CN118922211A - Cyclic polyribonucleotides encoding antifusogenic polypeptides - Google Patents

Abstract

The present disclosure generally relates to compositions and methods for producing, purifying, and using circular RNA encoding anti-fusion polypeptides.

Description

Cyclic polyribonucleotide encoding anti-fusion polypeptide

Background Art

The delivery of polynucleotides and proteins is important for a wide range of therapeutic areas. However, current delivery methods are often ineffective. For example, the delivery of short polypeptides (such as polypeptides encoding anti-fusion polypeptides) generally results in a short half-life and rapid clearance of the polypeptides. Therefore, there is a need for improved compositions and methods for delivering anti-fusion polypeptides, for example, for the treatment or prevention of viral infections.

Summary of the invention

The present disclosure provides compositions and methods for producing, purifying, and using circular RNA encoding anti-fusion polypeptides.

In one aspect, the invention features a cyclic polyribonucleotide comprising a polyribonucleotide cargo encoding an anti-fusion polypeptide. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding an anti-fusion polypeptide.

In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity with a polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity with any one of SEQ ID NOs: 1-324.

In some embodiments, the circular polyribonucleotide comprises a splice junction connecting a 5' exonic fragment and a 3' exonic fragment.

In some embodiments, the polyribonucleotide cargo includes an IRES operably connected to an expression sequence encoding an anti-fusion polypeptide. The cyclic polyribonucleotide may further include a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The length of the spacer region may be at least 5 ribonucleotides. For example, the length of the spacer region may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more ribonucleotides. In some embodiments, the length of the spacer region is 5 to 500 ribonucleotides. The spacer region may include poly-A, poly-A-C, poly-A-U or poly-A-G sequences. The spacer region may be a random sequence.

In certain embodiments, the length of the cyclic polyribonucleotide is at least 500 ribonucleotides.For example, the cyclic polyribonucleotide can be at least 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000 or more ribonucleotides.In certain embodiments, the length of the cyclic polyribonucleotide is 500 to 20,000 ribonucleotides.

On the other hand, it is characterized in that linear polyribonucleotide, described linear polyribonucleotide comprises (A) 3' intron fragment from 5' to 3'; (B) 3' splice site; (C) 3' exon fragment; (D) polyribonucleotide cargo encoding described anti-fusion polypeptide; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' intron fragment.

In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding an anti-fusion polypeptide.

In some embodiments, the polyribonucleotide cargo comprises an IRES operably connected to an expressed sequence encoding an anti-fusion polypeptide (e.g., a polypeptide of Table 1). The cyclic polyribonucleotide may further comprise a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The cyclic polyribonucleotide may further comprise a spacer region between one or more of (A), (B), (C), (D), (E), (F) and (G).

The length of the spacer region can be at least 5 ribonucleotides. For example, the length of the spacer region can be at least 10,20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900,1000 or more ribonucleotides. In certain embodiments, the length of the spacer region is 5 to 500 ribonucleotides. The spacer region can include poly-A, poly-A-C, poly-A-U or poly-A-G sequences. The spacer region can be a random sequence.

In some embodiments, the cyclic polyribonucleotide lacks an IRES. In some embodiments, the cyclic polyribonucleotide lacks one or both of a 5' cap and a poly A sequence.

In some embodiments, the cyclic polyribonucleotide comprises a protein translation initiation site. In some embodiments, the protein translation initiation site comprises a Kozak sequence.

In certain embodiments, the length of the linear polyribonucleotide is at least 500 ribonucleotides. For example, the linear polyribonucleotide can be at least 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000 or more ribonucleotides. In certain embodiments, the length of the linear polyribonucleotide is 500 to 20,000 ribonucleotides.

In another aspect, a DNA vector encoding a polyribonucleotide (eg, a linear or circular polyribonucleotide) as described herein is featured.

In another aspect, a method of expressing an anti-fusion polypeptide (e.g., a polypeptide of Table 1) in a cell is featured. The method comprises providing a circular, linear polyribonucleotide or DNA vector as described herein to the cell under conditions suitable for expressing the anti-fusion polypeptide.

On the other hand, it is characterised in that the method for producing cyclic polyribonucleotides by linear polyribonucleotides as described herein.Described method is included in and provides described linear polyribonucleotides under the condition that is suitable for described linear polyribonucleotides self-splicing to produce described cyclic polyribonucleotides.

In another aspect, it is characterized by a pharmaceutical composition comprising the cyclic polyribonucleotide, linear polyribonucleotide or DNA vector of any one of the above embodiments, and a diluent, carrier or excipient.

In another aspect, a method is characterized by expressing an anti-fusion polypeptide (e.g., a polypeptide of Table 1) in a subject. The method comprises expressing an anti-fusion polypeptide (e.g., a polypeptide of Table 1) in a subject at a concentration sufficient to produce 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, The first dose of the pharmaceutical composition is administered in an amount of an anti-fusion polypeptide (e.g., a polypeptide of Table 1) that achieves a serum concentration of 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).

In some embodiments, the method may further comprise administering a second dose of the pharmaceutical composition. The method may further comprise administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more dose of the pharmaceutical composition.

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, ten months, eleven months, one year, or more) after the first dose of the pharmaceutical composition.

In some embodiments, the second dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, such as 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, such as one day to one week, such as two days, three days, four days, five days, six days, or one week, such as one week to one month, such as two weeks, three weeks or one month, such as one month to one year, such as 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 1 to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days, or 90 days to 180 days).

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, ten months, eleven months, one year, or more) after the second dose of the pharmaceutical composition.

In some embodiments, the third dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, such as 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, such as one day to one week, such as two days, three days, four days, five days, six days, or one week, such as one week to one month, such as two weeks, three weeks or one month, such as one month to one year, such as 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 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days, or 90 days to 180 days).

In some embodiments, the second dose is administered before the serum concentration of the anti-fusion polypeptide in the serum of the subject is less than about 500 ng/mL.

In some embodiments, the methods maintain 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, ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL or more), for example, 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, 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, ten months, eleven months, one year or more).

In some embodiments, the method treats or prevents a viral infection in the subject. For example, the pharmaceutical composition can be administered to the subject in an amount and duration sufficient to treat or prevent a viral infection. The pharmaceutical composition can be administered to the subject to reduce the risk of a viral infection.

In some embodiments, the methods treat or prevent human immunodeficiency virus (HIV) infection.

In some embodiments, the methods treat or prevent coronavirus infection (e.g., betacoronavirus infection, e.g., SARS-CoV-2 infection, such as SARS-CoV-2 infection that produces COVID-19 symptoms).

In some embodiments, the methods treat or prevent hepatitis C virus (HCV) infection.

In some embodiments, circular polynucleotides encoding anti-fusion polypeptides (eg, polypeptides of Table 1) are used to reduce viral entry.

On the other hand, it is characterized in that the cyclic polyribonucleotide comprises a polyribonucleotide cargo encoding multiple anti-fusion polypeptides. The polyribonucleotide cargo may comprise an expression sequence encoding an anti-fusion polypeptide. In some embodiments, the anti-fusion polypeptide is directed against the same virus. Alternatively, the anti-fusion polypeptide may be directed against more than one virus.

definition

In order to promote the understanding of the present disclosure, a plurality of terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the field relevant to the present disclosure. Terms such as "a, an" and "said" are not intended to refer only to a single entity, but include general categories that can be illustrated using specific examples. The term "or" is used to mean "and/or", unless explicitly stated to refer only to alternatives or alternatives are mutually exclusive, although the present disclosure supports the definition of referring only to alternatives and "and/or". The terms herein are used to describe specific embodiments, but their use should not be considered as limiting, unless listed in the claims.

As used herein, any value provided within a range of values includes the upper and lower limits, and any value contained within the upper and lower limits.

As used herein, the term "about" refers to a value within ±10% of the recited value.

As used herein, the term "carrier" is a compound, composition, agent or molecule that promotes the transport or delivery of a composition (e.g., cyclic polyribonucleotide) to a cell by covalent modification of cyclic polyribonucleotides, via a partial or complete encapsulating agent or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-based materials), nanoparticles (e.g., nanoparticles encapsulated or covalently linked/bound to cyclic polyribonucleotides), liposomes, fusogens, reticulocytes differentiated in vitro, exosomes, protein carriers (e.g., proteins covalently linked to cyclic polyribonucleotides) or cationic carriers (e.g., cationic lipopolymers or transfection agents).

As used herein, the terms "cyclic polyribonucleotide", "circular RNA" and "circRNA" are used interchangeably, and refer to polyribonucleotide molecules with a structure without free ends (i.e., without free 3' or 5' ends), such as polyribonucleotide molecules that form a circular or endless structure by covalent or non-covalent bonds. Circular polyribonucleotides can be, for example, covalently closed polyribonucleotides.

As used herein, the term "cyclization efficiency" is a measure of the resulting circular polyribonucleotide relative to its non-cyclic starting material.

The term "diluent" means a medium comprising an inactive solvent, in which the compositions described herein (e.g., compositions comprising cyclic polyribonucleotides) can be diluted or dissolved. The diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof. The diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizers and emulsifiers (such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and 1,3-butylene glycol. Non-limiting 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, corn starch or powdered sugar.

As used herein, the terms "disease," "disorder," and "condition" each refer to a sub-health state, eg, a state that is or would normally be diagnosed or treated by a medical professional.

As used herein, the term "expressed sequence" is a nucleic acid sequence encoding a product such as a peptide or polypeptide (e.g., an anti-fusion polypeptide). An exemplary expressed sequence encoding a peptide or polypeptide may include multiple nucleotide triplets, each of which may encode an amino acid and is referred to as a "codon."

As used herein, the term "fragment" with respect to a polypeptide or nucleic acid sequence (e.g., an anti-fusion polypeptide or a nucleic acid sequence encoding an anti-fusion polypeptide) refers to a continuous, less than complete portion of a polypeptide or nucleic acid sequence. For example, a fragment of a polypeptide or a nucleic acid sequence encoding a polypeptide refers to a continuous, less than complete portion of a sequence (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the entire length) of a sequence (such as a sequence disclosed herein). It should be understood that all contents of the present disclosure contemplate fragments of any anti-fusion polypeptide disclosed herein.

As used herein, the term "Fc domain" refers to a polypeptide chain comprising at least a hinge domain and a second and third antibody constant domain ( _CH2 and _CH3 ) or a functional fragment thereof (e.g., a fragment capable of dimerization and binding to an Fc receptor). The Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA or IgD (e.g., IgG). In addition, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3 or IgG4) (e.g., IgG1). The Fc domain does not include any part of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementary determining region (CDR). The Fc domain in a conjugate as described herein can include one or more changes (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or deletions) in a wild-type Fc domain sequence that alter the interaction between the Fc domain and the Fc receptor. Examples of suitable changes are known in the art. Unless otherwise indicated herein, the numbering of amino acid residues in an IgG or Fc domain is according to the EU numbering system for antibodies, also known as the Kabat EU index, as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991.

As used herein, the term "GC content" refers to the percentage of guanine (G) and cytosine (C) in a nucleic acid sequence. The formula for calculating GC content is (G+C)/(A+G+C+U)×100% (for RNA) or (G+C)/(A+G+C+T)×100% (for DNA). Similarly, the term "uridine content" refers to the percentage of uridine (U) in a nucleic acid sequence. The formula for calculating uridine content is U/(A+G+C+U)×100%. Similarly, the term "thymidine content" refers to the percentage of thymidine (T) in a nucleic acid sequence. The formula for calculating thymidine content is T/(A+G+C+T)×100%.

"Heterologous" means occurring in a context different from that which occurs in nature (native). A "heterologous" polynucleotide sequence indicates that the polynucleotide sequence is used in a manner different from that found in the natural genome of the sequence. For example, a "heterologous promoter" is used to drive transcription of a sequence that is not a sequence that is naturally transcribed by the promoter; thus, "heterologous promoter" sequences are typically included in expression constructs by recombinant nucleic acid techniques. The term "heterologous" is also used to refer to a given sequence that is placed in a non-naturally occurring relationship with another sequence; for example, a heterologous coding or non-coding nucleotide sequence is typically inserted into a genome by genome transformation techniques, resulting in a genetically modified genome or a recombinant genome.

As used herein, the term "intron fragment" refers to a portion of an intron, wherein a first intron fragment and a second intron fragment together form an intron, such as a catalytic intron. An intron fragment can be a 5' portion of an intron (e.g., a 5' portion of a catalytic intron) or a 3' portion of an intron (e.g., a 3' portion of a catalytic intron), such that the 5' intron fragment and the 3' intron fragment together form a functional intron, such as a functional intron capable of catalyzing self-splicing. The term intron fragment means that an intron is divided into two parts. The term intron fragment is not intended to indicate, imply, or indicate that the two parts or halves are of equal length. The term intron fragment is used synonymously with the term split intron and can be used in place of the term "half intron".

As used herein, term " linear counterpart " is to have the nucleotide sequence identical or similar to cyclic polyribonucleotide (for example, 100%, 95%, 90%, 85%, 80%, 75% or the sequence identity of any percentage between) and have two free ends (that is, the uncyclized form (and its fragment) of cyclic polyribonucleotide).In certain embodiments, linear counterpart (for example, form before cyclization) is to have the nucleotide sequence identical or similar to cyclic polyribonucleotide (for example, 100%, 95%, 90%, 85%, 80%, 75% or any percentage sequence identity between) and have the polyribonucleotide molecule (and its fragment) of identical or similar nucleic acid modification and have two free ends (that is, the uncyclized form (and its fragment) of cyclic polyribonucleotide). In some embodiments, the linear counterpart is a polyribonucleotide molecule (and its fragment) having a nucleotide sequence identical or similar to a cyclic polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75% or any percentage of sequence identity therebetween) and different nucleic acid modifications or no nucleic acid modifications and having two free ends (i.e., the uncyclized form (and its fragment) of a cyclic polyribonucleotide). In some embodiments, the fragment of a polyribonucleotide molecule as a linear counterpart is any portion of a linear counterpart polyribonucleotide molecule 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 polyadenosine 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 polyribonucleotides" and "linear polyribonucleotide molecules" are used interchangeably and refer to polyribonucleotide molecules with 5' and 3' ends. One or both of the 5' and 3' ends can be free ends or can be connected to another part. Linear RNA includes RNA that has not yet undergone cyclization (e.g., before cyclization), and can be used as starting material to be cyclized by, for example, splint connection or chemical, enzymatic, ribozyme or splicing catalyzed cyclization methods.

As used herein, the term "modified ribonucleotide" means a nucleotide having at least one modification to the sugar, the nucleobase, or the internucleoside linkage.

As used herein, the term "naked delivery" is for delivery to cells without the help of a carrier and without the need for a covalently modified formulation that helps to deliver to the part of the cell. The naked delivery formulation does not contain any transfection reagent, cationic carrier, carbohydrate carrier, nanoparticle carrier or protein carrier. For example, the naked delivery formulation of cyclic polyribonucleotides includes cyclic polyribonucleotides without covalent modifications and does not contain a formulation of a carrier.

As used herein, the terms "nicked RNA" and "nicked linear polyribonucleotide" and "nicked linear polyribonucleotide molecule" can be used interchangeably and mean the polyribonucleotide molecule with 5' end and 3' end produced due to circular RNA nicking or degradation.

The term "pharmaceutical composition" is intended to disclose that the cyclic or linear polyribonucleotides included in the pharmaceutical composition can be used for treating human or animal bodies through therapy. Therefore, this means being equal to "polyribonucleotides for use in therapy".

As used herein, the term "polynucleotide" means a molecule comprising one or more nucleic acid subunits or nucleotides, and can be used interchangeably with "nucleic acid" or "oligonucleotide". A polynucleotide may include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. A nucleotide may include a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate (PO ₃ ) groups. A nucleotide may include a nucleobase, a pentose (ribose or deoxyribose) and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. A polyribonucleotide or ribonucleic acid or RNA may refer to a macromolecule comprising a plurality of ribonucleotides polymerized via a phosphodiester bond. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose. As used herein, a polyribonucleotide sequence describing thymine (T) should be understood to represent uracil (U).

As used herein, the term "polyribonucleotide cargo" herein includes any sequence comprising at least one polyribonucleotide. In an embodiment, the polyribonucleotide cargo includes one or more expressed sequences, wherein each expressed sequence encodes a polypeptide. In an embodiment, the polyribonucleotide cargo includes one or more non-coding sequences, such as polyribonucleotides with regulatory or catalytic functions. In an embodiment, the polyribonucleotide cargo includes a combination of expressed sequences and non-coding sequences. In an embodiment, the polyribonucleotide cargo includes one or more polyribonucleotide sequences described herein, such as one or more regulatory elements, internal ribosome entry site (IRES) elements or spacer sequences.

