1 -5 mM, 0.1 -2 mM, 0.5- 50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5 mM, 0.5-2 mM, or 0.1 -ATTORNEY DOCKET: 51719-011 WO2 PATENT10 mM). In some embodiments, suitable conditions include between 0.1 mM and 100 mM ribonucleoside triphosphate (NTP) (e.g., 0.1 -100 mM, 0.1 -50 mM, 0.1 -10 mM, 1 - 100 mM, 1 -50 mM, or 1 -10 mM). In some embodiments, suitable conditions include a pH of 4 to 10 (e.g., pH of 5 to 9, pH of 6 to 9, or pH of 6.5 to 8.5). In some embodiments, suitable conditions include a temperature of 4°C to 50°C (e.g., 10°C to 40°C, 15 °C to 40°C, 20°C to 40°C, or 30°C to 40°C),In some embodiments the linear polyribonucleotide is produced from a deoxyribonucleic acid, e.g., a deoxyribonucleic acid described herein, such as a DNA vector, a linearized DNA vector, or a cDNA. In some embodiments, the linear polyribonucleotide is transcribed from the deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).Methods of production in a cellThe disclosure also provides methods of producing a circular RNA in a cell, e.g., a prokaryotic cell or a eukaryotic cell. In some embodiments, an exogenous polyribonucleotide is provided to a cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding for the transcription of a linear polyribonucleotide described here). The linear polyribonucleotides may be transcribed in the cell from an exogenous DNA molecule provided to the cell. The linear polyribonucleotide may be transcribed in the cell from an exogenous recombinant DNA molecule transiently provided to the cell. In some embodiments, the exogenous DNA molecule does not integrate into the cell’s genome. In some embodiments, the linear polyribonucleotide is transcribed in the cell from a recombinant DNA molecule that is incorporated into the cell’s genome.In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell including the polyribonucleotides described herein may be a bacterial cell or an archaeal cell. For example, the prokaryotic cell including the polyribonucleotides described herein may be E coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp. (e.g., Bacillus subtilis, Bacillus anthracis, Bacillus cereus), betaproteobacteria (e.g., Burkholderia), alphaproteobacterial (e.g., Agrobacterium), Pseudomonas (e.g., Pseudomonas putida), and enterobacteria. The prokaryotic cells may be grown in a culture medium. The prokaryotic cells may be contained in a bioreactor.In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell including the polyribonucleotides described herein is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic is a unicellular fungal cell such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp, and Pichia spp.). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. A unicellular animal cell may be a cell isolated from a multicellular animal and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular animal cell may be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. A unicellular plant cell may be a cell isolated from a multicellular plant and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular plant cell may be dedifferentiated. In some embodiments, the unicellular plant cell is from a plant callus. In embodiments, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algal cell, such as a unicellular green alga, a diatom, a euglenid, or a dinoflagellate. Non-limiting examples of unicellular eukaryotic algae of interest includeATTORNEY DOCKET: 51719-011 WO2 PATENTDunaliella salina, Chlorella vulgaris, Chlorel la zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris spp., Protosiphon botryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell is a protozoan cell.In some embodiments, the eukaryotic cell is a cell of a multicellular eukaryote. For example, the multicellular eukaryote may be selected from the group consisting of a vertebrate animal, an invertebrate animal, a multicellular fungus, a multicellular alga, and a multicellular plant. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate animal. In some embodiments, the eukaryotic organism is an invertebrate animal. In some embodiments, the eukaryotic organism is a multicellular fungus. In some embodiments, the eukaryotic organism is a multicellular plant. In embodiments, the eukaryotic cell is a cell of a human or a cell of a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., bovids including cattle, buffalo, bison, sheep, goat, and musk ox; pig; camelids including camel, llama, and alpaca; deer, antelope; and equids including horse and donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare). In embodiments, the eukaryotic cell is a cell of a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the eukaryotic cell is a cell of an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc. In embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte. In embodiments, the eukaryotic cell is a cell of a eukaryotic multicellular alga.The eukaryotic cells may be grown in a culture medium. The eukaryotic cells may be contained in a bioreactor.Methods of purificationOne or more purification steps may be included in the methods described herein. For example, in some embodiments, the linear polyribonucleotide is substantively enriched or pure (e.g., purified) prior to self-splicing the linear polyribonucleotide. In other embodiments, the linear polyribonucleotide is not purified prior to self-splicing the linear polyribonucleotide. In some embodiments, the resulting circular RNA is purified.Purification may include separating or enriching the desired reaction product from one or more undesired components, such as any unreacted stating material, byproducts, enzymes, or other reaction components. For example, purification of linear polyribonucleotide following transcription in a cell-free system (e.g., in vitro transcription) may include separation or enrichment from the DNA template prior to self-splicing the linear polyribonucleotide. Purification of the circular RNA product following splicing may be used to separate or enrich the circular RNA from its corresponding linear RNA. Methods of purification of RNA are known to those of skill in the art and include enzymatic purification or by chromatography.In some embodiments, the methods of purification result in a circular polyribonucleotide that has less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) linear polyribonucleotides.ATTORNEY DOCKET: 51719-011 WO2 PATENTBioreactorsIn some embodiments, any method of producing a circular polyribonucleotide described herein may be performed in a bioreactor. A bioreactor refers to any vessel in which a chemical or biological process is carried out which involves organisms or biochemically active substances derived from such organisms. Bioreactors may be compatible with the cell-free methods for production of circular RNA described herein. A vessel for a bioreactor may include a culture flask, a dish, or a bag that may be single use (disposable), autoclavable, or sterilizable. A bioreactor may be made of glass, or it may be polymer- based, or it may be made of other materials.Examples of bioreactors include, without limitation, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors. The mode of operating the bioreactor may be a batch or continuous processes. A bioreactor is continuous when the reagent and product streams are continuously being fed and withdrawn from the system. A batch bioreactor may have a continuous recirculating flow, but no continuous feeding of reagents or product harvest.Some methods of the present disclosure are directed to large-scale production of circular polyribonucleotides. For large-scale production methods, the method may be performed in a volume of 1 liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more). In some embodiments, the method may be performed in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15L to 40 L, 15 L to 45 L, or 15 to 50 L.In some embodiments, a bioreactor may produce at least 1g of circular RNA. In some embodiments, a bioreactor may produce 1 -200 g of circular RNA (e.g., 1 -10 g, 1 -20 g, 1 -50 g, 10-50 g, 10-100 g, 50-100 g, of 50-200 g of circular RNA). In some embodiments, the amount produced is measured per liter (e.g., 1 -200 g per liter), per batch or reaction (e.g., 1 -200 g per batch or reaction), or per unit time (e.g., 1 -200 g per hour or per day).In some embodiments, more than one bioreactor may be utilized in series to increase the production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine bioreactors may be used in series).Methods of UseIn some embodiments, a circular polyribonucleotide encoding a plasmodium circumsporozoite polypeptide (e.g., a polypeptide of SEQ ID NO: 9) is used for the treatment or prevention of malaria, which is caused by the Plasmodium falciparum pathogen.In some embodiments, a circular polynucleotide encoding a plasmodium circumsporozoite polypeptide (e.g., a polypeptide of SEQ ID NO: 9) may be administered to a subject to reduce the risk of malaria.For example, a circular polyribonucleotide as described herein may be administered to a subject (e.g., in a pharmaceutical composition). In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep,ATTORNEY DOCKET: 51719-011 WO2 PATENT goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk.In some embodiments, the disclosure provides a method of modifying a subject by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes or a eukaryotic or prokaryotic cell including a nucleic acid described herein.In some embodiments, the disclosure provides a method of treating or preventing malaria in a subject in need thereof by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes a eukaryotic or prokaryotic cell including a nucleic acid described herein. In some embodiments, the polyribonucleotide is provided in an amount and for a duration sufficient to treat malaria in a subject, e.g., in need thereof.Methods of DosingA method of dosing to produce a level of circular polyribonucleotide encoding a plasmodium circumsporozoite polypeptide (e.g., a polypeptide of SEQ ID NO: 9) or express a level of a plasmodium circumsporozoite polypeptide (e.g., a polypeptide of SEQ ID NO: 9) in a cell after providing the cell with at least two doses or compositions of circular polyribonucleotide is disclosed herein. A method of dosing to produce a level of circular polyribonucleotide or express a level of a plasmodium circumsporozoite polypeptide (e.g., a polypeptide of SEQ ID NO: 9) in a subject (e.g., a mammal, e.g., a human) after providing (e.g., administering to) the subject with at least two doses or compositions of circular polyribonucleotide is disclosed herein. The composition includes a circular polyribonucleotide encoding a plasmodium circumsporozoite polypeptide as described herein. A method of dosing can include administering two or more doses of a composition of circular polyribonucleotides, e.g., over short time period or over an extended period. In some embodiments, the composition containing a circular polyribonucleotide further includes a pharmaceutically acceptable carrier or excipient. The circular polyribonucleotide encodes a plasmodium circumsporozoite polypeptide, which can be expressed in a cell, e.g., following administration.The methods described herein may include administering a first dose of the pharmaceutical composition in an amount sufficient to produce a serum concentration of at least 500 ng / mL (e.g., at least 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1 ,000 ng / mL, 1 ,100 ng / mL, 1 ,200 ng / mL, 1 ,300 ng / mL, 1 ,400 ng / mL, 1 ,500 ng / mL, 1 ,600 ng / mL, 1 ,700 ng / mL, 1 ,800 ng / mL, 1 ,900 ng / mL, 2,000 ng / mL, 2,100 ng / mL, 2,200 ng / mL, 2,300 ng / mL, 2,400 ng / mL, 2,500 ng / mL, 2,600 ng / mL, 2,700 ng / mL, 2,800 ng / mL, 2,900 ng / mL, 3,000 ng / mL, or more) of a plasmodium circumsporozoite polypeptide in the subject.