As used interchangeably herein, the terms "poly A" and "poly A sequence" refer to a non-translated continuous region of a nucleic acid molecule that is at least 5 nucleotides long and consists of adenosine residues. In some embodiments, the length of the poly A sequence is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides. In some embodiments, the poly A sequence is located 3' (e.g., downstream) of an open reading frame (e.g., an open reading frame encoding a polypeptide), and the poly A sequence is located 3' of a termination element (e.g., a stop codon) such that the poly A is not translated. In some embodiments, the poly A sequence is located 3' of the termination element and is a 3' non-translated region.

As used herein, elements of a nucleic acid are "operably connected" or "operably linked" if they are located on a vector such that they can be transcribed to form a linear RNA, which can then be circularized into a circular RNA using the methods provided herein.

"Polydeoxyribonucleotide", "deoxyribonucleic acid" and "DNA" mean a macromolecule comprising a plurality of deoxyribonucleotides polymerized via phosphodiester bonds. Nucleotides may be monophosphate nucleosides or polyphosphate nucleosides. Nucleotides mean polyphosphate deoxyribonucleosides including detectable labels (such as luminescent labels) or markers (e.g., fluorophores), such as deoxyribonucleoside triphosphates (dNTPs), which may be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs. Nucleotides may include any subunit that may be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T or U, or any other subunit that is specific for one or more complementary A, C, G, T or U or complementary to a purine (i.e., A or G or a variant thereof) or a pyrimidine (i.e., C, T or U or a variant thereof). In some instances, polynucleotides are deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or derivatives or variants thereof. In some cases, polynucleotides are short interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), and encompass nucleotide sequences and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, spiral, hairpin, etc. In some cases, polynucleotide molecules are circular. Polynucleotides can have various lengths. Nucleic acid molecules can have 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), 2kb, 3kb, 4kb, 5kb, 10kb, 50kb or a greater length. Polynucleotides can be isolated from cells or tissues. Embodiments of polynucleotides include isolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules, and synthetic DNA/RNA analogs.

Embodiments of polynucleotides (eg, polyribonucleotides or polydeoxyribonucleotides) can include one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, 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-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl Q nucleoside, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy Aminomethyl-2-thiouracil, β-D-mannosyl Q nucleoside, 5'-methoxycarboxymethyl uracil, 5-methoxy uracil, 2-methylthio-D46-isopentenyl adenine, uracil-5-hydroxyacetic acid, wybutoxosine, pseudouracil, Q nucleoside (queosine), 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2,6-diaminopurine, etc. In some cases, the nucleotide can include modifications in its phosphate portion, including modifications to the triphosphate portion. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., α-thiotriphosphates and β-thiotriphosphates). In an embodiment, the nucleic acid molecule is modified at the base moiety (e.g., at one or more atoms that are normally available for hydrogen bonding with a complementary nucleotide or at one or more atoms that are normally unable to hydrogen bond with a complementary nucleotide), the sugar moiety, or the phosphate backbone. In an embodiment, the nucleic acid molecule contains amine-modified groups such as aminoallyl 1-dUTP (aa-dUTP) and aminohexyl acrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine-reactive moieties such as N-hydroxysuccinimide ester (NHS). Substitution of standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure may provide higher density (in bits/m3), greater safety (against accidental or purposeful synthesis of natural toxins), easier discrimination of photo-programmed polymerases, or lower secondary structure. Such alternative base pairs that are compatible with natural and mutant polymerases for de novo 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 incorporated herein by reference for all purposes.

As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) most often linked together by peptide bonds. As used herein, the term 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 of the foregoing substances and other equivalents, variants and analogs. Polypeptides can be unimolecular or multimolecular complexes, such as dimers, trimers or tetramers. They can also include single-chain or multi-chain polypeptides (such as antibodies or insulin), and can be associated or connected. The most common disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

As used herein, the term "prevention" means reducing the likelihood of developing a disease, disorder or condition (e.g., a viral infection, such as HIV, SARS-CoV-2, HCV, influenza or RSV), or alternatively, reducing the severity or frequency of symptoms of a disease or disorder that subsequently develops. Therapeutic agents can be administered to subjects who have an increased risk of developing a viral infection relative to members of the general population in order to prevent the development of a disease or condition or to mitigate the severity of a disease or condition. Therapeutic agents can be administered as preventive agents, for example, before any symptoms or manifestations of a viral infection develop.

As used herein, the term "regulatory element" is a moiety, such as a nucleic acid sequence, that modifies the expression of an expressed sequence within a circular or linear polyribonucleotide.

As used herein, "spacer" refers to any contiguous nucleotide sequence (eg, a contiguous nucleotide sequence of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.

A "signal sequence" refers to a polypeptide sequence, for example, between 10 and 45 amino acids in length, which is present at the N-terminus of the polypeptide sequence of a nascent protein and targets the polypeptide sequence to the secretory pathway.

As used herein, the term "sequence identity" is determined by comparing two peptides or two nucleotide sequences using a global or local alignment algorithm. When a sequence shares at least a certain minimum percentage of sequence identity when optimally aligned (for example, when compared using default parameters by a program such as GAP or BESTFIT), the sequence is referred to as "substantially identical" or "substantially similar". GAP uses the Needleman and Wunsch global alignment algorithm to compare the entire length of the two sequences, thereby maximizing the number of matches and minimizing the number of gaps. Typically, using the GAP default parameters, gaps generate penalty points = 50 (nucleotides) / 8 (proteins), and gap extension penalty points = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is Blosum62 (Henikoff and Henikoff, 1992, PNAS [Proceedings of the National Academy of Sciences of the United States] 89, 915-919). Sequence alignments and percentage sequence identity scores are determined, for example, using computer programs such as GCG Wisconsin Software Package Version 10.3 or EmbossWin Version 2.10.0 (using program "needle") obtained from Accelrys Inc., 9685 Scranton Road, San Diego, CA, USA 92121-3752. Alternatively or in addition, percent identity is determined by searching a database using, for example, algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

As used herein, the term "subject" refers to an organism, such as an animal, plant or microorganism. In an embodiment, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile or an amphibian). In an embodiment, the subject is a human. In an embodiment, the subject is a non-human mammal. In an embodiment, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, ape), an ungulate (e.g., a cattle, a buffalo, a bison, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse) or a lagomorph (e.g., a rabbit). In an embodiment, the subject is a bird, such as a member of the avian taxonomic group Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathus (e.g., ostrich, emu), Columbiformes (e.g., pigeon, pigeon) or Psittaciformes (e.g., parrot). In an embodiment, the subject is an invertebrate, such as an arthropod (for example, insect, arachnid, crustacean), nematode, annelid, worm or mollusk. In an embodiment, the subject is an invertebrate agricultural harmful organism or an invertebrate parasitized on an invertebrate or vertebrate host. In an embodiment, the subject is a plant, such as angiosperm (which can be a dicotyledon or monocotyledon) or gymnosperm (for example, conifer, cycad, Gnetum plant, Ginkgo), fern, horsetail plant, lycophyte or bryophyte. In an embodiment, the subject is a eukaryotic algae (single cell or multicellular). In an embodiment, the subject is a plant with agricultural or horticultural importance, such as row crops, plants and trees, vegetables, trees and ornamental plants (including ornamental flowers, shrubs, trees, ground cover plants and lawn grass) produced by fruit production.

As used herein, the term "anti-fusion polypeptide" refers to a polypeptide that inhibits viral fusion-related events (such as viral entry or viral fusion), such as a polypeptide between 10 and 200 amino acids. Anti-fusion polypeptides include, for example, the polypeptides of Table 1. Anti-fusion polypeptides include polypeptides and any biologically active fragments thereof (e.g., fragments of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 amino acids). Anti-fusion polypeptides include, for example, polypeptides targeting HIV, SARS-CoV-2, HCV or RSV. In some embodiments, anti-fusion polypeptides include polypeptides having at least 70%, such as at least 80%, such as at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity with any one of SEQ ID NO: 1-324. Anti-fusion polypeptides also refer to polynucleotides (eg, polyribonucleotides, eg, cyclic polyribonucleotides) encoding anti-fusion polypeptides (eg, polypeptides of Table 1) or biologically active fragments thereof.

As used herein, the terms "treat" and "treating" refer to the prophylactic or therapeutic treatment of a viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV) in a subject. The effect of treatment can include reversing, alleviating, reducing the severity of one or more symptoms or manifestations of a disease or viral infection, curing one or more symptoms or manifestations of a disease or viral infection, inhibiting the progression of one or more symptoms or manifestations of a disease or viral infection, reducing the likelihood of recurrence of one or more symptoms or manifestations of a disease or viral infection, or stabilizing (i.e., not worsening) the state of a viral infection or preventing the spread of a viral infection compared to the state or condition of the viral infection in the absence of therapeutic treatment.

As used herein, the term "termination element" is a moiety, such as a nucleic acid sequence, that terminates translation of an expressed sequence in a circular or linear polyribonucleotide.

As used herein, the term "translation efficiency" is the rate or amount of protein or peptide production from ribonucleotide transcripts. In some embodiments, translation efficiency can be expressed as the amount of protein or peptide produced by a given amount of transcripts encoding a protein or peptide, such as within a given time period, such as in a given translation system (e.g., a cell-free translation system, such as rabbit reticulocyte lysate).

As used herein, the term "translation initiation sequence" is a nucleic acid sequence in a circular or linear polyribonucleotide that initiates translation of an expressed sequence.

As used herein, "vector" means a section of DNA, which is synthesized (e.g., using PCR) or taken from a cell of a virus, plasmid or higher organism, into which an exogenous DNA fragment can be or has been inserted for cloning or expression purposes. In certain embodiments, the vector can be stably maintained in an organism. Selectable marker vectors can include, for example, an origin of replication, a selectable marker or a reporter gene (such as antibiotic resistance or GFP) or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, clay midds, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) and the like. In one embodiment, the vector provided herein includes a multiple cloning site (MCS). In another embodiment, the vector provided herein does not include an MCS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the protein domains or regions of various coronavirus S proteins and the sequences of the HR1 and HR2 regions.

FIG. 2 is a schematic diagram showing various anti-fusion polypeptides and sequences derived from the HR2 region of SARS CoV-2.

FIG. 3 is a schematic diagram showing an exemplary embodiment of a multi-ORF anti-fusion polypeptide construct.

Figures 4A and 4B are graphs showing the inhibitory efficacy of fusion using Omicron and Delta pseudoviruses. Figure 4A shows the % inhibition of Delta and Omicron using full length HR2 or full length HR2 with HiBiT tag. Figure 4B shows the relative expression of anti-fusion polypeptides compared to mock controls.

FIG5 is a graph showing in vivo expression of HR2 full-length anti-fusion polypeptide with and without HiBiT tag at 6 hours and 24 hours.

Figures 6A and 6B are graphs showing the in vitro neutralization of pseudoviruses of Wuhan and Omicron strains of SARS CoV-2 using HR2A polypeptides. Figure 6A shows the neutralization of the Wuhan strain of SARS CoV-2 using HR2A. Figure 6B shows the neutralization of the Omicron strain of SARS CoV-2 using HR2A.

Figures 7A and 7B are graphs showing the inhibition rate (%) of SARS CoV-2 pseudovirus Omicron BA4 and BA.5 (Figure 7A) or SARS CoV-1 pseudovirus (Figure 7B) strains.

Figures 8A-8D are graphs showing the inhibition rate (%) of SARS CoV-2 pseudovirus using full-length HR2 (HR2 Complete). Figure 8A shows the inhibition of the Wuhan strain. Figure 8B shows the inhibition of the Omicron BA.4 and BA.5 strains. Figure 8C shows the inhibition of the Omicron BA.1 strain. Figure 8D shows the inhibition of the SARS CoV-1 pseudovirus.

FIG. 9 is a schematic diagram showing the construct design and sequence of various HIV anti-fusion polypeptides.

10A and 10B are a graph ( FIG. 10A ) and a table ( FIG. 10B ) showing the expression of various HIV anti-fusion polypeptides from circular RNA.

Figures 11A and 11B are graphs showing the expression of various HIV anti-fusion polypeptides from circular RNA (Figure 11A) or plasmid DNA (Figure 11B)

FIG. 12 is a table showing the expression of various HIV anti-fusion polypeptides from circular RNA.

DETAILED DESCRIPTION

The invention features compositions containing cyclic polyribonucleotides (circRNAs) encoding anti-fusion polypeptides and methods of using the same. The cyclic polyribonucleotides described herein are particularly useful for delivering polynucleotide cargo encoding anti-fusion polypeptides to target cells.

The cyclic polyribonucleotide may include a polyribonucleotide cargo encoding a polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity with a polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity with any one of SEQ ID NOs: 1-324.

Cyclic polyribonucleotides can be produced from precursors, such as linear deoxyribonucleotides, cyclic deoxyribonucleotides or cyclic polyribonucleotides.

The circular polyribonucleotide may comprise, for example, a splice junction joining a 5' exonic fragment and a 3' exonic fragment.

The linear RNA molecules described herein are polyribonucleotides encoding anti-fusion polypeptides. In some embodiments, the linear RNA molecules include (A) 3' catalytic intron fragments from 5' to 3'; (B) 3' splice sites; (C) 3' exon fragments; (D) polyribonucleotide cargoes encoding anti-fusion polypeptides (e.g., polyribonucleotide cargoes encoding IRES operably connected to the expression sequence encoding the anti-fusion polypeptide); (E) 5' exon fragments; (F) 5' splice sites; and (G) 5' catalytic intron fragments. Then, the catalytic intron fragments and splice sites can allow the linear polyribonucleotides to self-splice, thereby forming a cyclic polyribonucleotide encoding an anti-fusion polypeptide.

The invention also features a method using a cyclic polyribonucleotide as described herein. For example, the cyclic polyribonucleotide can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a subject (e.g., a human subject). The pharmaceutical composition can be administered with a composition of one or more dosages. The composition can be administered to a subject to treat or prevent viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).

Polynucleotide

The present disclosure is characterized in that the cyclic polyribonucleotide composition of coding anti-fusion polypeptide, its purposes and the method for preparing the cyclic polyribonucleotide of coding anti-fusion polypeptide.In certain embodiments, cyclic polyribonucleotide is produced by linear polyribonucleotide (for example, by the compatible end of self-scissoring linear polyribonucleotide).In certain embodiments, linear polyribonucleotide is transcribed from deoxyribonucleotide template (for example, carrier, linearization carrier or cDNA).Therefore, the present disclosure is characterized in that deoxyribonucleotide, linear polyribonucleotide and cyclic polyribonucleotide and composition thereof can be used for producing the cyclic polyribonucleotide of coding anti-fusion polypeptide.

Template deoxyribonucleotide

The present invention is characterized in that a template deoxyribonucleotide for preparing a circular RNA as described herein. In an embodiment, the deoxyribonucleotide comprises the following items operably linked in a 5' to 3' orientation: (A) 3' catalytic intron fragment; (B) 3' splice site; (C) 3' exon fragment; (D) polyribonucleotide cargo encoding anti-fusion polypeptide; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' catalytic intron fragment. In an embodiment, the deoxyribonucleotide comprises additional elements, for example, outside or between any one of elements (A), (B), (C), (D), (E), (F) or (G). In an embodiment, any one of 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 linear DNA (eg, cDNA, such as produced from a DNA vector).

In some embodiments, the deoxyribonucleotide further comprises an RNA polymerase promoter operably linked to a sequence encoding a linear RNA as described herein. In an embodiment, 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 viral promoter, or an SP3 promoter.

In some embodiments, the deoxyribonucleotide comprises a multiple cloning site (MCS).

In some embodiments, the deoxyribonucleotides are used to generate circular RNAs ranging in size from 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 polyribonucleotide

The present invention is also characterized in that the linear polyribonucleotides encoding anti-fusion polypeptides. Linear polyribonucleotides can be used to produce cyclic polyribonucleotides, for example, by connecting or splicing (for example, self-splicing) linear polyribonucleotides to produce cyclic polyribonucleotides. In an embodiment, the linear polyribonucleotides include the following items operably connected in a 5' to 3' orientation: (A) 3' catalytic intron fragments; (B) 3' splicing sites; (C) 3' exon fragments; (D) polyribonucleotide cargo encoding anti-fusion polypeptides; (E) 5' exon fragments; (F) 5' splicing sites; and (G) 5' catalytic intron fragments. In an embodiment, the linear polyribonucleotides include other elements, for example, outside or between any one of element (A), (B), (C), (D), (E), (F) or (G). For example, any one of element (A), (B), (C), (D), (E), (F) or (G) can be separated by a spacer sequence as described herein.

In certain embodiments, provided herein is a method for generating a linear RNA encoding an anti-fusion polypeptide by transcribing (e.g., in vitro transcription) in a cell-free system using a deoxyribonucleotide encoding an anti-fusion polypeptide as provided herein (e.g., a vector, a linearized vector, or a cDNA) as a template (e.g., a vector, a linearized vector, or a cDNA as provided herein having an RNA polymerase promoter located upstream of a region encoding the linear RNA).

In an embodiment, the deoxyribonucleotide template is transcribed to produce the linear RNA containing components as described herein.When expressed, the linear polyribonucleotide produces a splicing-compatible polyribonucleotide, and the splicing-compatible polyribonucleotide can be self-splicing to produce a circular polyribonucleotide.

In some embodiments, the length of the linear polyribonucleotide is 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, In an embodiment, the linear polyribonucleotide has a length of 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.

Cyclic polyribonucleotide

In some embodiments, the present invention is characterized in that the cyclic polyribonucleotide comprises the expression sequence encoding the anti-fusion polypeptide. In an embodiment, the polyribonucleotide comprises an IRES operably connected to the expression sequence encoding the anti-fusion polypeptide. The cyclic polyribonucleotide may include, for example, a splice junction connecting a 5' exon fragment and a 3' exon fragment. The cyclic polyribonucleotide may include any one or more elements described herein. In some embodiments, the cyclic polyribonucleotide includes any feature or any combination of features disclosed in International Patent Disclosure No. WO 2019/118919, which is incorporated herein by reference in its entirety.

In an embodiment, the cyclic polynucleotide further includes a polyribonucleotide cargo. In an embodiment, 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 an embodiment, the polyribonucleotide cargo includes an expression (coding) sequence of a coded polypeptide. In an embodiment, the polyribonucleotide comprises an IRES operably connected to an expression sequence of a coded 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 cyclic polyribonucleotide further includes an IRES and a spacer region between a 3' exon fragment or a 5' exon fragment. The length of the spacer region can be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides. In some embodiments, the subregion of the present invention can be 5 to 500 (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500) ribonucleotides. In some embodiments, the subregion of the present invention includes a poly-A sequence. In some embodiments, the subregion of the present invention includes a poly-A-C sequence. In some embodiments, the subregion of the present invention includes a poly-A-G sequence. In some embodiments, the subregion of the present invention includes a poly-A-T sequence. In some embodiments, the subregion of the present invention includes a random sequence. In some embodiments, the first annealing region and the second annealing region are connected to form a circular polyribonucleotide.

In some embodiments, the circular RNA is produced by a deoxyribonucleotide template or a linear RNA as described herein. In some embodiments, the circular RNA is produced by any one 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, or at least about 75 nucleotides. 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 certain embodiments, cyclic polyribonucleotide is 500 nucleotide to 20,000 nucleotide, 1,000 nucleotide to 20,000 nucleotide, 2,000 nucleotide to 20,000 nucleotide or 5,000 nucleotide to 20,000 nucleotide.In certain embodiments, cyclic polyribonucleotide is 500 nucleotide to 10,000 nucleotide, 1,000 nucleotide to 10,000 nucleotide, 2,000 nucleotide to 10,000 nucleotide or 5,000 nucleotide to 10,000 nucleotide.

As a result of its cyclization, cyclic polyribonucleotide may comprise some features that make it different from linear RNA.For example, compared with linear RNA, cyclic polyribonucleotide is not easily degraded by exonuclease.Like this, cyclic polyribonucleotide is more stable than linear RNA, especially when hatched in the presence of exonuclease.The stability of the increase of cyclic polyribonucleotide compared with linear RNA makes cyclic polyribonucleotide more useful as the cell transformation reagent producing polypeptide, and compared with linear RNA, storage is easier and the time is longer.The stability of the cyclic polyribonucleotide treated with exonuclease can be tested using the method of this area standard, and the method determines whether RNA degradation (for example, by gel electrophoresis) has occurred.In addition, different from linear RNA, when cyclic polyribonucleotide is hatched together with phosphatase (such as calf intestine phosphatase), cyclic polyribonucleotide is less likely to dephosphorylate.

Cyclic polyribonucleotide as herein described and composition thereof or pharmaceutical composition can be used for treatment and veterinary administration method, to produce a certain level of cyclic polyribonucleotide, a certain level of combination with target or a certain level of protein in described multiple cells after providing at least two doses of cyclic polyribonucleotide to multiple cells.In certain embodiments, cyclic polyribonucleotide is non-immunogenic in mammals such as people.In certain embodiments, cyclic polyribonucleotide can be from the cell of aquaculture animals (fish, crab, shrimp, oyster etc.), mammalian cell (for example, from the cell of pet or zoo animal (cat, dog, lizard, bird, lion, tiger and bear etc.), from the cell of farm or working animal (horse, cattle, pig, chicken etc.), human cell), cultivated cell, primary cell or cell line, stem cell, progenitor cell, differentiated cell, germ cell, cancer cell (for example, tumorigenic, transfer), non-tumorigenic cell (normal cell), fetal cell, embryonic cell, adult cell, mitotic cell, non-mitotic cell or its any combination and replicate or replicate therein. In some embodiments, the present invention includes cells comprising the cyclic polyribonucleotides described herein, wherein the cells are cells from aquaculture animals (fish, crab, shrimp, oysters, etc.), mammalian cells (e.g., cells from pets or zoo animals (cats, dogs, lizards, birds, lions, tigers and bears, etc.), cells from farm or working animals (horses, cattle, pigs, chickens, etc.), human cells, 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 some embodiments, the cells are modified to comprise cyclic polyribonucleotides.

In certain embodiments, the cyclic polyribonucleotide comprises the sequence of expression product.In certain embodiments, the cyclic polyribonucleotide comprises the binding site for being combined with target.In certain embodiments, via any dosage regimen as described herein, cyclic polyribonucleotide is provided to a plurality of cells.In certain embodiments, cyclic polyribonucleotide as described herein induces response or response level in experimenter.In certain embodiments, the expression product encoded by the sequence included in the cyclic polyribonucleotide is expressed in one or more cells in the plurality of cells.

In certain embodiments, the half-life that cyclic polyribonucleotide has is at least the half-life of linear counterpart (for example, linear expression sequence or linear polyribonucleotide).In certain embodiments, the half-life that cyclic polyribonucleotide has is extended relative to the half-life of linear counterpart.In certain embodiments, the half-life increases by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.In certain embodiments, the half-life or persistence of cyclic polyribonucleotide in cell is at least about 1 hour, for example 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 time. In certain embodiments, the half-life of cyclic polyribonucleotide in cell or persistence are about 1 hour to about 60 days, for example, 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 certain embodiments, cyclic polyribonucleotide has half-life or persistence in the cell just carrying out cell division.In certain embodiments, cyclic polyribonucleotide has half-life or persistence in the cell after division. In certain embodiments, the half-life or persistence of the cyclic polyribonucleotide in the cell undergoing division is at least about 10 minutes, for example at least about 1 hour, for example 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 half-life or persistence of the cyclic polyribonucleotide in a dividing cell is about 10 minutes to about 60 days, for example, 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 certain embodiments, cyclic polyribonucleotide for example regulates cell function instantaneously or for a long time.In certain embodiments, there is stable change in cell function, as continuing to exist the regulation of following time: at least about 10 minutes, for example at least about 1 hour, for example 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 time. In certain embodiments, the cellular function is stably altered, e.g., the modulation persists for about 1 hour to about 60 days, e.g., about 1 hour to about 30 days, e.g., 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 polynucleotides

The polynucleotides described herein (eg, circular polyribonucleotides) may include any one or more of the elements described herein and an expression sequence encoding an anti-fusion polypeptide.

Anti-fusion peptide

The present disclosure provides a cyclic polyribonucleotide encoding at least one expressed sequence, wherein the at least one expressed sequence encodes an anti-fusion polypeptide. In some embodiments, the anti-fusion polypeptide inhibits viral entry. In some embodiments, the anti-fusion polypeptide inhibits viral fusion.

In some embodiments, the anti-fusion polypeptide is a polypeptide or variant thereof comprising an amino acid sequence selected from a sequence in Table 1. In some embodiments, the anti-fusion polypeptide is a polypeptide comprising a continuous stretch of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids of a sequence in Table 1. In some embodiments, the anti-fusion polypeptide is a polypeptide comprising a continuous stretch of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the amino acids of a sequence in Table 1. In some embodiments, the anti-fusion polypeptide is a polypeptide comprising a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with a sequence in Table 1. In certain embodiments, the anti-fusion polypeptide is a variant of a sequence in Table 1, comprising no more than one, two, three, four, five, six, seven, eight, nine or ten mutations (e.g., substitutions, deletions or insertions). In certain embodiments, the cyclic polyribonucleotide comprises an expressed sequence encoding more than one anti-fusion polypeptide. In certain embodiments, the cyclic polyribonucleotide encoding more than one fusion protein reduces the chance of viral escape.

In some embodiments, the anti-fusion polypeptide targets one or more (e.g., two, three, four, or five) viruses. Anti-fusion polypeptides may target viruses including, but not limited to, all of the virus strains listed in Table 1. In some embodiments, the virus does not infect humans. In some embodiments, the virus infects humans.

Table 1. Exemplary anti-fusion polypeptides

In some embodiments, the virus is human immunodeficiency virus (HIV) (e.g., the anti-fusion polypeptide inhibits viral entry of HIV). In some embodiments, HIV is HIV-1. In some embodiments, HIV is a strain of HIV-1 (e.g., HIV-1 _HE , HIV-1 _IIIB , HIV-1 _MN , HIV-1 _NDK , HIV-1 _NL4-3 , HIV-1 _RF , or HIV-1 _SF2 ). In some embodiments, HIV is HIV-2. In some embodiments, HIV is a strain of HIV-2 (e.g., HIV-2 _EHO or HIV-2 _ROD ). In some embodiments, HIV is an HIV pseudovirion. In some embodiments, the anti-fusion polypeptide prevents HIV viral fusion by specifically binding to HIV glycoprotein 120 (gp120). In some embodiments, the anti-fusion polypeptide prevents binding of gp120 to the cluster of differentiation 4 (CD4) co-receptor. In some embodiments, the anti-fusion polypeptide reduces the affinity of gp120 for co-receptors (e.g., CC chemokine receptor type 5 (CCR5) and CXC chemokine receptor type 4 (CXCR4)). In some embodiments, the anti-fusion polypeptide prevents binding of gp120 to co-receptors (e.g., CCR5 and CXCR4). In some embodiments, the anti-fusion polypeptide prevents HIV virus fusion by specifically binding to HIV glycoprotein 41 (gp41). In some embodiments, the anti-fusion polypeptide inhibits HIV virus core entry into cells. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide of any one of SEQ ID NO: 1, 5, 11, 13-18, 22-38, 40-46, 49-56, 58, 59, 61, 66-78, 80-85, 87-91, 95-100, 112, 113, 153, 163, 64, 168, 169, 171-184, 211-215, 242, 243, 247, 255, 256, 258, 261, 261, 265, 267, 271, 272, 274, 276, 286, 287 or 312-324 (e.g., a polypeptide that inhibits viral entry of HIV). In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity to any of the polypeptides of SEQ ID NO: 1, 5, 11, 13-18, 22-38, 40-46, 49-56, 58, 59, 61, 66-78, 80-85, 87-91, 95-100, 112, 113, 153, 163, 64, 168, 169, 171-184, 211-215, 242, 243, 247, 255, 256, 258, 261, 261, 265, 267, 271, 272, 274, 276, 286, 287 or 312-324.

In some embodiments, the virus is a hepatitis virus (e.g., an anti-fusion polypeptide inhibits viral entry of a hepatitis virus). In some embodiments, the hepatitis virus is hepatitis A virus (HAV). In some embodiments, the hepatitis virus is hepatitis B virus (HBV). In some embodiments, the hepatitis virus is hepatitis C virus (HCV). In some embodiments, the hepatitis virus is hepatitis D virus (HDV). In some embodiments, the hepatitis virus is hepatitis E virus (HEV). In some embodiments, the hepatitis virus is duck hepatitis virus (DHV). In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a polypeptide of any one of SEQ ID NO: 104, 109, 112, 113, 141-145, 158-160 or 284 (e.g., a polypeptide that inhibits viral entry of a hepatitis virus such as HCV). In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity to any one of SEQ ID NOs: 104, 109, 112, 113, 141-145, 158-160 or 284.

In some embodiments, the virus is a coronavirus such as a betacoronavirus (e.g., the anti-fusion polypeptide inhibits viral entry of a coronavirus). In some embodiments, the betacoronavirus is SARS-CoV type 1 (SARS-CoV-1). In some embodiments, the betacoronavirus is SARS-CoV type 2 (SARS-CoV-2). In some embodiments, the betacoronavirus is a pseudotyped virus expressing the SARS-CoV-2 spike protein. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide of any one of SEQ ID NOs: 6-9, 119-123, 165-167, or 288-311 (e.g., a polypeptide that inhibits viral entry of a betacoronavirus such as SARS-CoV-1 or SARS-CoV-2). In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 6-9, 119-123, 165-167, or 288-311.

In some embodiments, the virus is respiratory syncytial virus (RSV) (e.g., the anti-fusion polypeptide inhibits the virus entry of RSV). In some embodiments, RSV is RSV A subtype (RSVA). In some embodiments, RSV is RSV B subtype (RSVB). In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a polypeptide of any one of SEQ ID NO: 11, 13, 39, 112, 113, 153, 185-188, 216-219, 244, 246, 247-249, 251, 252, 257, 259, 264, 266, 268, 270, 273, 282 or 285 (e.g., a polypeptide that inhibits the virus entry of RSV). In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity to any of the polypeptides of SEQ ID NO: 11, 13, 39, 112, 113, 153, 185-188, 216-219, 244, 246, 247-249, 251, 252, 257, 259, 264, 266, 268, 270, 273, 282 or 285.

In some embodiments, the virus is influenza (e.g., seasonal influenza, pandemic influenza, influenza A, H1N1 subtype influenza, influenza B, influenza C, influenza D). In some embodiments, influenza is influenza A. In some embodiments, influenza is influenza B. In some embodiments, influenza is influenza C. In some embodiments, influenza is influenza D. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide of any one of SEQ ID NO: 1, 4, 11, 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281 or 283 (e.g., a polypeptide that inhibits viral entry of influenza). In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity to any of the polypeptides of SEQ ID NO: 1, 4, 11, 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281 or 283.

In some embodiments, the virus is an influenza virus (e.g., the anti-fusion polypeptide inhibits viral entry of influenza). In some embodiments, the influenza virus is an influenza A virus (IAV). In some embodiments, the influenza virus is an influenza B virus (IBV). In some embodiments, the influenza virus is an influenza virus that expresses hemagglutinin (HA).

In some embodiments, the virus is herpes simplex virus (HSV) (e.g., the anti-fusion polypeptide inhibits viral entry of HSV). In some embodiments, the HSV is HSV-1. In some embodiments, the HSV is HSV-2.

In some embodiments, the virus is a human papillomavirus (HPV) (e.g., the anti-fusion polypeptide inhibits viral entry of HPV). In some embodiments, the HPV is a high-risk HPV strain (e.g., HPV 16, HPV 18, HPV 31, HPV 33, HPV45, HPV 52, or HPV 58). In some embodiments, the HPV is a low-risk HPV strain (e.g., HPV 6, HPV 11, HPV42, HPV 43, HPV, or 44).

In some embodiments, the virus is any virus listed in Table 1.

In some embodiments, the GC content of the nucleic acid sequence encoding the anti-fusion polypeptide is at least 51% (e.g., at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%). In some embodiments, the GC content of the nucleic acid sequence encoding the anti-fusion polypeptide is at most 52%, 53%, 54%, 55%, 56%, 57%, 58% or 59%, or 60%. In some embodiments, the GC content of the nucleic acid sequence encoding the anti-fusion polypeptide is 51% to 60%, 52% to 60%, 53% to 60%, 54% to 60%, 55% to 60%, 52% to 58%, 53% to 58%.