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some embodiments, the method may further include administering a second dose of the pharmaceutical composition. The method may further include administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more doses of the pharmaceutical composition. In some embodiments, a subsequent dose helps maintain a serum concentration of at least 500 ng / mL (e.g., at least 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1 ,000 ng / mL, 1 ,100 ng / mL, 1 ,200 ng / mL, 1 ,300 ng / mL, 1 ,400 ng / mL, 1 ,500 ng / mL, 1 ,600 ng / mL, 1 ,700 ng / mL, 1 ,800 ng / mL, 1 ,900 ng / mL, 2,000 ng / mL, 2,100 ng / mL, 2,200 ng / mL, 2,300 ng / mL, 2,400 ng / mL, 2,500 ng / mL, 2,600 ng / mL, 2,700 ng / mL, 2,800 ng / mL, 2,900 ng / mL, 3,000 ng / mL, or more) of a plasmodium circumsporozoite polypeptide in the subject. In some embodiments, a subsequent dose is administered before the serum concentration drops below 500 ng / mL of a plasmodium circumsporozoite polypeptide in the subject.In some embodiments, multiple doses are provided to produce a level of the composition or express a level of the plasmodium circumsporozoite polypeptide in a cell, tissue or subject. In some embodiments, multiple doses are provided to produce or maintain a level of the composition, or to produce or maintain a level of the plasmodium circumsporozoite polypeptide, in a cell, tissue or subject for a period of time, for instance, for at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 days, or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 21 , or 24 months, or at least 1 , 2, 3, 4, or 5 years.In some embodiments, the second dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the first dose of the pharmaceutical composition.In some embodiments, the second dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g., from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days, from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45 days, from 14 days to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days, from 21 days to 30 days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days, from 45 to 180ATTORNEY DOCKET: 51719-011 WO2 PATENT days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from 60 to 180 days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from 90 to 120 days, or from 90 to 180 days) after the first dose of the pharmaceutical composition.In some embodiments, the third dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the second dose of the pharmaceutical composition.In some embodiments, the third dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g., from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days, from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45 days, from 14 days to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days, from 21 days to 30 days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days, from 45 to 180 days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from 60 to 180 days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from 90 to 120 days, or from 90 to 180 days) after the second dose of the pharmaceutical composition.In some embodiments, the second dose is administered before a serum concentration of a plasmodium circumsporozoite polypeptide is less than about 500 ng / mL in serum of the subject.In some embodiments, the method maintains a serum concentration of at least 500 ng / mL (e.g., at least 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1 ,000 ng / mL, 1 ,100 ng / mL, 1 ,200 ng / mL, 1 ,300 ng / mL, 1 ,400 ng / mL, 1 ,500 ng / mL, 1 ,600 ng / mL, 1 ,700 ng / mL, 1 ,800 ng / mL, 1 ,900 ng / mL, 2,000 ng / mL, 2,100 ng / mL, 2,200 ng / mL, 2,300 ng / mL, 2,400 ng / mL, 2,500 ng / mL, 2,600 ng / mL, 2,700 ng / mL, 2,800 ng / mL, 2,900 ng / mL, 3,000 ng / mL, or more) of a plasmodium circumsporozoite polypeptide in the subject, e.g., for at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, fourATTORNEY DOCKET: 51719-011 WO2 PATENT days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer).A method of administering multiple doses of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide) includes providing two or more compositions over a period of time, to a cell, tissue or subject (e.g., a mammal). According to certain embodiments, multiple doses of a composition of a nucleic acid molecule described herein may be administered to a subject over a defined time course. The methods according to this aspect of the invention include sequentially administering to a subject multiple doses of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical or veterinary composition). As used herein, “sequentially administering” means that each dose of composition of a nucleic acid molecule described herein is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). In some embodiments, the present invention provides methods which include sequentially administering to the subject a single initial dose of a composition of a nucleic acid molecule described herein, followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition.The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of a composition of a nucleic acid molecule described herein. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen; the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of a composition of a nucleic acid molecule described herein, and in certain embodiments, may differ from one another in terms of frequency of administration. In certain embodiments, the amount of a composition of a nucleic acid molecule described herein contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, one or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).In certain embodiments, each secondary and / or tertiary dose is administered after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of the composition of a nucleic acid molecule described herein which is administered to a subject prior to the administration of the very next dose in the sequence with no intervening doses. In certain embodiments, each secondary and / or tertiary dose is administered every day, every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the immediately preceding dose. In certain embodiments, each secondary and / or tertiary dose is administered every 0.5 weeks, 1 week, 2 weeks, 3 weeks, or 4 weeks after the immediately preceding dose.The methods according to this aspect of the invention may include administering to a subject any number of secondary and / or tertiary doses of a composition of a nucleic acid molecule described herein. For example, in certain embodiments, only a single secondary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject. Likewise, in certain embodiments, only a single tertiary dose is administered to the subject. InATTORNEY DOCKET: 51719-011 WO2 PATENT other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the subject.In certain embodiments, the frequency at which the secondary and / or tertiary doses are administered to a subject can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment.In some embodiments, the method includes providing (e.g., administering) at least a first composition and a second composition to the cells, tissue, or subject (e.g., a mammal, e.g., a human). In some embodiments, the method further includes providing (e.g., administering) a third composition, fourth composition, fifth composition, sixth composition, seventh composition, eighth composition, ninth composition, tenth composition, or more. In some embodiments, additional compositions are provided for the duration of the life of the cell. In some embodiments, additional compositions are provided (e.g., administered) while the cell, tissue or subject obtains a benefit from the composition.In some embodiments, a first composition in a multiple dosing regimen includes a first amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, a second composition in a multiple dosing regimen includes a second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, a third composition, a fourth composition, a fifth composition, a sixth composition, a seventh composition, an eighth composition, a ninth composition, a tenth composition, or more in a multiple dosing regimen includes a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of theATTORNEY DOCKET: 51719-01 1 WO2 PATENT second composition is no more than 1 %, 5%, 10%, 15%, 20%, or 25% less than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1 -fold to 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., a composition administered after a first composition) is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1 -fold to 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., a composition administered after a first composition) is 0.1 -fold, 1 -fold, 5- fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)) is from 0.1 -fold to 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)) is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. For example, a first composition includes 1 -fold nucleic acid molecule (e.g., circular polyribonucleotide), a second composition includes 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition, and a third composition includes 0.2-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition. In some embodiments, the second composition includes at least 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to an amount of nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.In some embodiments, the first composition includes a higher amount of the nucleic acid molecule (e.g., circular polyribonucleotide) than the second composition. In some embodiments, the first composition includes a higher amount of the nucleic acid molecules (e.g., circular polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition.In some embodiments, the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding a plasmodium circumsporozoite polypeptide, which are administered in a multiple dosing regimen as described herein, are the same compositions. In some embodiments, the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding a plasmodium circumsporozoite polypeptide, which are administered in aATTORNEY DOCKET: 51719-011 WO2 PATENT multiple dosing regimen as described herein, are different compositions. In some embodiments, the same compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding the same plasmodium circumsporozoite polypeptide. In some embodiments, the different compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding different plasmodium circumsporozoite polypeptides, or a combination thereof.In some embodiments, in a multiple dosing regimen, the method of administering the nucleic acid molecule (e.g., circular polyribonucleotide) provided herein includes administering to a subject in need thereof the nucleic acid molecule for multiple times (multiple doses), e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 100, 150, 200, or 500 times, with an interval of from 1 day to 56 days, such as about 49 days, 42 days, 35 days, 28 days, 21 days, 14 days, or 7 days. In some embodiments, in a multiple dosing regimen, the method provided herein includes administering to a subject in need thereof the nucleic acid molecule for at least 3 times, with an interval of about 7 days. In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses) provided herein, a level of the plasmodium circumsporozoite polypeptide (e.g., a plasma plasmodium circumsporozoite polypeptide) is maintained at a level with variation of less than 50%, 40%, 30%, 20%, or 10% for a period of longer than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 18, or 20 weeks after the last dose. In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses) provided herein, a level of the plasmodium circumsporozoite polypeptide (e.g., a plasma plasmodium circumsporozoite polypeptide level) is maintained at a first level for a period of longer than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the plasmodium circumsporozoite polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose). In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3 doses) provided herein with an interval of about 7 days, a level of the plasmodium circumsporozoite polypeptide (e.g., a plasma plasmodium circumsporozoite polypeptide level) is maintained at a first level for a period of longer than 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the plasmodium circumsporozoite polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).Methods of DeliveryA circular polyribonucleotide encoding a plasmodium circumsporozoite polypeptide (e.g., a polypeptide of SEQ ID NO: 9) described herein may be included in pharmaceutical compositions with a carrier or without a carrier.Pharmaceutical compositions described herein may be formulated for example including a carrier, such as a pharmaceutical carrier and / or a polymeric carrier, e.g., a liposome, and delivered by known methods to a subject in need thereof (e.g., a human or non-human agricultural or domestic animal, e.g., cattle, dog, cat, horse, poultry). Such methods include, but not limited to, transfection (e.g., lipid- mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, microprojectile bombardment (“gene gun”), FuGENE®, direct sonic loading, cellATTORNEY DOCKET: 51719-011 WO2 PATENT squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Methods of delivery are also described, e.g., in Gori et al., Delivery and Specificity of CRISPR / Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy. July 2015, 26(7): 443-451 . doi :10.1089 / hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(1 ):73-80.In some embodiments, circular polyribonucleotides may be delivered in a “naked” delivery formulation. A naked delivery formulation delivers a circular polyribonucleotide to a cell without the aid of a carrier and without covalent modification of the circular polyribonucleotide or partial or complete encapsulation of the circular polyribonucleotide.A naked delivery formulation is a formulation that is free from a carrier and wherein the circular polyribonucleotide is without a covalent modification that binds a moiety that aids in delivery to a cell and the circular polyribonucleotide is not partially or completely encapsulated. In some embodiments, a circular polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may be a polyribonucleotide that is not covalently bound to a moiety, such as a protein, small molecule, a particle, a polymer, or a biopolymer that aids in delivery to a cell. In some embodiments, circular polyribonucleotides may be delivered in a delivery formulation with protamine or a protamine salt (e.g., protamine sulfate).A polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may not contain a modified phosphate group. For example, a polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may not contain phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.In some embodiments, a naked delivery formulation may be free of any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation may be free from phytoglycogen octenyl succinate, phytoglycogen betadextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy- diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1 ,2-Dioleoyl-3- Trimethylammonium-Propane (DOTAP), N-[1 -(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HCI), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N- (l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL), high- density lipoprotein (HDL), or globulin.A naked delivery formulation may include a non-carrier excipient. In some embodiments, a noncarrier excipient may include an inactive ingredient that does not exhibit an active cell-penetrating effect. In some embodiments, a non-carrier excipient may include a buffer, for example PBS. In some embodiments, a non-carrier excipient may be a solvent, a non-aqueous solvent, a diluent, a suspensionATTORNEY DOCKET: 51719-011 WO2 PATENT aid, a surface-active agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a granulating agent, a disintegrating agent, a binding agent, a buffering agent, a lubricating agent, or an oil.In some embodiments, a naked delivery formulation may include a diluent, such as a parenterally acceptable diluent. A diluent (e.g., a parenterally acceptable diluent) may be a liquid diluent or a solid diluent. In some embodiments, a diluent (e.g., a parenterally acceptable diluent) may be an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of an RNA solubilizing agent include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of a buffer include 2-(N- morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N- morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of an isotonic agent include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.In some embodiments, the formulation includes a cell-penetrating agent. In some embodiments, the formulation is a topical formulation and includes a cell-penetrating agent. The cell-penetrating agent can include organic compounds such as alcohols having one or more hydroxyl function groups. In some cases, the cell-penetrating agent includes an alcohol such as, but not limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic alcohols, and alicyclic alcohols. The cell-penetrating agent can include one or more of methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl alcohol, specially denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycols (PEG)-400, ethoxylated fatty acids, or hydroxyethylcellulose. In certain embodiments, the cellpenetrating agent includes ethanol. The cell-penetrating agents can include any cell-penetrating agent in any amount or in any formulation as described in WO 2020 / 180751 or WO 2020 / 180752, which are hereby incorporated by reference in their entirety.In some embodiments, the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein is in parenteral nucleic acid delivery system. The parental nucleic acid delivery system may include the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein, and a parenterally acceptable diluent. In some embodiments, the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein in the parenteral nucleic acid delivery system is free of any carrier.The disclosure is further directed to a host or host cell including the circular polyribonucleotide described herein. In some embodiments, the host or host cell is a vertebrate, mammal (e.g., human), or other organism or cell.In some embodiments, the circular polyribonucleotide has a decreased, or fails to produce a, undesired response by the host’s immune system as compared to the response triggered by a reference compound, e.g., a linear polynucleotide corresponding to the described circular polyribonucleotide. In embodiments, the circular polyribonucleotide is non-immunogenic in the host. Some immune responsesATTORNEY DOCKET: 51719-011 WO2 PATENT include, but are not limited to, humoral immune responses (e.g., production of immunogen-specific antibodies) and cell-mediated immune responses (e.g., lymphocyte proliferation).In some embodiments, a host or a host cell is contacted with (e.g., delivered to or administered to) the circular polyribonucleotide. In some embodiments, the host is a mammal, such as a human. The amount of the circular polyribonucleotide or linear, expression product, or both in the host can be measured at any time after administration. In certain embodiments, a time course of host growth in a culture is determined. If the growth is increased or reduced in the presence of the circular polyribonucleotide or linear, the circular polyribonucleotide or expression product or both is identified as being effective in increasing or reducing the growth of the host.A method of delivering a circular polyribonucleotide molecule as described herein to a cell, tissue, or subject, includes administering the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein to the cell, tissue, or subject.In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an ungulate cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an immune cell. In some embodiments, the tissue is a connective tissue, a muscle tissue, a nervous tissue, or an epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.).In some embodiments, the method of delivering is an in vivo method. For example, a method of delivery of a circular polyribonucleotide as described herein includes parenterally administering to a subject in need thereof, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein to the subject in need thereof. As another example, a method of delivering a circular polyribonucleotide to a cell or tissue of a subject, includes administering parenterally to the cell or tissue the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein. In some embodiments, the circular polyribonucleotide is in an amount effective to elicit a biological response in the subject. In some embodiments, the circular polyribonucleotide is an amount effective to have a biological effect on the cell or tissue in the subject. In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein includes a carrier. In some embodiments the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein includes a diluent and is free of any carrier.In some embodiments the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered parenterally. In some embodiments the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphaticly, subcutaneously, or intramuscularly. In some embodiments, parenteral administration is intravenously, intramuscularly, ophthalmically, subcutaneously, intradermally, by inhalation (e.g., after nebulization), or topically. In some embodiments, the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered by inhalation. In some embodiments, the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered by inhalation after nebulization.In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered intramuscularly. In some embodiments,ATTORNEY DOCKET: 51719-011 WO2 PATENT the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered subcutaneously. In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered topically. In some embodiments, the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered intratracheally.In some embodiments the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product is administered by injection. The administration can be systemic administration or local administration. In some embodiments, any of the methods of delivery as described herein are performed with a carrier. In some embodiments, any methods of delivery as described herein are performed without the aid of a carrier or cell penetrating agent.In some embodiments, the circular polyribonucleotide or a product translated from the circular polyribonucleotide is detected in the cell, tissue, or subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days after the administering step. In some embodiments, the presence of the circular polyribonucleotide or a product translated from the circular polyribonucleotide is evaluated in the cell, tissue, or subject before the administering step. In some embodiments, the presence of the circular polyribonucleotide or a product translated from the circular polyribonucleotide is evaluated in the cell, tissue, or subject after the administering step.FormulationsIn some embodiments, a pharmaceutical formulation disclosed herein can include: (i) a compound (e.g., circular polyribonucleotide) disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and / or (v) a stabilizer. In some embodiments, a pharmaceutical formulation disclosed herein can include: (i) a compound (e.g., linear polyribonucleotide) disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and / or (v) a stabilizer. In some embodiments, the pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical formulation. In some embodiments, the pharmaceutical formulation disclosed herein includes protamine or a protamine salt (e.g., protamine sulfate).The disclosure provides immunogenic compositions including a circular polyribonucleotide described above. The disclosure provides immunogenic compositions including a linear polyribonucleotide described above. Immunogenic compositions of the disclosure may include a diluent or a carrier, adjuvant, or any combination thereof. Immunogenic compositions of the disclosure may also include one or more immunoregulatory agents, e.g., one or more adjuvants. The adjuvants may include a TH1 adjuvant and / or a TH2 adjuvant, further discussed below. In some embodiments, the immunogenic composition includes a diluent free of any carrier and is used for naked delivery of the circular polyribonucleotide to a subject. In some embodiments, the immunogenic composition includes a diluent free of any carrier and is used for naked delivery of the linear polyribonucleotide to a subject.Immunogenic compositions of the disclosure are used to raise an immune response in a subject. The immune response is preferably protective and preferably involves an antibody response (usually including IgG) and / or a cell-mediated immune response. For example, a subject is immunized with an immunogenic composition including a circular polyribonucleotide of the disclosure to induce an immune response. In another example, a subject is immunized with an immunogenic composition including a linear polyribonucleotide including an immunogen to stimulate production of antibodies that bind to theATTORNEY DOCKET: 51719-011 WO2 PATENT immunogen. By raising an immune response in the subject by these uses and methods, the subject can be protected against malaria. In certain embodiments, the immunogenic compositions are vaccine compositions. Vaccines according to the disclosure may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection) but will typically be prophylactic. In some embodiments, the subject is a mammal. In some embodiments, the subject is an animal, preferably a mammal, e.g., a human. In one embodiment, the subject is a human. In other embodiments the subject is a non-human mammal, e.g., selected from a cow (e.g., dairy and beef cattle), a sheep, a goat, a pig, a horse, a dog, or a cat. In other embodiments the subject is a bird, e.g., a hen or rooster, turkey, parrot. In some embodiments, the animal is not a mouse or a rabbit or a cow. In a particular embodiment, where the immunogenic composition is for prophylactic use, the human is a child (e.g. a toddler or infant) or a teenager. In another embodiment, where the immunogenic composition is for therapeutic use, the human is a teenager or an adult. An immunogenic composition intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.Immunogenic composition prepared according to the disclosure may be used to treat both children and adults. A human subject may be less than 1 year old, less than 5 years old, 1 -5 years old, 5- 15 years old, 15-55 years old, or at least 55 years old. In a particular embodiment, a human subject for receiving the immunogenic compositions are the elderly (e.g., >50 years old, >60 years old, and >65 years), the young (e.g., <5 years old), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, or immunodeficient patients. The immunogenic compositions are not suitable solely for these groups, however, and may be used more generally in a population.In some embodiments, the subject is further immunized with an adjuvant. In some embodiments the subject is further immunized with a vaccine.ImmunizationIn some embodiments, methods of the disclosure include immunizing a subject with an immunogenic composition including a circular polyribonucleotide as disclosed herein. In some embodiments, an immunogen is expressed from the circular polyribonucleotide. In some embodiments, immunization induces an immune response in a subject against the immunogen expressed from the circular polyribonucleotide. In some embodiments, immunization induces an immune response in a subject (e.g., induces the production of antibodies that bind to the immunogen expressed from the circular polyribonucleotide). In some embodiments, an immunogenic composition includes the circular polyribonucleotide and a diluent, carrier, first adjuvant or a combination thereof in a single composition. In some embodiments, the subject is further immunized with a second adjuvant. In some embodiments, the subject is further immunized with a vaccine.In some embodiments, methods of the disclosure include immunizing a subject with an immunogenic composition including a linear polyribonucleotide as disclosed herein. In some embodiments, an immunogen is expressed from the linear polyribonucleotide. In some embodiments, immunization induces an immune response in a subject against the immunogen expressed from the linear polyribonucleotide. In some embodiments, immunization induces the production of antibodies that bind to the immunogen expressed from the linear polyribonucleotide. In some embodiments, immunization induces a cell-mediated immune response. In some embodiments, an immunogenicATTORNEY DOCKET: 51719-01 1 WO2 PATENT composition includes the linear polyribonucleotide and a diluent, carrier, first adjuvant or a combination thereof in a single composition. In some embodiments, the subject is further immunized with a second adjuvant. In some embodiments, the subject is further immunized with a vaccine.The subject is immunized with one or more immunogenic composition(s) including any number of circular polyribonucleotides. The subject is immunized with, for example, one or more immunogenic composition(s) including at least 1 circular polyribonucleotide. A non-human animal having a nonhumanized immune system is immunized with, for example, one or more immunogenic composition(s) including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 20 different circular polyribonucleotides, or more different circular polyribonucleotides. In some embodiments, a subject is immunized with one or more immunogenic composition(s) including at most 1 circular polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is immunized with one or more immunogenic composition(s) including at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 1 1 , at most 12, at most 13, at most 14, at most 15, at most 20 different circular polyribonucleotides, or less than 21 different circular polyribonucleotides. In some embodiments, a subject is immunized with one or more immunogenic composition(s) including about 1 circular polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is immunized with one or more immunogenic composition(s) including about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, or about 20 different circular polyribonucleotides. In some embodiments, a subject is immunized with one or more immunogenic composition(s) including about 1 -20, 1 -15, 1 -10, 1 -9, 1 -8, 1 -7, 1 -6, 1 -5, 1 -4, 1 -3, 1 -2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3- 6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, I Q- 15, or 15-20 different circular polyribonucleotides. Different circular polyribonucleotides have different sequences from each other. For example, they can include or encode different immunogens, overlapping immunogens, similar immunogens, or the same immunogens (for example, with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the disclosure). In cases where a subject is immunized with one or more immunogenic composition(s) including two or more different circular polyribonucleotides, the two or more different circular polyribonucleotides can be in the same or different immunogenic compositions and immunized at the same time or at different times. The immunogenic compositions including two or more different circular polyribonucleotides can be administered to the same anatomical location or different anatomical locations.The subject can be immunized with one or more immunogenic composition(s) including any number of linear polyribonucleotides. The subject is immunized with, for example, one or more immunogenic composition(s) including at least 1 linear polyribonucleotide. A non-human animal having a non-humanized immune system is immunized with, for example, one or more immunogenic composition(s) including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 20 different linear polyribonucleotides, or more different linear polyribonucleotides. In some embodiments, a subject is immunized with one or more immunogenic composition(s) including at most 1 linear polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is immunized with one orATTORNEY DOCKET: 51719-01 1 WO2 PATENT more immunogenic composition(s) including at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 1 1 , at most 12, at most 13, at most 14, at most 15, at most 20 different linear polyribonucleotides, or less than 21 different linear polyribonucleotides. In some embodiments, a subject is immunized with one or more immunogenic composition(s) including about 1 linear polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is immunized with one or more immunogenic composition(s) including about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, or about 20 different linear polyribonucleotides. In some embodiments, a subject is immunized with one or more immunogenic composition(s) including about 1 -20, 1 -15, 1 -10, 1 -9, 1 -8, 1 -7, 1 -6, 1 -5, 1 -4, 1 -3, 1 -2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-15, or 15-20 different linear polyribonucleotides. Different linear polyribonucleotides can have different sequences from each other. For example, they can include or encode different immunogens, overlapping immunogens, similar immunogens, or the same immunogens (for example, with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the disclosure). In cases where a subject is immunized with one or more immunogenic composition(s) including two or more different linear polyribonucleotides, the two or more different linear polyribonucleotides can be in the same or different immunogenic compositions and immunized at the same time or at different times. The immunogenic compositions including two or more different linear polyribonucleotides can be administered to the same anatomical location or different anatomical locations.The two or more different linear polyribonucleotides can include or encode immunogens from the same source, different source, or different combinations of sources disclosed herein. The two or more different linear polyribonucleotides can include or encode immunogens from the same virus or from different viruses, for example, different isolates.In some embodiments, the subject is immunized with one or more immunogenic composition(s) including any number of circular polyribonucleotides and one or more immunogenic composition(s) including any number of linear polyribonucleotides as disclosed herein. In some embodiments, an immunogenic composition disclosed herein includes one or more circular polyribonucleotides and one or more linear polyribonucleotides as disclosed herein.In some embodiments, an immunogenic composition includes a circular polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof. In a particular embodiment, an immunogenic composition includes a circular polyribonucleotide described herein and a carrier or a diluent free of any carrier. In some embodiments, an immunogenic composition including a circular polyribonucleotide with a diluent free of any carrier is used for naked delivery of the circular polyribonucleotide to a subject. In another particular embodiment, an immunogenic composition includes a circular polyribonucleotide described herein and a first adjuvant.In certain embodiments, a subject is further administered a second adjuvant. An adjuvant enhances the innate immune response, which in turn, enhances the adaptive immune response in a subject. An adjuvant can be any adjuvant as discussed below. In certain embodiments, an adjuvant is formulated with the circular polyribonucleotide as a part of an immunogenic composition. In certain embodiments, an adjuvant is not part of an immunogenic composition including the circular polyribonucleotide. In certain embodiments, an adjuvant is administered separately from an immunogenicATTORNEY DOCKET: 51719-011 WO2 PATENT composition including the circular polyribonucleotide. In this aspect, the adjuvant is co-administered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the circular polyribonucleotide to the subject. For example, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the circular polyribonucleotide. In some embodiments, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the circular polyribonucleotide. For example, the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenic composition including the circular polyribonucleotide. In some embodiments, the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the circular polyribonucleotide. The adjuvant is administered to the same anatomical location or different anatomical location as the immunogenic composition including the circular polyribonucleotide.In some embodiments, an immunogenic composition includes a linear polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof. In a particular embodiment, an immunogenic composition includes a linear polyribonucleotide described herein and a carrier or a diluent free of any carrier. In some embodiments, an immunogenic composition including a linear polyribonucleotide with a diluent free of any carrier is used for naked delivery of the linear polyribonucleotide to a subject. In another particular embodiment, an immunogenic composition includes a linear polyribonucleotide described herein and a first adjuvant.In certain embodiments, a subject is further administered a second adjuvant. An adjuvant enhances the innate immune response, which in turn, enhances the adaptive immune response in a subject. An adjuvant can be any adjuvant as discussed below. In certain embodiments, an adjuvant is formulated with the linear polyribonucleotide as a part of an immunogenic composition. In certain embodiments, an adjuvant is not part of an immunogenic composition including the linear polyribonucleotide. In certain embodiments, an adjuvant is administered separately from an immunogenic composition including the linear polyribonucleotide. In this aspect, the adjuvant is co-administered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the linear polyribonucleotide to the subject. For example, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the linear polyribonucleotide. In some embodiments, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the linear polyribonucleotide. For example, the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenicATTORNEY DOCKET: 51719-011 WO2 PATENT composition including the linear polyribonucleotide. In some embodiments, the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the linear polyribonucleotide. The adjuvant is administered to the same anatomical location or different anatomical location as the immunogenic composition including the linear polyribonucleotide.In some embodiments, a subject is further immunized with a second agent, e.g., a vaccine (as described below) that is not a circular polyribonucleotide. The vaccine is co-administered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the circular polyribonucleotide to the subject. For example, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the circular polyribonucleotide. In some embodiments, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the circular polyribonucleotide. For example, the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenic composition including the circular polyribonucleotide. In some embodiments, the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the circular polyribonucleotide.In some embodiments, a subject is further immunized with a second agent, e.g., a vaccine (as described below) that is not a linear polyribonucleotide. The vaccine is co-administered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the linear polyribonucleotide to the subject. For example, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the linear polyribonucleotide. In some embodiments, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the linear polyribonucleotide. For example, the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenic composition including the linear polyribonucleotide. In some embodiments, the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the linear polyribonucleotide.A subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof any suitable number of times to achieve a desired response. For example, a prime-boost immunization strategy can be utilized to elicit systemic and / or mucosal immunity. A subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., proteinATTORNEY DOCKET: 51719-011 WO2 PATENT subunit vaccine), or a combination thereof, of the disclosure, for example, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 15 times, or more.In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, or at most 20 times, or less.In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times.In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure once. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure twice. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure three times. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure four times. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure five times. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure seven times.Suitable time intervals can be selected for spacing two or more immunizations. The time intervals can apply to multiple immunizations with the same immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, for example, the same immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, can be administered in the same amount or a different amount, via the same immunization route or a different immunization route. The time intervals can apply to multiple immunizations with a different immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, for example, a different immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, can be administered in the same amount or a different amount, via the same immunization route or a different immunization route. The time intervals can apply to immunizations with different agents, for example, a first immunogenic composition including a first circular polyribonucleotide and a second immunogenic composition including a second circular polyribonucleotide. The time intervals can apply to immunizations with different agents, for example, a first immunogenic composition including a first circular polyribonucleotide and a second immunogenic composition including a protein immunogen (e.g., a protein subunit). The time intervals can apply to a first immunogenic composition including a first linear polyribonucleotide and a second immunogenic composition including a second linear polyribonucleotide. For regimens including three or more immunizations, the time intervals between immunizations can be the same or different. In some examples, about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 16, 17 ,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 48, or 72 hours elapse between two immunizations. In some embodiments, about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 16, 17, 18, 20, 21 , 24, 28, or 30 days elapse between two immunizations. In some embodiments, about 1 , 2, 3, 4, 5, 6, 7, or 8 weeks elapse between twoATTORNEY DOCKET: 51719-011 WO2 PATENT immunizations. In some embodiments, about 1 , 2, 3, 4, 5, 6, 7, or 8 months elapse between two immunizations.In some embodiments, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 24, at least 36, or at least 72 hours, or more elapse between two immunizations. In some embodiments, at most 1 , at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 24, at most 36, or at most 72 hours, or less elapse between two immunizations.In some embodiments, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26 at least 27, at least 28, at least 29, or at least 30 days, or more, elapse between two immunizations. In some embodiments, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 21 , at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 32, at most 34, or at most 36 days, or less elapse between two immunizations.In some embodiments, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 weeks, or more elapse between two immunizations. In some embodiments, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8 weeks, or less elapse between two immunizations.In some embodiments, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 months, or more elapse between two immunizations. In some embodiments, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8 months, at most 9 months, at most 10 months, at most 11 months, or at most 12 months or less elapse between two immunizations.In some embodiments, the method includes pre-administering to the subject an agent to improve immunogenic responses to a circular polyribonucleotide including a sequence encoding an immunogen. In some embodiments, the agent is the immunogen as disclosed herein (e.g., a protein immunogen). For example, the method includes administering the protein immunogen from 1 to 7 days prior to administration of the circular polyribonucleotide including the sequence encoding the protein immunogen. In some embodiments, the protein immunogen is administered 1 , 2, 3, 4, 5, 6, or 7 days prior to administration of the circular polyribonucleotide including the sequence encoding the protein immunogen. For example, the method includes administering the protein immunogen from 1 to 7 days prior to administration of the linear polyribonucleotide including the sequence encoding the protein immunogen. In some embodiments, the protein immunogen is administered 1 , 2, 3, 4, 5, 6, or 7 days prior to administration of the linear polyribonucleotide including the sequence encoding the protein immunogen. The protein immunogen may be administered as a protein preparation, encoded in a plasmid (pDNA), presented in a virus-like particle (VLP), formulated in a lipid nanoparticle, or the like.In some embodiments, the method includes administering to the subject an agent to improve immunogenic responses to a circular polyribonucleotide including a sequence encoding an immunogen after the subject has been administered the circular polyribonucleotide including a sequence encoding an immunogen. In some embodiments, the agent is the immunogen as disclosed herein (e.g., a protein immunogen). In some embodiments, the circular polyribonucleotide includes a sequence encoding a protein immunogen. For example, the method includes administering the protein immunogen within 1 year (e.g., within 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3ATTORNEY DOCKET: 51719-011 WO2 PATENT months, 2 months, and 1 month) of administering the circular polyribonucleotide including a sequence encoding the immunogen to the subject. In some embodiments, the method includes administering any one of the circular polyribonucleotides described herein or any one of the immunogenic compositions described herein and a protein subunit to the subject.In some embodiments, the protein immunogen has the same amino acid sequence as the immunogen encoded by circular polyribonucleotide. For example, the polypeptide immunogen may correspond to (e.g., shares 90%, 95%, 96%, 97%, 98%, or 100%) amino acid sequence identity with a polypeptide immunogen encoded by a sequence of the circular polyribonucleotide. In some embodiments, the protein immunogen has a different amino acid sequence from the amino acid sequence of the immunogen encoded by the circular polyribonucleotide. For example, the polypeptide immunogen may share less than 90% (e.g., 80%, 70%, 30%, 20%, or 10%) amino acid sequence identity with the polypeptide immunogen encoded by a sequence of the circular polyribonucleotide.A subject can be immunized with an immunogenic composition, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, at any suitable number anatomical sites. The same immunogenic composition, an adjuvant, a vaccine (e.g., protein subunit vaccine), or a combination thereof can be administered to multiple anatomical sites, different immunogenic compositions including the same or different circular polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccine) or a combination thereof can be administered to different anatomical sites, different immunogenic compositions including the same or different circular polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccines) or a combination thereof can be administered to the same anatomical site, or any combination thereof. For example, an immunogenic composition including a circular polyribonucleotide can be administered in to two different anatomical sites, and / or an immunogenic composition including a circular polyribonucleotide can be administered to one anatomical site, and an adjuvant can be administered to a different anatomical site. The same immunogenic composition, an adjuvant, a vaccine (e.g., protein subunit vaccine), or a combination thereof can be administered to multiple anatomical sites, different immunogenic compositions including the same or different linear polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccine) or a combination thereof can be administered to different anatomical sites, different immunogenic compositions including the same or different linear polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccines) or a combination thereof can be administered to the same anatomical site, or any combination thereof. For example, an immunogenic composition including a linear polyribonucleotide can be administered in to two different anatomical sites, and / or an immunogenic composition including a linear polyribonucleotide can be administered to one anatomical site, and an adjuvant can be administered to a different anatomical site.Immunization at any two or more anatomical routes can be via the same route of immunization (e.g., intramuscular) or by two or more routes of immunization. In some embodiments, an immunogenic composition including a circular polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 anatomical sites of a subject. In some embodiments, an immunogenic composition including a circular polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 anatomical sites of the subject, or less. In some embodiments, an immunogenic composition including a circular polyribonucleotide or an adjuvantATTORNEY DOCKET: 51719-011 WO2 PATENT of the disclosure is immunized to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 anatomical sites of a subject. In some embodiments, an immunogenic composition including a linear polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at least1 , at least 2, at least 3, at least 4, at least 5, or at least 6 anatomical sites of a subject. In some embodiments, an immunogenic composition including a linear polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at most2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 anatomical sites of the subject, or less. In some embodiments, an immunogenic composition including a linear polyribonucleotide or an adjuvant of the disclosure is immunized to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 anatomical sites of a subject.Immunization can be by any suitable route. Non-limiting examples of immunization routes include intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intracerebroventricular, intracisternal, or intraparenchymal, e.g., injection and infusion. In some cases, immunization can be via inhalation. Two or more immunizations can be done by the same route or by different routes.Any suitable amount of a circular polyribonucleotide can be administered to a subject of the disclosure. For example, a subject can be immunized with at least about 1 ng, at least about 10 ng, at least about 100 ng, at least about 1 pg, at least about 10 pg, at least about, at least about 100 pg, at least about 1 mg, at least about 10 mg, at least about 100 mg, or at least about 1 g of a circular polyribonucleotide. In some embodiments, a subject can be immunized with at most about 1 ng, at most about 10 ng, at most about 100 ng, at most about 1 pg, at most about 10 pg, at most about, at most about 100 pg, at most about 1 mg, at most about 10 mg, at most about 100 mg, or at most about 1 g of a circular polyribonucleotide. In some embodiments, a subject can be immunized with about 1 ng, about 10 ng, about 100 ng, about 1 pg, about 10 pg, about, about 100 pg, about 1 mg, about 10 mg, about 100 mg, or about 1 g of a circular polyribonucleotide.In some embodiments, the method further includes evaluating the subject for antibody response to the immunogen. In some embodiments, the evaluating is before and / or after administration of the circular polyribonucleotide including a sequence encoding an immunogen. In some embodiments, the evaluating is before and / or after administration of the linear