In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the anti-fusion polypeptide is greater than 10% (e.g., greater than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the anti-fusion polypeptide is at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the anti-fusion polypeptide is 20% to 28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23%, or 22% to 23%.

The GC content of the expressed sequence encoding the anti-fusion polypeptide refers to the GC content of the expressed sequence encoding only the anti-fusion polypeptide, without encoding other coding regions for polypeptides other than the anti-fusion polypeptide. Similarly, the uridine content or thymidine content of the expressed sequence encoding the anti-fusion polypeptide refers to the uridine content of the expressed sequence encoding only the anti-fusion polypeptide, without encoding other coding regions for polypeptides other than the anti-fusion polypeptide. In some embodiments, the calculation of the GC content or uridine (or thymidine) content of the expressed sequence encoding the anti-fusion polypeptide only considers the continuous nucleic acid sequence starting from the first nucleoside of the start codon of the open reading frame encoding the anti-fusion polypeptide to the last nucleoside of the stop codon of the same open reading frame in the 5' to 3' direction. In other embodiments, the calculation of the GC content or uridine (or thymidine) content of the expressed sequence encoding the anti-fusion polypeptide only considers the continuous nucleic acid sequence starting from the first nucleoside of the codon encoding the N-terminal amino acid residue of the anti-fusion polypeptide to the last nucleoside of the codon encoding the C-terminal amino acid residue of the anti-fusion polypeptide in the 5' to 3' direction.

In some embodiments, the nucleic acid sequence encoding the anti-fusion polypeptide has a uridine content of more than 20%. In some embodiments, the uridine content of the nucleic acid sequence encoding the anti-fusion polypeptide is greater than 10% (e.g., greater than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content of the nucleic acid sequence encoding the anti-fusion polypeptide is at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the uridine content of the nucleic acid sequence encoding the anti-fusion polypeptide is 20% to 28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23%, or 22% to 23%. In some embodiments, the nucleic acid sequence encoding the anti-fusion polypeptide has a uridine content of 20% to 28%.

Multiple anti-fusion peptides

In certain embodiments, the cyclic polyribonucleotide encodes a plurality of expressed sequences (e.g., two or more, e.g., 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20 or 5 to 10 expressed sequences) that each encodes an anti-fusion polypeptide.

In some embodiments, the circular polyribonucleotide encodes two or more (e.g., 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, or 5 to 10) copies of the same anti-fusion polypeptide.

In some embodiments, the cyclic polyribonucleotide encodes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) different (e.g., sharing less than 100% sequence identity) anti-fusion polypeptides. In some embodiments, two or more different anti-fusion polypeptides are each selected from the anti-fusion polypeptides of Table 1. In some embodiments, two or more different anti-fusion polypeptides each inhibit different viruses. For example, the cyclic polyribonucleotide can encode a first anti-fusion polypeptide that inhibits influenza and a second anti-fusion polypeptide that inhibits RSV. The cyclic polyribonucleotide can include a first anti-fusion polypeptide that inhibits influenza and a second anti-fusion polypeptide that inhibits SARS-CoV-2. The cyclic polyribonucleotide can include a first anti-fusion polypeptide that inhibits HIV and a second anti-fusion polypeptide that inhibits SARS-CoV-2. The cyclic polyribonucleotide can include a first anti-fusion polypeptide that inhibits HIV and a second anti-fusion polypeptide that inhibits HCV. When the first or second anti-fusion polypeptide inhibits multiple viruses, the first and second anti-fusion polypeptides can have different viral specificities.

Wherein the cyclic polyribonucleotide encodes two or more anti-fusion polypeptides, the anti-fusion polypeptides may be encoded in a single open reading frame or in multiple open reading frames.

In some embodiments, the disclosure provides a cyclic polyribonucleotide comprising an open reading frame (e.g., an open reading frame operably connected to an IRES), comprising two or more expressed sequences, wherein each expressed sequence encodes an anti-fusion polypeptide. In some embodiments, the translation of the open reading frame produces a polypeptide fusion comprising two or more anti-fusion polypeptides. The anti-fusion polypeptide can be connected, for example, by a joint as described herein (e.g., a peptide joint encoded by an open reading frame, such as the glycine-serine joint described below about peptide-Fc fusions). In some embodiments, the anti-fusion polypeptide can be separated by a cleavage domain (e.g., staggered sequence), for example, as described herein.

In some embodiments, the disclosure provides a circular polyribonucleotide comprising a first open reading frame encoding a first anti-fusion polypeptide (e.g., operably linked to a first IRES) and a second open reading frame encoding a second anti-fusion polypeptide (e.g., operably linked to a second IRES).

Anti-fusion peptide-Fc fusion

In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding a fusion protein (comprising an anti-fusion polypeptide). In some embodiments, the fusion protein comprises an anti-fusion polypeptide fused to the Fc domain of an immunoglobulin (e.g., a single chain of an Fc domain). In some embodiments, the anti-fusion polypeptide is selected from Table 1. In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding more than one fusion protein (comprising an anti-fusion polypeptide). In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding more than one anti-fusion polypeptide. In some embodiments, the cyclic polyribonucleotide encoding more than one fusion protein reduces the chance of viral escape.

In some embodiments, the Fc domain is an IgG4 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG1 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG2 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG2a Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG2b Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG3 Fc domain or a fragment thereof.

In some embodiments, the C-terminal amino acid residue of the anti-fusion polypeptide is fused to the N-terminal amino acid residue of the Fc domain, optionally through a peptide linker. In some embodiments, the N-terminal amino acid residue of the anti-fusion polypeptide is fused to the C-terminal amino acid residue of the Fc domain, optionally through a peptide linker. In some embodiments, the peptide linker between the anti-fusion polypeptide and the Fc domain comprises at least two amino acid residues (e.g., 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, or at least 20 amino acid residues). In some embodiments, the peptide linker between the anti-fusion polypeptide and the Fc domain comprises 2-200 amino acid residues (e.g., 2-200, 2-180, 2-160, 2-140, 2-120, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-2 In some embodiments, the peptide linker consists of glycine (Gly) and serine (Ser) residues. In some embodiments, the peptide linker comprises an amino acid sequence of any one of (GS) _x , (GGS) _x , (GGGGS) _x , (GGSG) _x , or (SGGG) _x , wherein x is an integer from 1 to 50 (e.g., 1-40, 1-30, 1-20, 1-10, or 1-5). In some embodiments, the peptide linker comprises an amino acid sequence of any one of (GS) _x , (GGS) _x , (GGGGS) _x , (GGSG) _x , or (SGGG) _x , wherein x is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the peptide linker comprises 6 to 36 amino acids. In some embodiments, the peptide linker comprises 21 to 31 amino acids.

Polyribonucleotide cargo

The polyribonucleotide cargo described herein includes any sequence comprising at least one polyribonucleotide. In certain embodiments, the polyribonucleotide cargo includes an expressed sequence, a non-coding sequence, or an expressed sequence and a non-coding sequence. In certain embodiments, the polyribonucleotide cargo includes an expressed sequence encoding an anti-fusion polypeptide. In certain embodiments, the polyribonucleotide cargo includes an IRES operably connected to an expressed sequence encoding an anti-fusion polypeptide. In certain embodiments, the polyribonucleotide cargo includes an expressed sequence encoding an anti-fusion polypeptide having a biological effect on a subject.

For example, the polyribonucleotide cargo can 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 comprises 1-20,000 nucleotides, 1-10,000 nucleotides, 1-5,000 nucleotides, 100-20,000 nucleotides, 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 an embodiment, the polyribonucleotide cargo includes one or more expression (or coding) sequences, wherein each expression (or coding) sequence encoding polypeptide (e.g., anti-fusion polypeptide). In an embodiment, the polyribonucleotide cargo includes one or more non-coding sequences. In an embodiment, the polyribonucleotide cargo is completely composed of one or more non-coding sequences. In an embodiment, the polyribonucleotide cargo includes a combination of expression (or coding) and non-coding sequences.

In some embodiments, the polyribonucleotide comprises any feature or any combination of features as disclosed in International Patent Publication No. WO 2019/118919, which is hereby incorporated by reference in its entirety.

Peptide expression sequence

In certain embodiments, polyribonucleotides as described herein (for example, polyribonucleotide goods of cyclic polyribonucleotides) include one or more expression (or coding) sequences, wherein each expression sequence encodes anti-fusion polypeptide.In certain embodiments, cyclic polyribonucleotides include two, three, four, five, six, seven, eight, nine, ten or more expression (or coding) sequences.

Each encoded polypeptide can be linear or branched. In various embodiments, the polypeptide has a length of 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 length of the polypeptide is 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 10,000 amino acids, less than about 1 ... In some embodiments, polypeptides of less than about 1,000 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.

The 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 an anti-fusion polypeptide). For example, the polypeptide may be a functionally active variant of any polypeptide described herein, for example, having 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 to the sequence of a polypeptide described herein or a naturally occurring polypeptide in a specified region or throughout the sequence. In some cases, a polypeptide can be at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identical to a protein of interest.

In an embodiment, the polypeptide includes a plurality of polypeptides, for example, multiple copies of a polypeptide sequence, or a plurality of different polypeptide sequences. In an embodiment, the plurality of polypeptides are connected by a linker amino acid or a spacer amino acid.

In an embodiment, 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 "twin arginine" motif, which is used to translocate a folded protein containing such a Tat signal peptide across a lipid bilayer. See also, for example, the publicly available signal peptide database at www[dot]signalpeptide[dot]de. Signal peptides can also be used to direct proteins to specific organelles; see, for example, the experimentally determined and computationally predicted signal peptides disclosed in the Spdb signal peptide database, which is publicly available at proline.bic.nus.edu.sg/spdb.

In some embodiments, the expressed (or coding) sequence includes a poly-A sequence (e.g., at the 3' end of the expressed sequence). In some embodiments, the 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 A sequence is designed according to the description of the poly A sequence in [0202]-[0204] of International Patent Publication No. WO 2019/118919A1, which is incorporated herein by reference in its entirety. In some embodiments, the expressed sequence lacks a poly A sequence (e.g., at the 3' end of the expressed sequence).

In certain embodiments, cyclic polyribonucleotide comprises poly-A, lacks poly-A or has modified poly-A to regulate one or more features of cyclic polyribonucleotide.In certain embodiments, lack poly-A or have modified cyclic polyribonucleotide of poly-A to improve one or more functional characteristics, such as immunogenicity (for example, the level of one or more marks of immunity or inflammatory response), half-life and/or expression efficiency.

Internal ribosome entry site

In certain embodiments, cyclic polyribonucleotides as herein described comprise one or more internal ribosome entry sites (IRES) elements. In certain embodiments, IRES is operably connected to one or more expressed sequences (e.g., each IRES is operably connected to one or more expressed sequences). In an embodiment, IRES is located between the 5' end of a heterologous promoter and a coding sequence.

Suitable IRES elements included in the polyribonucleotide include RNA sequences that can engage eukaryotic ribosomes. In certain embodiments, the IRES element is at least about 5nt, at least about 8nt, at least about 9nt, at least about 10nt, at least about 15nt, at least about 20nt, at least about 25nt, at least about 30nt, at least about 40nt, at least about 50nt, at least about 100nt, at least about 200nt, at least about 250nt, at least about 350nt or at least about 500nt.

In some embodiments, the IRES element is derived from the DNA of an organism, including but not limited to viruses, mammals, and fruit flies. Such viral DNA can be derived from, but not limited to, picornavirus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA, and poliovirus cDNA. In one embodiment, the fruit fly DNA from which the IRES element is derived includes but is not limited to the antennapedia gene from Drosophila melanogaster.

In some embodiments, the IRES sequence, if present, is an IRES sequence of a virus: Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Fumanpoliovirus 1, Plautia stall intestine virus, Kashmir honey 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, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea looper (Ectropis obliqua), picorna-like virus, encephalomyocarditis virus (EMCV), fruit fly C virus, crucifer tobacco 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 (Hibiscus chlorotic ringspot virus virus), classical swine fever virus, human FGF2, human SFTPA1, human AML1/RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila In another embodiment, the IRES is the IRES sequence of Coxsackievirus B3 (CVB3). In another embodiment, the IRES is the IRES sequence of encephalomyocarditis virus. In further embodiments, the IRES is the IRES sequence of Thiele's encephalomyelitis virus.

The IRES sequence may have a modified sequence compared 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 so that it is a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence, in which the terminal adenosine residue is modified to a cytosine residue. In some embodiments, the modified CVB3 IRES may have the following nucleic acid sequence:

TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGA CTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGG CCTGCCCATGGGGAAACCCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCC GTGTTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAC(SEQ ID NO:325).

In some embodiments, the IRES sequence is an enterovirus 71 (EV17) IRES. In some embodiments, the terminal guanosine residue of the EV17 IRES sequence is modified to a cytosine residue. In some embodiments, the modified EV71 IRES may have the following nucleic acid sequence:

ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCT TTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTC TGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGA AGGTACCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATA(SEQ ID NO:326).

In some embodiments, the polyribonucleotide comprises at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expressed sequence. In some embodiments, the IRES flanks at least one (e.g., 2, 3, 4, 5 or more) expressed sequence on both sides. In some embodiments, the polyribonucleotide comprises one or more IRES sequences on one or both sides of each expressed sequence, resulting in the separation of one or more peptides and or one or more polypeptides. For example, the polyribonucleotide described herein may include a first IRES operably connected to a first expressed sequence and a second IRES operably connected to a second expressed sequence.

In some embodiments, the polyribonucleotides described herein comprise an IRES (e.g., an IRES operably linked to a coding region). For example, the polyribonucleotides 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 [Nucleic Acids Research] 35(8):2473-82, 2007; Chen et al. Science [Science] 268:415-417, 1995; Fan et al. Nature Communication [Nature Communication] 13(1):3751-3765, 2022 and International Publication No. WO 2021/263124, each of which is hereby incorporated by reference in its entirety.

Signal sequence

In some embodiments, the anti-fusion polypeptide expressed by the cyclic polyribonucleotide disclosed herein includes secretory proteins, such as proteins that naturally include signal sequences, or proteins that usually do not encode signal sequences but are modified to contain signal sequences. In some embodiments, the anti-fusion polypeptide encoded by the cyclic polyribonucleotide includes a secretion signal. For example, the secretion signal can be a secretion signal of the natural encoding of the secretory protein. In another example, the secretion signal can be a modified secretion signal of the secretory protein. In other embodiments, the anti-fusion polypeptide encoded by the cyclic polyribonucleotide does not include a secretion signal.

In some embodiments, the signal sequence is selected from SecSP38 (MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO:327); SecD4 (MWWLLLLLLLLWPMVWA; SEQ ID NO:328), gLuc (MGVKVLFALICIAVAEAK; SEQ ID NO:329); INHC1 (MASRLTLLTLLLLLLAGDRASS; SEQ ID NO:330); Epo (MGVHECPAWLWLLLSLLSLPLGLPVLG; SEQ ID NO:331); and IL-2 (MYRMQLLSCIALSLALVTNS; SEQ ID NO:332).

In some embodiments, the cyclic polyribonucleotide encodes multiple copies of the same anti-fusion polypeptide (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more). In some embodiments, at least one copy of the anti-fusion polypeptide includes a signal sequence, and at least one copy of the anti-fusion polypeptide does not include a signal sequence. In some embodiments, the cyclic polyribonucleotide encodes multiple anti-fusion polypeptides (e.g., multiple different anti-fusion polypeptides or multiple anti-fusion polypeptides with less than 100% sequence identity), wherein at least one of the multiple anti-fusion polypeptides includes a signal sequence and at least one copy of the multiple anti-fusion polypeptides does not include a signal sequence.

In some embodiments, the signal sequence is a wild-type signal sequence, which is present at the N-terminus of the corresponding wild-type anti-fusion polypeptide, for example, when endogenously expressed. In some embodiments, the signal sequence is heterologous to the anti-fusion polypeptide, for example, it is not present when the wild-type anti-fusion polypeptide is endogenously expressed. The polyribonucleotide sequence encoding the anti-fusion polypeptide can be modified to remove the nucleotide sequence encoding the wild-type signal sequence and/or add a sequence encoding a heterologous signal sequence.

The polypeptide (for example, anti-fusion polypeptide) encoded by polyribonucleotide can include the signal sequence that anti-fusion polypeptide is directed to secretory pathway.In certain embodiments, the signal sequence can guide anti-fusion polypeptide to reside in certain organelles (for example, endoplasmic reticulum, Golgi apparatus or endosome).In certain embodiments, the signal sequence guides anti-fusion polypeptide to be secreted from cells.For secretory protein, the signal sequence can be cut after secretion, thereby producing mature protein.In other embodiments, the signal sequence can be embedded in cell membrane or certain organelles, thereby producing transmembrane segment, and the transmembrane segment anchors protein to cell membrane, endoplasmic reticulum or Golgi apparatus.In certain embodiments, the signal sequence of transmembrane protein is a short sequence at polypeptide N-terminal.In other embodiments, the first transmembrane domain serves as the first signal sequence, by protein targeting 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 having at least 90% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 332). In some embodiments, the IL-2 secretion signal has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 332. In some embodiments, the IL-2 secretion signal has an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 332. In some embodiments, the IL-2 secretion signal has an amino acid sequence having 100% sequence identity to SEQ ID NO: 332.

In some embodiments, the secretion signal is a Gaussia luciferase secretion signal. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence having at least 90% sequence identity to MGVKVLFALICIAVAEAK (SEQ ID NO: 329). In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 329. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 329. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence having 100% sequence identity to SEQ ID NO: 329.

In some embodiments, the secretion signal is an EPO (e.g., human EPO) secretion signal. In some embodiments, the EPO secretion signal has an amino acid sequence having at least 90% sequence identity to MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 333). In some embodiments, the EPO secretion signal has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, the EPO secretion signal has an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 333. In some embodiments, the EPO secretion signal has an amino acid sequence having 100% sequence identity to SEQ ID NO: 333.

In some embodiments, the secretion signal is a wild-type SARS-CoV-2 secretion signal. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 90% sequence identity to MFVFLVLLPLVSS (SEQ ID NO: 334). In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 334. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 334. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 334.

In some embodiments, the anti-fusion polypeptide encoded by the polyribonucleotide includes a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.

Regulatory elements

In certain embodiments, polyribonucleotide as described herein (for example, the polyribonucleotide goods of polyribonucleotide) comprises one or more regulating and controlling elements.In certain embodiments, polyribonucleotide comprises regulating and controlling elements, for example, the sequence of the expression of the expression sequence of modification polyribonucleotide.

A regulatory element may include a sequence adjacent to an expressed sequence encoding an expression product. A regulatory element may be operably connected to an adjacent sequence. A regulatory element may increase the amount of the product expressed as compared to the amount of the product expressed when the regulatory element is not present. In addition, a regulatory element may increase the amount of the product expressed by a plurality of expressed sequences connected in series. Therefore, a regulatory element may enhance the expression of one or more expressed sequences. A plurality of regulatory elements are well known to those of ordinary skill in the art.

In certain embodiments, the regulating and controlling element is a translation regulator. The translation regulator can regulate the translation of an expressed sequence in a polyribonucleotide. The translation regulator can be a translation enhancer or a translation repressor. In certain embodiments, the polyribonucleotide comprises at least one translation regulator adjacent to at least one expressed sequence. In certain embodiments, the polyribonucleotide comprises a translation regulator adjacent to each expressed sequence. In certain embodiments, the translation regulator is present in one or both sides of each expressed sequence, causing, for example, the separation of the expression product of one or more peptides and/or one or more polypeptides.

In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.

Other examples of regulatory elements are described, for example, in paragraphs [0154]-[0161] of International Patent Publication No. WO 2019/118919, which is hereby incorporated by reference in its entirety.

Cleavage domain

The cyclic polyribonucleotides of the present disclosure may include a cleavage domain (e.g., an interlaced element or a cleavage sequence).

The term "staggered element" refers to a moiety, such as a nucleotide sequence, that induces ribosome pauses during translation. In some embodiments, the staggered element is a non-conserved sequence of amino acids with a strong α-helical tendency, followed by a consensus sequence -D(V/I)ExNPGP, where x = any amino acid (SEQ ID NO: 335). In some embodiments, the staggered 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 certain embodiments, the cyclic polyribonucleotide comprises at least one staggered element adjacent to an expressed sequence. In certain embodiments, the cyclic polyribonucleotide comprises staggered elements adjacent to each expressed sequence. In certain embodiments, staggered elements are present on one or both sides of each expressed sequence, causing the separation of expression products such as one or more peptides and/or one or more polypeptides. In certain embodiments, staggered elements are a part for one or more expressed sequences. In certain embodiments, the cyclic polyribonucleotide comprises one or more expressed sequences, and each of the one or more expressed sequences is separated from the subsequent expressed sequence by the staggered elements on the cyclic polyribonucleotide. In certain embodiments, staggered elements prevent two rounds of translation of (a) a single expressed sequence or one or more rounds of translation of (b) two or more expressed sequences to generate a single polypeptide. In certain embodiments, staggered elements are sequences separated from the one or more expressed sequences. In certain embodiments, staggered elements include a part for the expressed sequence of the one or more expressed sequences.

In certain embodiments, the cyclic polyribonucleotide comprises staggered elements. In order to avoid producing continuous expression products while keeping rolling circle translation, for example peptide or polypeptide, staggered elements can be included to induce ribosome to pause during translation. In certain embodiments, staggered elements are at least one 3 ' end in one or more expressed sequences. Staggered elements can be configured to make ribosome stagnate during the rolling circle translation of cyclic polyribonucleotide. Staggered elements can include but are not limited to 2A sample or CHYSEL (SEQ ID NO:336) (cis-acting hydrolase element) sequence. In certain embodiments, staggered element encoding has the sequence of C-terminal consensus sequence, and the consensus sequence is X ₁ X ₂ X ₃ EX ₅ NPGP (SEQ ID NO:337), wherein X ₁ does not exist or is G or H, X ₂ does not exist or is D or G, X ₃ is D or V or I or S or M, and X ₅ is any amino acid. In some embodiments, the sequence comprises a non-conserved sequence of amino acids with strong alpha-helical propensity followed by the consensus sequence -D(V/I)EXNPGP (SEQ ID NO: 338), where x = any amino acid. Some non-limiting examples of interlaced elements include GDVESNPGP (SEQ ID NO: 339), GDIEENPGP (SEQ ID NO: 340), VEPNPGP (SEQ ID NO: 341), IETNPGP (SEQ ID NO: 342), GDIESNPGP (SEQ ID NO: 343), GDVELNPGP (SEQ ID NO: 344), GDIETNPGP (SEQ ID NO: 345), GDVENPGP (SEQ ID NO: 346), GDVEENPGP (SEQ ID NO: 347), GDVEQNPGP (SEQ ID NO: 348), IESNPGP (SEQ ID NO: 349), GDIELNPGP (SEQ ID NO: 350), HDIETNPGP (SEQ ID NO: 351), HDVETNPGP (SEQ ID NO: 352), HDVEMNPGP (SEQ ID NO: 353), GDMESNPGP (SEQ ID NO: 354), GDVETNPGP (SEQ ID NO: 355), NO:355), GDIEQNPGP (SEQ ID NO:356) and DSEFNPGP (SEQ ID NO:357).

In certain embodiments, staggered elements as herein described cut expression product, such as between G and P of consensus sequence as herein described.As a non-limiting example, cyclic polyribonucleotide comprises at least one staggered element to cut expression product.In certain embodiments, cyclic polyribonucleotide comprises the staggered element adjacent to at least one expressed sequence.In certain embodiments, cyclic polyribonucleotide is included in the staggered element after each expressed sequence.In certain embodiments, cyclic polyribonucleotide comprises the staggered element that is present in one side or both sides of each expressed sequence, causes one or more independent peptides and/or polypeptides to be translated by each expressed sequence.

In certain embodiments, the staggered elements include one or more modified nucleotides or non-natural nucleotides that induce ribosomes to pause during translation. Non-natural nucleotides can include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Examples such as these are different from naturally occurring DNA or RNA by changing the molecular backbone. Exemplary modifications can include modifications to sugar, core bases, internucleoside bonds (e.g., to connecting phosphate/phosphodiester bonds/phosphodiester backbones) and any combination thereof that can induce ribosomes to pause during translation. Some exemplary modifications provided herein are described elsewhere herein.

In certain embodiments, staggered elements are present in cyclic polyribonucleotides in other forms.For example, in some exemplary cyclic polyribonucleotides, staggered elements comprise the terminator element of the first expressed sequence in the cyclic polyribonucleotide, and the nucleotide spacer sequence that terminator element is separated from the first translation initiation sequence of the subsequent expression of the first expressed sequence.In some instances, the upstream (5 ') of the first translation initiation sequence of the subsequent expression of the first expressed sequence of the first staggered element of the first expressed sequence in the cyclic polyribonucleotide.In some cases, the first expressed sequence and the subsequent expression sequence of the first expressed sequence are two separated expressed sequences in the cyclic polyribonucleotide.The distance between the first staggered element and the first translation initiation sequence can enable the first expressed sequence and its subsequent expression sequence to be continuously translated.

In certain embodiments, the first staggered element includes a terminator, and the expression product of the first expressed sequence is separated from the expression product of its subsequent expressed sequence, thereby producing a discrete expression product. In some cases, the first translation initiation sequence upstream of the subsequent sequence in the cyclic polyribonucleotide comprises the cyclic polyribonucleotide of the first staggered element and is translated continuously, and the corresponding cyclic polyribonucleotide of the staggered element comprising the second expressed sequence upstream of the second translation initiation sequence of the second expressed sequence subsequent expressed sequence is not translated continuously. In some cases, there is only one expressed sequence in the cyclic polyribonucleotide, and the first expressed sequence and its subsequent expressed sequence are identical expressed sequences. In some exemplary cyclic polyribonucleotides, the staggered element comprises the first terminator of the first expressed sequence in the cyclic polyribonucleotide, and the nucleotide spacer sequence separating the terminator from the downstream translation initiation sequence. In some such examples, the first staggered element in the cyclic polyribonucleotide is upstream (5 ') of the first translation initiation sequence of the first expressed sequence. In some cases, the distance between the first staggered element and the first translation initiation sequence enables the first expressed sequence and any subsequent expressed sequence to be translated continuously.

In certain embodiments, the first staggered element separates a round expression product of the first expressed sequence from the next round expression product of the first expressed sequence, thereby produces discrete expression product.In some cases, the circular polyribonucleotide that comprises the first staggered element in the first translation initiation sequence upstream of the first sequence in the circular polyribonucleotide is translated continuously, and the corresponding circular polyribonucleotide that comprises the staggered element in the second translation initiation sequence upstream of the second expressed sequence in the corresponding circular polyribonucleotide is not translated continuously.In some cases, the distance between the second staggered element and the second translation initiation sequence in the corresponding circular polyribonucleotide is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times large of the distance between the first staggered element and the first translation initiation sequence in the circular polynucleotide. In some cases, the distance between the first staggered element and the first translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt or more. In certain embodiments, the distance between the second staggered element and the second translation initiation is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt or greater than the distance between the first staggered element and the first translation initiation.In certain embodiments, the circular polyribonucleotide comprises more than one expressed sequence.

Examples of interlaced elements are described in paragraphs [0172]-[0175] of International Patent Publication No. WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, a plurality of anti-fusion polypeptides encoded by cyclic ribonucleotides can be separated by an IRES between each anti-fusion polypeptide (e.g., each anti-fusion polypeptide is operably connected to a separate IRES). For example, a cyclic polyribonucleotide can include a first IRES operably connected to a first expression sequence and a second IRES operably connected to a second expression sequence. The IRES between all anti-fusion polypeptides can be the same IRES. The IRES between different anti-fusion polypeptides can be different.

In some embodiments, multiple anti-fusion polypeptides can be separated by a 2A self-cleaving peptide. For example, a cyclic polyribonucleotide can encode an IRES operably linked to an open reading frame encoding a first anti-fusion polypeptide, 2A, and a second anti-fusion polypeptide.

In certain embodiments, a plurality of anti-fusion polypeptides can be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a cyclic polyribonucleotide can encode an IRES that is operably connected to an open reading frame encoding a first anti-fusion polypeptide, a protease cleavage site (e.g., a furin cleavage site) and a second anti-fusion polypeptide.

In certain embodiments, a plurality of anti-fusion polypeptides can be separated by 2A self-cleavage peptide and protease cleavage site (e.g., furin cleavage site). For example, cyclic polyribonucleotides can encode an IRES operably connected to an open reading frame encoding a first anti-fusion polypeptide, 2A, a protease cleavage site (e.g., furin cleavage site) and a second anti-fusion polypeptide. Circular polyribonucleotides can also encode an IRES operably connected to an open reading frame encoding a first anti-fusion polypeptide, a protease cleavage site (e.g., furin cleavage site), 2A and a second anti-fusion polypeptide. 2A and furin cleavage site in series can be referred to as furin-2A (which includes furin-2A or 2A-furin arranged in any orientation).

In addition, multiple anti-fusion polypeptides encoded by cyclic ribonucleotides can be separated by both IRES and 2A sequences. For example, IRES can be between an anti-fusion polypeptide and a second anti-fusion polypeptide, and 2A peptides can be between the second anti-fusion polypeptide and a third anti-fusion polypeptide. The selection of specific IRES or 2A self-cleaving peptides can be used to control the expression level of anti-fusion polypeptides under the control of IRES or 2A sequences. For example, depending on the selected IRES and/or 2A peptides, the expression on the polypeptide can be higher or lower.

In some embodiments, the cyclic polyribonucleotide comprises at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to the expressed sequence. In some embodiments, the cleavage sequence is between two expressed sequences. In some embodiments, the cleavage sequence is included in the expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises 2 to 10 cleavage sequences. In some embodiments, the cyclic polyribonucleotide comprises 2 to 5 cleavage sequences. In some embodiments, multiple cleavage sequences are between multiple expressed sequences; for example, the cyclic polyribonucleotide may include three expressed sequences and two cleavage sequences, so that there is a cleavage sequence between each expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a cleavage sequence, such as in sacrificial circRNA or cleavable circRNA or self-cleaving circRNA. In some embodiments, the cyclic polyribonucleotide comprises two or more cleavage sequences, resulting in the cyclic polyribonucleotide being separated into multiple products, such as miRNA, linear RNA, smaller cyclic polyribonucleotides, etc.

In certain embodiments, the cleavage sequence includes a ribozyme RNA sequence. Ribozymes (from ribonucleases, also referred to as RNA enzymes or catalytic RNA) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze the hydrolysis of one of the phosphodiester bonds of themselves, or catalyze the hydrolysis of the bonds in other RNAs, but it is also found that natural ribozymes catalyze the aminotransferase activity of ribosomes. Catalytic RNA can be "evolved" by in vitro methods. Similar to the riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In certain embodiments, catalytic RNA or ribozymes are placed in larger non-coding RNAs, which makes ribozymes present in cells with many copies for the purpose of chemical conversion of bulky molecules. In certain embodiments, aptamers and ribozymes can be encoded in identical non-coding RNAs.

In some embodiments, the cleavage sequence encodes a cleavable polypeptide joint. For example, a polyribonucleotide can encode two or more anti-fusion polypeptides, for example, wherein the two or more anti-fusion polypeptides are encoded by a single open reading frame (ORF). For example, two or more anti-fusion polypeptides can be encoded by a single open reading frame, and the expression of the open reading frame is controlled by IRES. In some embodiments, ORF further encodes a polypeptide joint, for example, so that the expression product of ORF encodes two or more anti-fusion polypeptides, and each anti-fusion polypeptide is separated by a sequence encoding a polypeptide joint (for example, a joint of 5-200, 5 to 100, 5 to 50, 5 to 20, 50 to 100 or 50 to 200 amino acids). The polypeptide joint can include a cleavage site, for example, a cleavage site recognized and cut by a protease (for example, an endogenous protease in a subject after applying polyribonucleotides to the subject). In such an embodiment, a single expression product including the amino acid sequence of two or more anti-fusion polypeptides is cut when expressed, so that the two or more anti-fusion polypeptides are separated after expression. Exemplary protease cleavage sites are known to those skilled in the art, for example, amino acid sequences that serve as protease cleavage sites recognized by metalloproteinases (e.g., matrix metalloproteinases (MMPs), such as any one or more of MMP 1-28), disintegrins and metalloproteinases (ADAMs, such as any one or more of ADAM 2, 7-12, 15, 17-23, 28-30, and 33), serine proteases (furin), urokinase-type plasminogen activator, matriptase, cysteine proteases, aspartic proteases, or cathepsins. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is a matriptase.