polyribonucleotide including a sequence encoding an immunogen.Cell-Penetrating AgentsThe cell-penetrating agent described herein can include any substance that enhances delivery of a polyribonucleotide into a cell. The cell-penetrating agent can include an organic compound or an inorganic molecule. In some cases, the cell-penetrating agent is an organic compound having one or more functional groups such as, but not limited to, alkane, alkene, and arene; halogen-substituted alkane, alkenes, and arenes; alcohols, phenols (derivatives of benzene), ethers, aldehydes, ketones, and carboxylic acids; amines and nitriles; and organosulfurs (e.g., dimethyl sulfoxide). In some embodiments, the cell-penetrating agent is soluble in polar solvents. In some embodiments, the cell-penetrating agent is insoluble in polar solvents. The polyribonucleotide can be present in either linear or circular form.ATTORNEY DOCKET: 51719-01 1 WO2 PATENTThe cell-penetrating agent can include organic compounds such as alcohols having one or more hydroxyl function groups. In some cases, the cell-penetrating agent includes an alcohol such as, but not limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic alcohols, and alicyclic alcohols. The cell-penetrating agent can include one or more of methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl alcohol, specially denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycols (PEG)-400, ethoxylated fatty acids, or hydroxyethylcellulose. In certain embodiments, the cell-penetrating agent comprises ethanol.In other cases, the compositions and methods provided herein only include an alcohol as the cellpenetrating agent, and do not have or use any other agent to enhance the delivery of the polyribonucleotide into a cell. In some cases, the cell-penetrating agent comprises ethanol and any other alcohol that can enhance delivery of polyribonucleotide into a cell. In some cases, the cell-penetrating agent comprises ethanol and any other organic or inorganic molecules that can enhance delivery of polyribonucleotide into a cell. In some cases, the cell-penetrating agent comprises ethanol and liposome or nanoparticles such as those described in International Publication Nos. WO2013 / 006825, WO2016 / 036735, WO2018 / 1 12282A1 , and WO2012 / 031043A1 , each of which is incorporated herein by reference in its entirety. In some cases, the cell-penetrating agent comprises ethanol and cell-penetrating peptides or proteins such as those described in Bechara et al, Cell-penetrating peptides: 20 years later, where do we stand? FEBS Letters 587(12):1693-1702 (2013); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al., Curr. Pharm. Des. 1 1 (28):3597-61 1 (2003); Deshayes et al, Cell. Mol. Life Sci. 62(16):1839-49 (2005), US Patent Publication Nos. US20130129726, US20130137644 and US20130164219, each of which is herein incorporated by reference in its entirety). In some cases, the ratio of ethanol versus other cell-penetrating agent is about 1 :0.001 , 1 :0.002, 1 : 005, 1 :008, 1 :0.01 , 1 :0.02, 1 :0.05, 1 :0.08, 1 : 0.1 , 1 : 0.2, 1 : 0.3, 1 :0.4, 1 :0.5, 1 :0.6, 1 :0.7, 1 :0.8, 1 :0.9, 1 :1 , 1 :1 .2, 1 : 1 .5, 1 : 1 .8, 1 : 2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :15, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 :100, 1 :120, 1 :150, 1 :200, 1 :250, 1 :500, or 1 :1000. In some cases, the ratio of ethanol versus other cell-penetrating agent is at least about 1 :0.001 , 1 :0.002, 1 : 005, 1 :008, 1 :0.01 , 1 :0.02, 1 :0.05, 1 :0.08, 1 : 0.1 , 1 : 0.2, 1 : 0.3, 1 :0.4, 1 :0.5, 1 :0.6, 1 :0.7, 1 :0.8, 1 :0.9, 1 :1 , 1 :1 .2, 1 : 1 .5, 1 : 1 .8, 1 : 2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :15, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 :100, 1 :120, 1 :150, 1 :200, 1 :250, or 1 :500.The composition disclosed herein can include a mixture of a cell-penetrating agent and a polyribonucleotide. In some cases, the polyribonucleotide is present in a pre-mixed mixture with the cellpenetrating agent. In some cases, the polyribonucleotide is provided separately from the cell-penetrating agent prior to contact to a cell. In these instances, the polyribonucleotide is contacted with the cellpenetrating agent when being applied to a cell and becomes mixed together for delivery of the polyribonucleotide into the cell. Without being bound to a certain theory, the concentration of the cellpenetrating agent in the mixture can contribute to the efficiency of delivery. Therefore, in some cases, the cell-penetrating agent is provided at a predetermined concentration in the mixture. In some other cases, when the cell-penetrating agent and the polyribonucleotide are separate initially but mixed together when being applied for delivery, the cell-penetrating agent is provided at a sufficient amount relative to the polyribonucleotide that would ensure it reach a minimum predetermined concentration in the mixture.ATTORNEY DOCKET: 51719-011 WO2 PATENTIn some cases, the cell-penetrating agent constitutes at least about 0.01 %, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% volume per volume (v / v) of the mixture. In some cases, the cell-penetrating agent constitutes at most about 0.01%, at most about 0.02%, at most about 0.03%, at most about 0.04%, at most about 0.05%, at most about 0.06%, at most about 0.07%, at most about 0.08%, at most about 0.09%, at most about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% v / v of the mixture. In some cases, the cell-penetrating agent constitutes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% v / v of the mixture.In some cases, the cell-penetrating agent constitutes at least about 0.01 %, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% weight per weight (w / w) of the mixture. In some cases, the cell-penetrating agent constitutes at most about 0.01%, at most about 0.02%, at most about 0.03%, at most about 0.04%, at most about 0.05%, at most about 0.06%, at most about 0.07%, at most about 0.08%, at most about 0.09%, at most about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% w / w of the mixture. In some cases, the cell-penetrating agent constitutes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98% w / w of the mixture. In some cases, the cell-penetrating agent constitutes about 10% v / v of the mixture.In some cases, the mixture described herein is a liquid solution. For instance, the cell-penetrating agent is a liquid substance itself. Alternatively, the cell-penetrating agent is a solid, liquid, or gas substance and dissolved in a liquid carrier, e.g., water. In these cases, the polyribonucleotide can also be dissolved in the liquid solution.In some cases, ethanol constitutes at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% volume per volume (v / v) of the mixture. In some cases, ethanol constitutes at most about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,ATTORNEY DOCKET: 51719-011 WO2 PATENT0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% v / v of the mixture. In some cases, ethanol constitutes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% v / v of the mixture. In some cases, ethanol constitutes about 10% v / v of the mixture.PreservativesA composition or pharmaceutical composition provided herein can comprise material for a single administration, or can comprise material for multiple administrations (e.g., a “multidose” kit). The polyribonucleotide can be present in either linear or circular form. The composition or pharmaceutical composition can include one or more preservatives such as thiomersal or 2-phenoxyethanol. Preservatives can be used to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M, or other agents known to those skilled in the art. In ophthalmic products, e.g., such preservatives can be employed at a level of from 0.004% to 0.02%. In the compositions described herein the preservative, e.g., benzalkonium chloride, can be employed at a level of from 0.001 % to less than 0.01 %, e.g., from 0.001 % to 0.008%, preferably about 0.005% by weight.Polyribonucleotides can be susceptible to RNase that can be abundant in ambient environment. Compositions provided herein can include reagents that inhibit RNase activity, thereby preserving the polyribonucleotide from degradation. In some cases, the composition or pharmaceutical composition includes any RNase inhibitor known to one skilled in the art. Alternatively or additionally, the polyribonucleotide, and cell-penetrating agent and / or pharmaceutically acceptable diluents or carriers, vehicles, excipients, or other reagents in the composition provided herein can be prepared in RNase-free environment. The composition can be formulated in RNase-free environment.In some cases, a composition provided herein can be sterile. The composition can be formulated as a sterile solution or suspension, in suitable vehicles, known in the art. The composition can be sterilized by conventional, known sterilization techniques, e.g., the composition can be sterile filtered.SaltsIn some cases, a composition or pharmaceutical composition provided herein comprises one or more salts. For controlling the tonicity, a physiological salt such as sodium salt can be included a composition provided herein. Other salts can comprise potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, or the like. In some cases, the composition is formulated with one or more pharmaceutically acceptable salts. The one or more pharmaceutically acceptable salts can comprise those of the inorganic ions, such as, for example, sodium, potassium, calcium, magnesium ions, and the like. Such salts can comprise salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. The polyribonucleotide can be present in either linear or circular form.ATTORNEY DOCKET: 51719-011 WO2 PATENTBuffers / pHA composition or pharmaceutical composition provided herein can comprise one or more buffers, such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (e.g., with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included in the 5-20 mM range.A composition or pharmaceutical composition provided herein can have a pH between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8. The composition or pharmaceutical composition can have a pH of about 7. The polyribonucleotide can be present in either linear or circular form.Detergents / surfactantsA composition or pharmaceutical composition provided herein can comprise one or more detergents and / or surfactants, depending on the intended administration route, e.g., polyoxyethylene sorbitan esters surfactants (commonly referred to as “Tweens”), e.g., polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, e.g., octoxynol-9 (Triton X-100, or t- octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as “SPANs”), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, an octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide (“CTAB”), or sodium deoxycholate. The one or more detergents and / or surfactants can be present only at trace amounts. In some cases, the composition can include less than 1 mg / ml of each of octoxynol-10 and polysorbate 80. Non-ionic surfactants can be used herein. Surfactants can be classified by their “HLB” (hydrophile / lipophile balance). In some cases, surfactants have a HLB of at least 10, at least 15, and / or at least 16. The polyribonucleotide can be present in either linear or circular form.DiluentsIn some embodiments, an immunogenic composition of the disclosure includes a circular polyribonucleotide and a diluent. In some embodiments, an immunogenic composition of the disclosure includes a linear polyribonucleotide and a diluent.A diluent can be a non-carrier excipient. A non-carrier excipient serves as a vehicle or medium for a composition, such as a circular polyribonucleotide as described herein. A non-carrier excipient serves as a vehicle or medium for a composition, such as a linear polyribonucleotide as described herein. Nonlimiting examples of a non-carrier excipient include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersing agents, granulating agents, disintegrating agents, binding agents, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof. A non-carrier excipient can be any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database that does not exhibit a cell-penetrating effect. A non-ATTORNEY DOCKET: 51719-011 WO2 PATENT carrier excipient can be any inactive ingredient suitable for administration to a non-human animal, for example, suitable for veterinary use. Modification of 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.In some embodiments, the circular polyribonucleotide may be delivered as a naked delivery formulation, such as including a diluent. A naked delivery formulation delivers a circular polyribonucleotide, to a cell without the aid of a carrier and without modification or partial or complete encapsulation of the circular polyribonucleotide, capped polyribonucleotide, or complex thereof.A naked delivery formulation is a formulation that is free...