In some embodiments, the cyclic polyribonucleotides described herein are sacrificial cyclic polyribonucleotides, cleavable cyclic polyribonucleotides or self-cleaved cyclic polyribonucleotides. Circular polyribonucleotides can deliver cell components, including, for example, RNA, lncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA or shRNA. In some embodiments, the cyclic polyribonucleotides include the following separated miRNA: (i) self-cleavable elements; (ii) cutting recruitment sites; (iii) degradable joints; (iv) chemical joints; and/or (v) spacer sequences. In some embodiments, circRNA includes the following separated siRNA: (i) self-cleavable elements; (ii) cutting recruitment sites (such as ADAR); (iii) degradable joints (such as glycerol); (iv) chemical joints; and/or (v) spacer sequences. Non-limiting examples of self-cleavable elements include hammerhead structures, splicing elements, hairpins, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozyme.

Translation start sequence

In certain embodiments, polyribonucleotides as described herein (e.g., polyribonucleotide goods of polyribonucleotides) include at least one translation initiation sequence. In certain embodiments, polyribonucleotides include a translation initiation sequence operably connected to an expressed sequence.

In some embodiments, polyribonucleotides encode polypeptides and may include a translation initiation sequence, such as a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotides include a translation initiation sequence adjacent to an expressed sequence, such as a Kozak sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, a translation initiation sequence (such as a Kozak sequence) is present on one or both sides of each expressed sequence, resulting in the separation of an expression product. In some embodiments, the polyribonucleotides include at least one translation initiation sequence adjacent to an expressed sequence. In some embodiments, the translation initiation sequence provides conformational flexibility for the polyribonucleotides. In some embodiments, the translation initiation sequence is in the substantially single-stranded region of the polyribonucleotide. Other examples of translation initiation sequences are described in paragraphs [0163]-[0165] of International Patent Publication No. WO 2019/118919, which are hereby incorporated by reference in their entirety.

Polyribonucleotide can comprise 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 can start on first start codon or can start in the downstream of first start codon.

In certain embodiments, polyribonucleotide can be started at the codon (such as AUG) that is not the first start codon.The translation of polyribonucleotide can start at alternative translation initiation sequence, such as but not limited to ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG.In certain embodiments, translation starts at alternative translation initiation sequence under selective condition (such as, stress induction condition).As non-limiting example, the translation of polyribonucleotide can start at alternative translation initiation sequence (such as ACG).As another non-limiting example, polyribonucleotide translation can start at alternative translation initiation sequence CTG/CUG.As another non-limiting example, polyribonucleotide translation can start at alternative translation initiation sequence GTG/GUG.As another non-limiting example, polyribonucleotide can repeat related non-AUG (RAN) sequence, such as including the alternative translation initiation sequence of short segment repeat RNA (such as CGG, GGGGCC, CAG, CTG) and start translation.

Termination element

In certain embodiments, polyribonucleotides as described herein (e.g., polyribonucleotide goods of polyribonucleotides) include at least one termination element. In certain embodiments, polyribonucleotides include a termination element operably connected to an expressed sequence. In certain embodiments, polynucleotides lack a termination element.

In certain embodiments, polyribonucleotide comprises one or more expressed sequences, and each expressed sequence may or may not have a terminator. In certain embodiments, polyribonucleotide comprises one or more expressed sequences, and expressed sequence lacks a terminator so that polyribonucleotide is translated continuously. The exclusion of the terminator can cause rolling circle translation or the continuous expression of expression product.

In certain embodiments, the cyclic polyribonucleotide comprises one or more expressed sequences, and each expressed sequence may or may not have a termination element. In certain embodiments, the cyclic polyribonucleotide comprises one or more expressed sequences, and the expressed sequence lacks a termination element so that the cyclic polyribonucleotide is translated continuously. Due to lack of ribosome stagnation or falling off, the exclusion of the termination element may cause rolling circle translation or continuous expression of expression products such as peptides or polypeptides. In such embodiments, rolling circle translation expresses continuous expression products by each expressed sequence. In some other embodiments, the termination element of an expressed sequence can be a part for an interlaced element. In certain embodiments, one or more expressed sequences in the cyclic polyribonucleotide include a termination element. However, rolling circle translation or subsequent (for example, the second, third, fourth, fifth, etc.) expression of an expressed sequence is carried out in the cyclic polyribonucleotide. In such cases, when ribosome encounters a termination element (for example, a stop codon) and stops translation, the expression product can fall off from the ribosome. In certain embodiments, translation stops when ribosome such as at least one subunit of a ribosome keeps contact with the cyclic polyribonucleotide.

In certain embodiments, the cyclic polyribonucleotide includes a terminator at the end of one or more expressed sequences. In certain embodiments, one or more expressed sequences include two or more continuous terminators. In such embodiments, translation stops and rolling circle translation stops. In certain embodiments, ribosomes are completely separated from cyclic polyribonucleotides. In some such embodiments, the generation of subsequent (for example, the second, third, fourth, fifth, etc.) expressed sequences in cyclic polyribonucleotides may require ribosomes to reengage with cyclic polyribonucleotides before initiating translation. Usually, terminator includes a nucleotide triplet in the frame that sends a translation termination signal, such as UAA, UGA, UAG. In certain embodiments, one or more terminators in cyclic polyribonucleotides are terminators of reading frame shifts, such as, but not limited to, the off-frame (off-frame) or -1 and +1 shifted reading frames (for example, hidden termination) that can terminate translation. The terminator of frame shifts includes nucleotide triplet, TAA, TAG and TGA that appear in the second reading frame and the third reading frame of the expressed sequence. The terminator of frame shifts may be important to prevent the mRNA misreading that is usually harmful to cells. In some embodiments, the termination element is a stop codon.

Other examples of termination elements are described in paragraphs [0169]-[0170] of International Patent Publication No. WO 2019/118919, which is hereby incorporated by reference in its entirety.

Untranslated region

In certain embodiments, the cyclic polyribonucleotide comprises an untranslated region (UTR). The UTR comprising the genomic region of a gene can be transcribed but not translated. In certain embodiments, the UTR may be included in the upstream of the translation initiation sequence of an expressed sequence as described herein. In certain embodiments, the UTR may be included in the downstream of an expressed sequence as described herein. In some cases, a UTR of the first expressed sequence is identical or continuous or overlapping with another UTR of the second expressed sequence. In certain embodiments, the intron is a human intron. In certain embodiments, the intron is a full-length human intron, such as ZKSCAN1.

Exemplary untranslated regions are described in paragraphs [0197]-[201] of International Patent Publication No. WO 2019/118919, which is hereby incorporated by reference in its entirety.

In certain embodiments, the cyclic polyribonucleotide comprises a poly-A sequence. Exemplary poly-A sequences are described in paragraphs [0202]-[0205] of International Patent Publication No. WO 2019/118919, which are hereby incorporated by reference in their entirety. In certain embodiments, the cyclic polyribonucleotide lacks a poly-A sequence.

In some embodiments, the cyclic polyribonucleotide comprises a UTR with one or more stretches of adenosine and uridine embedded therein. These AU-enriched signatures may increase the conversion rate of the expression product.

The introduction, removal or modification of the element (ARE) rich in UTR AU can be used for regulating the stability or immunogenicity (for example, the level of one or more markers of immune or inflammatory response) of cyclic polyribonucleotides. During the specific cyclic polyribonucleotides of engineering approaches, one or more copies of ARE can be introduced into the cyclic polyribonucleotides, and the copy of ARE can regulate the translation and/or generation of the expression product. Similarly, ARE can be identified and removed or engineered to cyclic polyribonucleotides to regulate intracellular stability, thereby affecting the translation and generation of the resulting protein.

It will be appreciated that any UTR from any gene may be incorporated into the corresponding flanking regions of a circular polyribonucleotide.

In some embodiments, the cyclic polyribonucleotide lacks 5'-UTR, and can express proteins from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks 3'-UTR, and can express proteins from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a poly A sequence, and can express proteins from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a termination element, and can express proteins from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site, and can express proteins from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a cap, and can express proteins from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks 5'-UTR, 3'-UTR and IRES, and can express proteins from one or more expressed sequences. In certain embodiments, the cyclic polyribonucleotide further comprises one or more of the following sequences: sequences encoding one or more miRNAs, sequences encoding one or more replication proteins, sequences encoding exogenous genes, sequences encoding therapeutic agents, regulatory elements (e.g., translation regulators, e.g., translation enhancers or repressors), translation initiation sequences, one or more regulatory nucleic acids targeting endogenous genes (e.g., siRNA, lncRNA, shRNA) and sequences encoding therapeutic mRNA or proteins.

In some embodiments, the cyclic polyribonucleotide lacks 5'-UTR. In some embodiments, the cyclic polyribonucleotide lacks 3'-UTR. In some embodiments, the cyclic polyribonucleotide lacks a poly A sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination element. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks the degradation susceptibility of exonucleases. In some embodiments, the fact that the cyclic polyribonucleotide lacks degradation susceptibility may mean that the cyclic polyribonucleotide is not degraded by exonucleases, or the limited extent degraded when only exonucleases are present is, for example, comparable or similar to the absence of exonucleases. In some embodiments, the cyclic polyribonucleotide is not degraded by exonucleases. In some embodiments, when exposed to exonucleases, the degradation of the cyclic polyribonucleotides is reduced. In some embodiments, the cyclic polyribonucleotide lacks the combination with the cap-binding protein. In some embodiments, the cyclic polyribonucleotide lacks the 5' cap.

Protein binding sequence

In certain embodiments, cyclic polyribonucleotide comprises one or more protein binding sites so that protein such as ribosome can be attached to the internal site in the RNA sequence.By engineering protein binding site (such as ribosome bind site) into cyclic polyribonucleotide, cyclic polyribonucleotide can escape or less detected by host's immune system, regulate degradation or regulate translation by masking the cyclic polyribonucleotide in the host immune system composition.

In certain embodiments, cyclic polyribonucleotide comprises at least one immune protein binding site, for example, for escaping immune response, for example CTL (cytotoxic T lymphocyte) reaction.In certain embodiments, immune protein binding site is to be bonded to immune protein and help cyclic polyribonucleotide to be shielded as external nucleotide sequence.In certain embodiments, immune protein binding site is to be bonded to immune protein and help cyclic polyribonucleotide to be hidden as external or external nucleotide sequence.

The traditional mechanism that ribosome is engaged with linear RNA comprises the combination of ribosome and the capped 5 ' end of RNA.From 5 ' end, ribosome migrates to start codon, so forms the first peptide bond.According to the present disclosure, the internal start (that is, independent of cap) of the translation of cyclic polyribonucleotide does not need free end or capped end.To be precise, ribosome is bound to the internal site of uncapped, and ribosome begins polypeptide extension at start codon thus.In certain embodiments, cyclic polyribonucleotide comprises one or more RNA sequences, and described RNA sequence comprises ribosome bind site, for example start codon.

Natural 5'UTRs have features that play a role in translation initiation. They carry a signature similar to the Kozak sequence, which is well known to be involved in the process of ribosome initiation of translation of a variety of genes. The Kozak sequence has a consensus CCR(A/G)CCAUGG (SEQ ID NO: 358), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". It is also known that the 5'UTR forms a secondary structure that is involved in the binding of elongation factors.

In certain embodiments, cyclic polyribonucleotide encoding and protein-bound protein binding sequence.In certain embodiments, protein binding sequence targeting cyclic polyribonucleotide or it is positioned to specific target.In certain embodiments, protein binding sequence specificity is bound to the arginine-rich region of protein.

In some embodiments, protein binding sites include, but are not limited to, binding sites for proteins 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, EIF4 G2, ELAVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B 1. 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, S MNDC1, 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 sequence

In certain embodiments, polyribonucleotides as described herein include one or more spacer sequences. Spacer refers to any continuous nucleotide sequence (for example, a continuous nucleotide sequence of one or more nucleotides) providing distance or flexibility between two adjacent polynucleotide regions. Spacer can be present between any nucleic acid element as described herein. Spacer can also be present in nucleic acid elements as described herein.

For example, wherein the nucleic acid includes any two or more of the following elements: (A) 3' catalytic intron fragment; (B) 3' splice site; (C) 3' exon fragment; (D) polyribonucleotide cargo; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' catalytic intron fragment; a spacer may be present between any one or more of the elements. Any of the elements (A), (B), (C), (D), (E), (F) or (G) may be separated by a spacer sequence as described herein. For example, a spacer may be present 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 comprises a first spacer region between the 5' exon fragment of (C) and the polyribonucleotide cargo of (D). The length of the spacer can be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides. In some embodiments, the polyribonucleotide further comprises a second spacer region between the polyribonucleotide cargo of (D) and the 5' exon fragment of (E).

The spacer sequence can be used to separate the IRES from adjacent structural elements to ensure the structure and function of the IRES or adjacent elements. The spacer specificity can be engineered according to the IRES. In certain embodiments, RNA folding computer software (such as RNAFold) can be used to guide the design of various elements (including spacers) of the vector.

The length of the spacer can be, for example, at least 5 (for example, at least 10, at least 15, at least 20) ribonucleotides. In certain embodiments, the length of each spacer region is at least 5 (for example, at least 10, at least 15, at least 20) ribonucleotides. The length of each spacer region can be, for example, 5 to 500 (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500) ribonucleotides. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a poly-A sequence. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a poly-A-C sequence. In certain embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region include a poly-A-G sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a poly A-T sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a random sequence.

Spacers can also be present in nucleic acid regions described herein. For example, a polynucleotide cargo region can include one or more spacers. Spacers can separate regions within a polynucleotide cargo.

In certain embodiments, the length of the spacer sequence can be, for example, at least 10 nucleotides, at least 15 nucleotides, or at least 30 nucleotides. In certain embodiments, the length of 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 certain embodiments, the length of the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. In certain embodiments, the length of the spacer sequence is 20 to 50 nucleotides. 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 sequence can be a poly A sequence, a poly A-C sequence, a poly C sequence, or a poly U sequence.

In some embodiments, the spacer sequence can be poly A-T, poly A-C, poly A-G, or a random sequence.

Exemplary spacer sequences are described in paragraphs [0293]-[0302] of International Patent Publication No. WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the polyribonucleotide comprises a 5' spacer sequence (e.g., between a 5' annealing region and a polyribonucleotide cargo). In some embodiments, the length of the 5' spacer sequence is at least 10 nucleotides. In another embodiment, the length of the 5' spacer sequence is at least 15 nucleotides. In a further embodiment, the length of the 5' spacer sequence is at least 30 nucleotides. In some embodiments, the length of 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 some embodiments, the length of the 5' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. In some embodiments, the length of the 5' spacer sequence is between 20 and 50 nucleotides. In certain embodiments, the length of 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In one embodiment, the 5' spacer sequence is a poly A sequence. In another embodiment, the 5' spacer sequence is a poly A-C sequence. In some embodiments, the 5' spacer sequence includes a poly A-G sequence. In some embodiments, the 5' spacer sequence includes a poly A-T sequence. In some embodiments, the 5' spacer sequence includes a random sequence.

In some embodiments, the polyribonucleotide comprises a 3' spacer sequence (e.g., between the 3' annealing region and the polyribonucleotide cargo). In some embodiments, the length of the 3' spacer sequence is at least 10 nucleotides. In another embodiment, the length of the 3' spacer sequence is at least 15 nucleotides. In another embodiment, the length of the 3' spacer sequence is at least 30 nucleotides. In some embodiments, the length of 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 some embodiments, the length of the 3' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. In some embodiments, the length of the 3' spacer sequence is 20 to 50 nucleotides. In certain embodiments, the length of 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 one embodiment, the 3' spacer sequence is a poly A sequence. In another embodiment, the 5' spacer sequence is a poly A-C sequence. In some embodiments, the 5' spacer sequence includes a poly A-G sequence. In some embodiments, the 5' spacer sequence includes a poly A-T sequence. In some embodiments, the 5' spacer sequence includes a random sequence.

In one embodiment, the polyribonucleotide comprises a 5' spacer sequence, but does not comprise a 3' spacer sequence. In another embodiment, the polyribonucleotide comprises a 3' spacer sequence, but does not comprise a 5' spacer sequence. In another embodiment, the polyribonucleotide neither comprises a 5' spacer sequence nor a 3' spacer sequence. In another embodiment, the polyribonucleotide does not comprise an IRES sequence. In a further embodiment, the polyribonucleotide does not comprise an IRES sequence, a 5' spacer sequence or a 3' spacer sequence.

In some embodiments, the spacer sequence comprises 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. nucleotides, 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 about 100 ribonucleotides.

Modification

Relative to a reference sequence (particularly a parent polyribonucleotide), a polyribonucleotide as described herein (e.g., a cyclic polyribonucleotide) may include one or more substitutions, insertions and/or additions, deletions and covalent modifications included within the scope of the present disclosure.