Claims

ATTORNEY DOCKET: 51719-011 WO2 PATENTCLAIMS1 . A linear polyribonucleotide comprising, from 5’-to-3’, a first circularization element, an open reading frame (ORF) encoding a plasmodium falciparum circumsporozoite (CSP) polypeptide having at least 85% sequence identity to of SEQ ID NO: 9, and a second circularization element.

2. The linear polyribonucleotide of claim 1 , wherein the CSP polypeptide comprises the sequence of SEQ ID NO: 9.

3. The linear polyribonucleotide of claim 1 or 2, wherein the ORF comprises a sequence having at least 85% sequence identity to the any one of SEQ ID NOs: 1 -8.

4. The linear polyribonucleotide of claim 3, wherein the ORF comprises the sequence of any one of SEQ ID NOs: 1 -8.

5. The linear polyribonucleotide of any one of claims 1 -4, wherein the first circularization element comprises, from 5’-to-3’, a 3’ portion of a group I catalytic intron fragment, a 3’ splice site, and a 3’ exon fragment, and wherein the second circularization element comprises, from 5’-to-3’, a 5’ portion of a group I catalytic intron fragment, a 5’ splice site, and a 5’ exon fragment.

6. The linear polyribonucleotide of claim 5, wherein the first circularization element comprises a sequence having at least 85% sequence identity to AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAG ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGA AAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA (SEQ ID NO: 10).