In some embodiments, the cyclic polyribonucleotide comprises one or more post-transcriptional modifications (e.g., capping, cutting, polyadenylation, splicing, poly A 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 modifications, such as any of more than one hundred different nucleoside modifications identified in RNA (Rozenski, J, Crain, P and McCloskey, J. (1999). The RNA Modification Database: 1999update [RNA modification database: 1999 update]. Nucl Acids Res [nucleic acid research] 27: 196-197). In some embodiments, the first isolated nucleic acid includes messenger RNA (mRNA). In some embodiments, the polyribonucleotide comprises at least one nucleoside selected from the group consisting of: as described in [0311] of International Patent Publication No. WO 2019/118919A1, which are incorporated herein by reference in their entirety.

Polyribonucleotide can comprise any useful modification, for example, for sugar, core base or internucleoside bond (for example, for the phosphate ester connected/for the phosphodiester bond/for the phosphodiester backbone).One or more atoms of pyrimidine core base can be substituted or replaced by the amino optionally substituted, the thiol optionally substituted, the alkyl optionally substituted (for example, methyl or ethyl) or halo (for example, chloro or fluoro).In certain embodiments, there is modification (for example, one or more modifications) in each sugar and internucleoside bond.Modification can be to the ribonucleic acid (RNA) modification of deoxyribonucleic acid (DNA), threose nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA) or its hybrid.Other modifications are described herein.

In certain embodiments, polyribonucleotide comprises at least one N (6) methyladenosine (m6A) modification to increase translation efficiency.In certain embodiments, m6A modification can reduce the immunogenicity (for example, reduce the level of one or more markers of immune or inflammatory response) of cyclic polyribonucleotide.

In some embodiments, the modification may include chemical or cell-induced modification. For example, some non-limiting examples of RNA modification in cells are described in Lewis and Pan, "RNA modifications and structures cooperate to guide RNA-protein interactions", Nat Reviews Mol Cell Biol, 2017, 18: 202-210.

In certain embodiments, the chemical modification of the ribonucleotide of cyclic polyribonucleotide can enhance immune escape.Cyclic polyribonucleotide can be synthesized and/or modified by methods well known in the art, such as in Current protocols in nucleic acid chemistry [current scheme of nucleic acid chemistry], Beaucage, S.L et al. (editor), those methods described in John Wiley & Sons, Inc., New York, NY, USA (by citing its full text hereby incorporated herein).Modification includes for example end modification, such as 5' end modification (phosphorylation (single, di and triphosphorylation), conjugation, reverse connection, etc.), 3' end modification (conjugation, DNA nucleotide, reverse connection, etc.), base modification (for example, with stable base, unstable base or with the base of extended parental library base pairing) Replacement), base removal (absaccharide nucleotide) or base conjugation.Modified ribonucleotide base can also include 5-methylcytidine and pseudouridine. In some embodiments, base modification can regulate the expression, immune response, stability, and subcellular localization of cyclic polyribonucleotides, to name a few functional effects. In some embodiments, modification includes biorthogonal nucleotides, such as non-natural bases. See, for example, Kimoto et al., Chem Commun (Camb) [Chemical Communications (Cambridge)], 2017, 53: 12309, DOI: 10.1039/c7cc06661a, which is hereby incorporated by reference in its entirety.

In certain embodiments, the sugar modification (for example, at 2' position or 4' position) or sugar substitution and main chain modification of one or more ribonucleotides of cyclic polyribonucleotides may include modification or substitution of phosphodiester bond.The specific example of cyclic polyribonucleotides includes but is not limited to the cyclic polyribonucleotides including modified main chain or non-natural internucleoside bond (such as internucleoside modification, including modification or substitution of phosphodiester bond).The cyclic polyribonucleotides with modified main chain are especially included in those without phosphorus atom in the main chain.For the purpose of the application, and as sometimes mentioned in the art, the modified RNA without phosphorus atom in its internucleoside main chain may also be considered as oligonucleoside.In a specific embodiment, cyclic polyribonucleotides will include ribonucleotides with phosphorus atom in its internucleoside main chain.

Modified polyribonucleotide backbone can include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates (such as 3'-alkylene phosphonates and chiral phosphonates), phosphinate, phosphoramidate (such as 3'-amino phosphoramidate and aminoalkyl phosphoramidate), thionophosphoramidate, thionoalkyl phosphonates, thionoalkyl phosphotriester and the borane phosphate with normal 3'-5' key, the analog of these 2'-5' connections, and those with opposite polarity, wherein adjacent nucleoside unit is connected to 3'-5' to 5'-3' or 2'-5' to 5'-2'.Also comprise various salts, mixed salts and free acid form.In certain embodiments, cyclic polyribonucleotide can be negatively charged or positively charged.

The modified nucleotide that can be incorporated into the polyribonucleotide can be modified on the internucleoside bond (for example phosphate backbone).Here, in the context of the polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used interchangeably.The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents.In addition, modified nucleosides and nucleotides can include the overall replacement of the unmodified phosphate moiety by another internucleoside bonding as described herein.The example of modified phosphate group includes but is not limited to phosphorothioate, phosphite selenate, boric acid phosphate (boranophosphate/boranophosphate ester), hydrogen phosphonate, phosphoramidate, diaminophosphorate, alkyl or aryl phosphonate and phosphotriester.Two non-connected oxygens of dithiophosphate are replaced by sulfur.It is also possible to modify the phosphate joint by replacing the connection oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate) and carbon (bridged methylene phosphonate).

α-Thio-substituted phosphate moieties are provided to impart stability to RNA and DNA polymers through non-natural phosphorothioate backbone bonds. Phosphorothioate DNA and RNA have enhanced nuclease resistance and therefore have a longer half-life in the cellular environment. Phosphorothioates attached to cyclic polyribonucleotides are expected to reduce innate immune responses by attenuating the binding/activation of cellular innate immune molecules.

In particular embodiments, modified nucleosides include α-thio-nucleosides (e.g., 5'-0-(l-phosphothioate)-adenosine, 5'-0-(l-phosphothioate)-cytidine (a-thiocytidine), 5'-0-(l-phosphothioate)-guanosine, 5'-0-(l-phosphothioate)-uridine, or 5'-0-(1-phosphothioate)-pseudouridine).

Other internucleoside linkages that can be used in accordance with the present disclosure are described herein, including internucleoside linkages that do not include a phosphorus atom.

In certain embodiments, cyclic polyribonucleotides may include one or more cytotoxic nucleosides.For example, cytotoxic nucleosides may be incorporated into cyclic polyribonucleotides, such as bifunctional modifications.Cytotoxic nucleosides may include but are not limited to adenosine, 5-azacytidine, 4'-thioarabinoside, cyclopentenylcytosine, cladribine, clofarabine, cytosine arabinoside, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H)-dione), troxacitabine, tezcitabine, 2'-deoxy-2'-methylene cytidine (DMDC) and 6-mercaptopurine. Other examples include fludarabine phosphate, N4-behenoyl-l-β-D-arabinopentaofuranosyl cytosine, N4-octadecyl-l-β-D-arabinopentaofuranosyl cytosine, N4-palmitoyl-l-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-arachidate).

Polyribonucleotides can be uniformly modified along the entire length of the molecule or may not be so. For example, one or more or all types of nucleotides (for example, naturally occurring nucleotides, purines or pyrimidines, or any one or more or all of A, G, U, C, I, pU) in cyclic polyribonucleotides, or in its given predetermined sequence region can be uniformly modified or may not be so. In certain embodiments, cyclic polyribonucleotides include pseudouridine. In certain embodiments, cyclic polyribonucleotides include inosine, and relative to viral RNA, inosine can help the immune system to characterize cyclic polyribonucleotides as endogenous. The incorporation of inosine can also mediate to improve RNA stability/reduce degradation. See, for example, Yu, Z et al., (2015) RNA editing by ADAR1 marks dsRNA as "self". [RNA editing performed by ADAR1 marks dsRNA as "self"] Cell Res [cell research]. 25, 1283-1284, which is incorporated herein by reference in its entirety.

In some embodiments, all nucleotides in a polyribonucleotide (or a given sequence region thereof) are modified. In some embodiments, modifications may include m6A, which may enhance expression; inosine, which may attenuate immune responses; pseudouridine, which may increase RNA stability or translation read-through (interlaced elements); m5C, which may increase stability; and 2,2,7-trimethylguanosine, which may facilitate subcellular translocation (e.g., nuclear localization).

In each position of cyclic polyribonucleotide, can there be different sugar modifications, nucleotide modifications and/or internucleoside bonding (for example main chain structure).It will be appreciated by those of ordinary skill in the art that nucleotide analogs or other one or more modifications can be positioned at any one or more positions of cyclic polyribonucleotide so that the function of cyclic polyribonucleotide does not reduce substantially.Modification can also be non-coding region modification. Circular polyribonucleotides can comprise about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e. any one or more of A, G, U or C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 15%, 10% to 20%, 10% to 15%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 15%, 10% to 15% or more). % to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%).

Cyclization method

The present disclosure provides a method for producing a cyclic polyribonucleotide encoding an anti-fusion polypeptide (e.g., polypeptide of Table 1), including, for example, recombinant technology or chemical synthesis. For example, the DNA molecule used to produce an RNA ring may include a DNA sequence of a naturally occurring original nucleic acid sequence, a modified form thereof, or a DNA sequence (e.g., a chimeric molecule or fusion protein) encoding a synthetic polypeptide that is not usually present in nature. DNA and RNA molecules can be modified using a variety of techniques, including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, connection of nucleic acid fragments, polymerase chain reaction (PCR) amplification or selected regions of mutagenic nucleic acid sequences, synthetic oligonucleotide mixtures, and connection of mixture groups to "build" nucleic acid molecule mixtures and combinations thereof.

In certain embodiments, the linear polyribonucleotide for cyclization can be cyclized or concatenated.In certain embodiments, the linear polyribonucleotide for cyclization can be cyclized in vitro before preparing and/or sending.In certain embodiments, the cyclic polyribonucleotide can be a mixture with the linear polyribonucleotide.In certain embodiments, the linear polyribonucleotide has the nucleotide sequence identical with the cyclic polyribonucleotide.

In certain embodiments, chemical method cyclization or concatenation is used for the linear polyribonucleotide of cyclization to form cyclic polyribonucleotide.In some chemical methods, the 5'-end and 3'-end of nucleic acid (for example, the linear polyribonucleotide for cyclization) include chemical reactive groups, when the groups are close to each other, new covalent bonds can be formed between the 3'-end and the 5'-end of the molecule.5'-end can contain NHS ester reactive groups, and 3'-end can contain 3'-amino terminal nucleotides, so that in organic solvents, the 3'-amino terminal nucleotides on the 3'-end of linear RNA molecules will experience nucleophilic attack on 5'-NHS-ester moieties, forming new 5'-/3'-amide bonds.

In certain embodiments, a 5'-phosphorylated nucleic acid molecule (e.g., a linear polyribonucleotide for cyclization) is enzymatically connected to the 3'-hydroxyl of a nucleic acid (e.g., a linear nucleic acid) using a DNA or RNA ligase to form a new phosphodiester bond. In an exemplary reaction, the linear polyribonucleotide for cyclization is incubated at 37°C for 1 hour with 1-10 units of T4RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. Ligation can occur in the presence of linear nucleic acids that can base pair with 5'- and 3'-regions in parallel to assist enzymatic ligation. In certain embodiments, the connection is a splint connection. For example, a splint ligase, such as Ligase, can be used for splint connection, i.e. RNA ligase II, T4 RNA ligase or T4 DNA ligase. For splint connection, single-stranded polynucleotide (splint), such as single-stranded RNA, can be designed to hybridize with two ends of linear polyribonucleotide, so that two ends can be parallel when hybridizing with single-stranded splint. Therefore, splint ligase can catalyze the connection of two parallel ends of linear polyribonucleotide, generating cyclic polyribonucleotide.

In certain embodiments, DNA or RNA ligase is used for the synthesis of circular polynucleotide.In certain embodiments, the 5'-end or 3'-end of the linear polyribonucleotide for cyclization can encode the ligase ribozyme sequence, so that during in vitro transcription, the linear polyribonucleotide for cyclization of the gained comprises the active ribozyme sequence, and the active ribozyme sequence can connect the 5'-end of the linear polyribonucleotide for cyclization to the 3'-end of the linear polyribonucleotide for cyclization.Ligase ribozyme can be derived from Group I intron, hepatitis D virus, hairpin ribozyme, or can be selected by SELEX (ligand system evolution by index enrichment).Ribozyme ligase reaction may need 1 hour to 24 hours at a temperature between 0 ℃ and 37 ℃.

In certain embodiments, can be by using at least one non-nucleic acid part for the linear polyribonucleotide cyclization or concatenation of cyclization.On the one hand, described at least one non-nucleic acid part can be with the linear polyribonucleotide for cyclization near 5 ' end and/or 3 ' end near region or characteristic reaction, with cyclization or concatenation for the linear polyribonucleotide for cyclization.On the other hand, described at least one non-nucleic acid part can be located at or be connected to or be adjacent to the linear polyribonucleotide for cyclization 5 ' end and/or 3 ' end.The non-nucleic acid part of imagination can be homology or heterology.As a non-limiting example, the non-nucleic acid part can be a key, such as hydrophobic bond, ionic bond, biodegradable key and/or cleavable key.As another non-limiting example, the non-nucleic acid part is a connecting portion.As another non-limiting example, the non-nucleic acid part can be an oligonucleotide or peptide moiety, such as, aptamer or non-nucleic acid joint as described herein.

In some embodiments, linear polyribonucleotides for circularization are synthesized using IVT and RNA polymerase, wherein the nucleotide mixture used for IVT can contain an excess of guanosine monophosphate relative to guanosine triphosphate to preferentially produce RNA with a 5' monophosphate; the purified IVT product can be circularized using a splint DNA.

In certain embodiments, the linear polyribonucleotide for cyclization is cyclized or concatenated due to the non-nucleic acid part, causing attraction between being located at, adjacent to or being connected to the 5' end and 3' end of the linear polyribonucleotide for cyclization, the atom, the molecular surface. As a non-limiting example, one or more linear polyribonucleotides for cyclization can be cyclized or concatenated by intermolecular force or intramolecular force. The non-limiting examples of intermolecular force include dipole-dipole force, dipole-induced dipole force, induced dipole-induced dipole force, van der Waals force and dispersion force. The non-limiting examples of intramolecular force include covalent bond, metallic bond, ionic bond, resonance bond, agnostic bond, dipole bond, conjugation, super conjugation and reverse bond.

In certain embodiments, the linear polyribonucleotide for cyclization can comprise ribozyme RNA sequence near 5' end and 3' end.When sequence is exposed to the remainder of ribozyme, ribozyme RNA sequence can be covalently connected to peptide.On the one hand, the peptide covalently connected to ribozyme RNA sequence near 5' end and 3' end can associate with each other, causing linear polyribonucleotide cyclization or concatenation for cyclization.On the other hand, peptide covalently connected to ribozyme RNA sequence 5' end and 3' end can cause linear primary construct or linear mRNA to be connected after cyclization or concatenation using methods known in the art (such as but not limited to protein connection).The non-limiting examples of ribozyme in linear primary construct of the present invention or linear RNA, or the non-exhaustive list of the method for incorporating and/or covalently connecting peptide is described in U.S. Patent Application No. US20030082768, and the patent content is incorporated herein in its entirety by citing.

In certain embodiments, the linear polyribonucleotide for cyclization can include the 5' triphosphate of nucleic acid, for example, by making 5' triphosphate contact with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase (dephosphatase) and convert it into 5' monophosphate.In certain embodiments, the 5' end of at least a portion of linear polyribonucleotide includes a phosphate moiety.In certain embodiments, the polyribonucleotide colony including cyclic and linear polyribonucleotide is contacted with RppH before digesting at least a portion of linear polyribonucleotide with 5' exonuclease and/or 3' exonuclease. Alternatively, the 5' triphosphates of the linear polyribonucleotides used for cyclization can be converted into 5' monophosphates by a two-step reaction comprising: (a) contacting the 5' nucleotides of the linear polyribonucleotides used for cyclization with a phosphatase (e.g., a thermosensitive phosphatase, shrimp alkaline phosphatase or calf intestinal phosphatase) to remove all three phosphates; and (b) after step (a), contacting the 5' nucleotides with a kinase (e.g., a polynucleotide kinase) that adds a single phosphate.