7. The linear polyribonucleotide of claim 5, wherein the first circularization element comprises the sequence of AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAG ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGA AAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA (SEQ ID NO: 10).

8. The linear polyribonucleotide of any one of claims 5-7, wherein the second circularization element comprises a sequence having at least 85% sequence identity to CCCACACGACCGTTTAGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGA TGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTT (SEQ ID NO: 11 ).

9. The linear polyribonucleotide of claim 8, wherein the second circularization element comprises the sequence of CCCACACGACCGTTTAGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGAATTORNEY DOCKET: 51719-01 1 WO2 PATENTTGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTT (SEQ ID NO: 1 1 ).

10. The linear polyribonucleotide of any one of claims 1 -9, further comprising an internal ribosomal entry site (IRES) operatively linked to the ORF.1 1 . The linear polyribonucleotide of any one of claims 1 -10, further comprising a spacer region positioned between the first circularization element and the ORF.

12. The linear polyribonucleotide of claim 1 1 , wherein the spacer region comprises a polyA-C or a polyA- T sequence.

13. The linear polyribonucleotide of claim 1 1 or 12, wherein the spacer region is from 5 to 500 ribonucleotides in length.

14. The linear polyribonucleotide of claim 13, wherein the spacer region is from 50 to 150 ribonucleotides in length.

15. The linear polyribonucleotide of any one of claims 1 -14, further comprising a spacer region positioned between the ORF and the second circularization element.

16. The linear polyribonucleotide of claim 10, wherein the linear polyribonucleotide comprises a first spacer region positioned between the first circularization element and the IRES and a second spacer region positioned between the ORF and the second circularization element.

17. The linear polyribonucleotide of claim 1 1 , wherein the first spacer and / or the second spacer comprises a polyA-C or a polyA-T sequence.

18. The linear polyribonucleotide of any one of claims 10-17, wherein the IRES is a Coxsackievirus B3 (CVB3) IRES.

19. The linear polyribonucleotide of claim 18, wherein the CVB3 IRES comprises the sequence of TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA CTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG GCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTG CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAA TTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCATTORNEY DOCKET: 51719-01 1 WO2 PATENTTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACA GCAA (SEQ ID NO: 12).

20. The linear polyribonucleotide of claim 18, wherein the CVB3 IRES comprises the sequence ofCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGAT CAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTC GTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTG CGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTA TTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTG TTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGAT TGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAG AGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAA (SEQ ID NO: 96).21 . The linear polyribonucleotide of any one of claim 1 -20, wherein the linear polyribonucleotide is from 500 to 20,000 ribonucleotides.

22. The linear polyribonucleotide of any one of claim 1 -21 , wherein the linear polyribonucleotide is at least 1 ,000 ribonucleotides.

23. The linear polyribonucleotide of any one of claim 1 -22, wherein the ORF further encodes a signal sequence.

24. The linear polyribonucleotide of claim 24, wherein the signal sequence is an IL-2 signal sequence.

25. A DNA vector comprising an RNA polymerase promoter operably linked to a DNA sequence that encodes the linear polyribonucleotide of any one of claims 1 -24.

26. A circular polyribonucleotide produced from the linear polyribonucleotide of any one of claims 1 -24 or from the DNA vector of claim 25.

27. A circular polyribonucleotide comprising (a) a splice junction joining a 5’ exon fragment and a 3’ exon fragment and (b) an ORF encoding a CSP polypeptide having at least 85% sequence identity to SEQ ID NO: 9.

28. The circular polyribonucleotide of claim 27, wherein the CSP polypeptide comprises the sequence of SEQ ID NO: 9.

29. The circular polyribonucleotide of claim 27 or 28, wherein the ORF comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 -8.ATTORNEY DOCKET: 51719-011 WO2 PATENT30. The circular polyribonucleotide of claim 29, wherein the ORF comprises the sequence of any one of SEQ ID NOs: 1 -8.31 . The circular polyribonucleotide of any one of claims 27-30, wherein the circular polyribonucleotide further comprises an IRES operably linked to the ORF.

32. The circular polyribonucleotide of claim 31 , wherein the circular polyribonucleotide further comprises a spacer region between the ORF and the splice junction and / or the splice junction and the IRES.

33. The circular polyribonucleotide of claim 32, wherein the spacer region is at least 5 ribonucleotides in length.

34. The circular polyribonucleotide of claim 33, wherein the spacer region is from 5 to 500 ribonucleotides in length.

35. The circular polyribonucleotide of any one of claims 32-34, wherein the spacer region comprises a polyA-C or polyA-T sequence.

36. The circular polyribonucleotide of any one of claims 26-35, wherein the circular polyribonucleotide is at least 500 ribonucleotides in length.

37. The circular polyribonucleotide of claim 36, wherein the circular polyribonucleotide is from 500 to 20,000 ribonucleotides in length.

38. A polynucleotide comprising an ORF comprising a sequence having at least 95% sequence identity to the sequence of any one of SEQ ID NOs: 1 -8.

39. The polynucleotide of claim 38, wherein ORF comprises the sequence of any one of SEQ ID NOs: 1 - 8.

40. The polynucleotide of claim 38 or 39, further comprising an IRES operably linked to the ORF.41 . The polynucleotide of any one of claims 38-40, wherein the polynucleotide is a polyribonucleotide.

42. The polynucleotide of claim 41 , wherein the polyribonucleotide is a linear polyribonucleotide.

43. The polynucleotide of claim 41 , wherein the polyribonucleotide is a circular polyribonucleotide.

44. A DNA vector comprising an RNA polymerase promoter operably linked to a DNA sequence that encodes the polynucleotide of any one of claims 38-42.ATTORNEY DOCKET: 51719-011 WO2 PATENT45. An immunogenic composition comprising the circular polyribonucleotide of any one of claims 26-37 or the polynucleotide of claim 43.

46. A pharmaceutical composition comprising the circular polyribonucleotide of any one of claims 26-37, the polynucleotide of claim 43, or the immunogenic composition of claim 45, and a pharmaceutically acceptable excipient.

47. The pharmaceutical composition of claim 46, further comprising an adjuvant.

48. The pharmaceutical composition of claim 47, wherein the adjuvant is an inorganic adjuvant, a small molecule adjuvant, and oil in water emulsion, a lipid or polymer, a peptide or peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a viral particle, a bacterial adjuvant, a hybrid molecule, a fungal or oocyte microbe-associated molecular pattern (MAMP), an inorganic nanoparticle, or a multi-component adjuvant.

49. A method of treating or preventing malaria in a subject, the method comprising administering to the subject the circular polyribonucleotide of any one of claims 26-37, the polynucleotide of claim 43, the immunogenic composition of claim 45, or the pharmaceutical composition of claim 46.

50. A method of inducing an immune response in a subject, the method comprising administering to the subject the circular polyribonucleotide of any one of claims 26-37, the polynucleotide of claim 43, the immunogenic composition of claim 45, or the pharmaceutical composition of claim 46.51 . The method of claim 49 or 50, wherein the method further comprises administering an adjuvant to the subject.

52. The method of claim 51 , wherein the adjuvant is an inorganic adjuvant, a small molecule adjuvant, and oil in water emulsion, a lipid or polymer, a peptide or peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a viral particle, a bacterial adjuvant, a hybrid molecule, a fungal or oocyte microbe-associated molecular pattern (MAMP), an inorganic nanoparticle, or a multi-component adjuvant.

53. The method of any one of claims 49-52, wherein the circular polyribonucleotide is administered to the subject as a single dose.

54. The method of any one of claims 49-52, wherein the circular polyribonucleotide is administered to the subject two or more times, three or more times, four or more times, or five or more times.

55. The method of claim 54, wherein administration of the circular polyribonucleotide occurs about weekly, about every two weeks, about every three weeks, about every month, about every two months, about every three months, about every four months, about every five months, about every six months,ATTORNEY DOCKET: 51719-011 WO2 PATENT about every year, about every two years, about every three years, about every four years, about every five years, or about every ten years.

56. The method of any one of claims 49-55, wherein the method further comprises administering to the subject a plasmodium circumsporozoite polypeptide.

57. The method of claim 56, wherein the plasmodium circumsporozoite polypeptide is administered to the subject after administering the circular polyribonucleotide.

58. The method of claim 56 or 57, wherein administration of the plasmodium circumsporozoite polypeptide maintains or enhances an immune response in the subject against the plasmodium circumsporozoite polypeptide.

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