In some embodiments, the cyclization efficiency of the cyclization method 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 cyclization efficiency of the cyclization method provided herein is at least about 40%. In some embodiments, the cyclization efficiency of the cyclization method provided is between about 10% and about 100%; for example, the cyclization efficiency can 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 cyclization efficiency is between about 20% and about 80%. In some embodiments, the cyclization efficiency is between about 30% and about 60%. In some embodiments, the cyclization efficiency is about 40%.

In certain embodiments, cyclic polyribonucleotide comprises internal splicing element, and described internal splicing element is when being copied, and splicing end is connected together.Some examples comprise the miniature intron (<100nt) with splicing site sequence and short inverted repeat sequence (30-40nt), such as the reverse sequence in AluSq2, AluJr and AluSz, side joint intron, the Alu element in side joint intron and the motif found in (suptable4 enrichment motif) cis sequence element close to back splicing event, such as the sequence in (upstream) or (downstream) 200bp before the back splicing site (back splice site) with side joint exon.In certain embodiments, linear polyribonucleotide comprises at least one herein other place described repetitive nucleotide sequence as internal splicing element.In such embodiments, repetitive nucleotide sequence can include the repetitive sequence from Alu family intron.In certain embodiments, the biogenesis (for example, blind muscle protein and vibration protein (QKI) splicing factor) of the ribosome binding protein regulatable cyclic polyribonucleotide that splicing is relevant.

In some embodiments, the linear polyribonucleotide may comprise a canonical splice site at the head-to-tail junction flanking the circular polyribonucleotide.

In certain embodiments, linear polyribonucleotide can comprise ridge-helix-ridge motif, and described motif comprises the 4-base pair stem of two 3-nucleotide ridges of side joint.Cutting occurs at a site in the ridge region, generates the characteristic fragment of 5'-hydroxyl and 2',3'-cyclic phosphate ester at the end.Carry out cyclization on the 2',3'-cyclic phosphate ester of the same molecule that forms 3',5'-phosphodiester bridge by the nucleophilic attack of 5'-OH group.

In certain embodiments, linear polyribonucleotides may include poly-repeated RNA sequences with HPR elements. HPR includes 2', 3'-cyclic phosphate and 5'-OH terminal. HPR element self-processes the 5' and 3' ends of linear polyribonucleotides, thereby connecting the ends together.

In certain embodiments, linear polyribonucleotide can comprise the sequence that mediates self-connection.In one embodiment, linear polyribonucleotide can comprise HDV sequence, for example, HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUUCUGU AAAGAGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (Beeharry et al. 2004) (SEQ ID NO:359) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGA CUGCUGGACUCGCCGCCCGAGCC (SEQ ID NO:360), to carry out self-connection.In one embodiment, linear polyribonucleotide can comprise ring E sequence (for example in PSTVd) to carry out self-connection.In another embodiment, linear polyribonucleotide can comprise self-cyclization intron, for example 5 ' and 3 ' splice junction, or self-cyclization catalytic intron, as class I, class II or class III intron. Non-limiting examples of group I intron self-splicing sequences can include the self-splicing replacement intron-exon sequence derived from the T4 bacteriophage gene td and the insertion sequence (IVS) rRNA of Tetrahymena.

In certain embodiments, the linear polyribonucleotide for cyclization may include complementary sequences, including repetition or non-repetition nucleic acid sequences in individual introns or in flanking introns.Repetition nucleic acid sequences are sequences that occur in the section of linear polyribonucleotides. In certain embodiments, linear polyribonucleotides include repetition nucleic acid sequences. In certain embodiments, repetition nucleic acid sequences include poly-CA sequences or poly-UG sequences. In certain embodiments, linear polyribonucleotides include at least one repetition nucleic acid sequence hybridized with the complementary repetition nucleic acid sequence in another section of linear polyribonucleotides, and the section of its hybridization forms an internal double strand. In certain embodiments, linear polyribonucleotides include 1 to 10 (for example, 2,3,4,5,6,7,8,9 and 10) repetition nucleic acid sequences hybridized with the complementary repetition nucleic acid sequence in another section of linear polyribonucleotides, and the section of its hybridization forms an internal double strand. In certain embodiments, linear polyribonucleotides include 2 repetition nucleic acid sequences hybridized with the complementary repetition nucleic acid sequence in another section of linear polyribonucleotides, and the section of its hybridization forms an internal double strand. In certain embodiments, the repetitive nucleic acid sequence of two independent linear polyribonucleotides and complementary repetitive nucleic acid sequence hybridize to generate single cyclization polyribonucleotide, and the section of hybridization forms internal double-strand.In certain embodiments, complementary sequence is present in 5 ' end and 3 ' end of the linear polyribonucleotide for cyclization.In certain embodiments, complementary sequence comprises the nucleotide of 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 pairings.

In certain embodiments, cyclization chemical method can be used for generating cyclic polyribonucleotide.Such method can include but is not limited to click chemistry (for example, based on the method of alkynes and azide, or clickable base), olefin metathesis, phosphoramidate connection, hemiaminal-imine cross-linking, base modification and any combination thereof.

In certain embodiments, cyclase method can be used for generating cyclic polyribonucleotide.In certain embodiments, ligase, for example DNA or RNA ligase, can be used for generating the template of cyclic polyribonucleotide or complementary sequence, complementary strand of cyclic polyribonucleotide or cyclic polyribonucleotide.

Cyclization of linear polyribonucleotides can be accomplished by methods known in the art, for example, Petkovic and Muller, "RNA circularization strategies in vivo and in vitro," Nucleic Acids Res, 2015, 43(4):2454-2465, and Muller and Appel, "Invitro circularization of RNA," RNA Biol, 2017, 14(8):1018-1027.

Circular polyribonucleotides can encode sequences and/or motifs that can be used 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 certain embodiments, linear polyribonucleotide can include complementary sequence, including repetitive or non-repetitive nucleic acid sequence in individual intron or in flanking intron.Repetitive nucleic acid sequence is the sequence that occurs in the section of cyclic polyribonucleotide.In certain embodiments, linear polyribonucleotide comprises repetitive nucleic acid sequence.In certain embodiments, repetitive nucleotide sequence comprises poly-CA sequence or poly-UG sequence.In certain embodiments, linear polyribonucleotide comprises at least one repetitive nucleic acid sequence hybridized with complementary repetitive nucleic acid sequence in another section of linear polyribonucleotide, and the section of its hybridization forms internal double chain.In certain embodiments, the repetitive nucleic acid sequence of two independent linear polyribonucleotides and complementary repetitive nucleic acid sequence hybridize to generate single cyclization polyribonucleotide, and the section of hybridization forms internal double chain.In certain embodiments, complementary sequence is present in 5 ' end and 3 ' end of linear polyribonucleotide. In some embodiments, the complementary sequence includes 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 certain embodiments, cyclization chemical method can be used for generating cyclic polyribonucleotide.Such method can include but is not limited to click chemistry (for example, based on the method of alkynes and azide, or clickable base), olefin metathesis, phosphoramidate connection, hemiaminal-imine cross-linking, base modification and any combination thereof.

Methods for preparing the cyclic polyribonucleotides described herein are described in, for example, Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); Zhao, Synthetic Biology: Tools and Applications (1st ed.), Academic Press (2013); Muller and Appel, RNA Biol, 2017, 14(8): 1018-1027; and Egli and Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (1st ed.), Wiley-VCH (2012). For example, other methods for preparing cyclic polyribonucleotides are described in International Publication No. WO 2022/247943, U.S. Patent No. US 11000547, International Publication No. 2018/191722, International Publication No. WO 2019/236673, International Publication No. WO 2020/023595, International Publication No. WO 2022/204460, International Publication No. WO 2022/204464 and International Publication No. WO 2022/204466.

Various methods for synthesizing cyclic polyribonucleotides have also been described elsewhere (see, e.g., U.S. Patent No. 6210931, U.S. Patent No. 5773244, U.S. Patent No. 5766903, U.S. Patent No. 5712128, U.S. Patent No. 5426180, U.S. Publication No. US 20100137407, International Publication No. WO 1992001813 and International Publication No. WO2010084371, and Petkovic et al., Nucleic Acids Res [nucleic acid research]. 43: 2454-65 (2015); the contents of each of which are incorporated herein by reference in their entirety).

In certain embodiments, cyclic polyribonucleotide is purification, for example, has removed free ribonucleic acid, linear or nicked RNA, DNA, protein etc.In certain embodiments, can be by any known method purification cyclic polyribonucleotide commonly used in this area.The limiting examples of purification process comprises column chromatography, gel excision, size exclusion etc.

Generation method

Production methods in cell-free systems

The present disclosure also provides a method for producing circular RNA. For example, deoxyribonucleotide templates can be transcribed (for example, by in vitro transcription) in a cell-free system to produce linear RNA. Linear polyribonucleotides produce splicing-compatible polyribonucleotides, and the polyribonucleotides can be self-splicing to produce circular polyribonucleotides.

In some embodiments, present disclosure provides a method for producing cyclic polyribonucleotides (e.g., in a cell-free system) by: providing linear polyribonucleotides; and self-splicing linear polyribonucleotides under conditions suitable for splicing 3' and 5' splice sites of linear polyribonucleotides; thereby producing cyclic polyribonucleotides.

In some embodiments, the present disclosure provides a method for producing cyclic polyribonucleotides by: providing deoxyribonucleotides encoding linear polyribonucleotides; transcribing the deoxyribonucleotides in a cell-free system to produce linear polyribonucleotides; optionally purifying compatible linear polyribonucleotides for splicing; and self-splicing linear polyribonucleotides under conditions suitable for splicing 3' and 5' splice sites of linear polyribonucleotides, thereby producing cyclic polyribonucleotides.

In certain embodiments, the present disclosure provides a method for producing cyclic polyribonucleotides by: providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce a linear polyribonucleotide (wherein the transcription occurs in solution under the conditions of 3' and 5' splice sites suitable for splicing linear polyribonucleotides), thereby producing cyclic polyribonucleotides. In certain embodiments, the linear polyribonucleotides include 5' fracture introns and 3' fracture introns (e.g., for producing a self-splicing construct of a cyclic polyribonucleotide). In certain embodiments, the linear polyribonucleotides include 5' annealing regions and 3' annealing regions.

Suitable conditions for in vitro transcription and/or self-splicing may include any conditions (e.g., solutions or buffers, such as aqueous buffers or solutions) that mimic physiological conditions in one or more aspects. In some embodiments, suitable conditions include Mg2+ ions or salts thereof between 0.1-100 mM (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 K ⁺ ions or salts thereof between 1-1000 mM, such as KCl (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 Cl- ions or salts thereof between 1-1000 mM, such as KCl (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 Mn2+ ions or salts thereof between 0.1-100 mM, such as MnCl2 (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 μM, 1-500 μM, 1-200 μM, 50-500 μM, 100-500 μM, 100-300 μM, 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 ribonucleoside triphosphates (NTPs) between 0.1 mM and 100 mM (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., a pH of 5 to 9, a pH of 6 to 9, or a 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 certain embodiments, linear polyribonucleotide is produced from deoxyribonucleic acid (for example, deoxyribonucleic acid as described herein, as DNA vector, linearized DNA vector or cDNA).In certain embodiments, linear polyribonucleotide is transcribed from deoxyribonucleic acid by transcribing (for example, in vitro transcription) in a cell-free system.

Production methods in cells

The present disclosure also provides a method for producing circular RNA in a cell (e.g., a prokaryotic cell or a eukaryotic cell). In certain embodiments, an exogenous polyribonucleotide (e.g., a linear polyribonucleotide as described herein or a DNA molecule encoding the transcription of a linear polyribonucleotide as described herein) is provided to the cell. Linear polyribonucleotides can be transcribed in the cell from the exogenous DNA molecules provided to the cell. Linear polyribonucleotides can be transcribed in the cell from the exogenous recombinant DNA molecules provided to the cell instantaneously. In certain embodiments, the exogenous DNA molecules are not integrated into the genome of the cell. In certain embodiments, linear polyribonucleotides are transcribed in the cell from the recombinant DNA molecules integrated into the cell genome.

In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell comprising the polyribonucleotides described herein can be a bacterial cell or an archaeal cell. For example, the prokaryotic cell comprising the polyribonucleotides described herein can be Escherichia 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, Bacillus spp.), or Bacillus spp. cereus), betaproteobacteria (e.g., Burkholderia), alphaproteobacterial (e.g., Agrobacterium), Pseudomonas (e.g., Pseudomonas putida), and enterobacteria. Prokaryotic cells can be grown in culture medium. Prokaryotic cells can be contained in a bioreactor.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell comprising the polyribonucleotides described herein is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic cell is a unicellular fungal cell, such as a yeast cell (e.g., Saccharomyces cerevisiae and other yeasts (Saccharomyces spp.), Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp. and Pichia spp.). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. The unicellular animal cell can be a cell separated from a multicellular animal and grown in culture, or a daughter cell thereof. In some embodiments, the unicellular animal cell can be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. The unicellular plant cell can be a cell separated from a multicellular plant and grown in culture, or a daughter cell thereof. In some embodiments, the unicellular plant cell can be dedifferentiated. In some embodiments, the unicellular plant cell is from a plant callus. In an embodiment, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algae cell, such as a unicellular green algae, diatom, euglena, or dinoflagellates. Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella 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 eukaryotic organism. For example, a multicellular eukaryotic organism can be selected from the group consisting of vertebrates, invertebrates, multicellular fungi, multicellular algae, and multicellular plants. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate. In some embodiments, the eukaryotic organism is an invertebrate. In some embodiments, the eukaryotic organism is a multicellular fungi. In some embodiments, the eukaryotic organism is a multicellular plant. In some embodiments, the eukaryotic cell is a human cell or a non-human mammal cell, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., bovine, including cattle, buffalo, bison, sheep, goat, and musk ox; pig; camelid, including camel, llama, and alpaca; deer, antelope; and equine, 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 some embodiments, the eukaryotic cell is a cell of a bird, such as a member of the avian taxa Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathus (e.g., ostrich, emu), Columbiformes (e.g., pigeon, pigeon), or Psittaciformes (e.g., parrot). In some embodiments, the eukaryotic cell is a cell of an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, worm, or mollusk. In embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm (which may be a dicot or a monocot) or a gymnosperm (e.g., conifer, cycad, gnetum, ginkgo), a fern, a horsetail plant, a lycophyte, or a bryophyte. In embodiments, the eukaryotic cell is a cell of a eukaryotic multicellular algae.

The eukaryotic cells may be grown in a culture medium. The eukaryotic cells may be contained in a bioreactor.

Purification method

One or more purification steps may be included in the methods described herein. For example, in some embodiments, before self-splicing linear polyribonucleotides, linear polyribonucleotides are substantially enriched or pure (for example, purification). In other embodiments, before self-splicing linear polyribonucleotides, linear polyribonucleotides are not purified. In certain embodiments, the circular RNA obtained is purified.

Purification can include separation or enrichment of desired reaction products from one or more undesirable components (such as any unreacted starting material, by-product, enzyme or other reaction components). For example, purification of linear polyribonucleotides after transcription (such as in vitro transcription) in a cell-free system can include separation or enrichment from a DNA template before self-splicing linear polyribonucleotides. The purification of circular RNA products after scissoring can be used for separation or enrichment of circular RNA from its corresponding linear RNA. The purification method of RNA is well known to those skilled in the art, and includes enzyme purification or by chromatography.

In some embodiments, the purification method produces a circular polyribonucleotide having less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1%) linear polyribonucleotides.

Bioreactor

In certain embodiments, any method producing cyclic polyribonucleotide as herein described can be carried out in bioreactor.Bioreactor refers to any container carrying out chemical or biological process therein, and the process relates to organism or the biochemically active substance derived from such organism.Bioreactor can be compatible with the cell-free method for producing circular RNA as herein described.The container for bioreactor can include culture bottle, culture dish or culture bag, and they can be disposable (disposable), autoclave sterilizable or sterilizable.Bioreactor can be made of glass, or it can also be based on polymer, or it can also be made of other materials.

Examples of bioreactors include, but are not limited to, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, rotary filter stirred tanks, vibrating mixers, fluidized bed reactors, and membrane bioreactors. The mode of operating a bioreactor can be an intermittent or continuous process. A bioreactor is continuous when reagents and product streams continuously enter and exit the system. An intermittent bioreactor can have a continuous recirculation flow, but without continuous reagent feed or product harvesting.

Some methods of this disclosure relate to large-scale production of cyclic polyribonucleotides.For large-scale production method, described method can be carried out in the volume of 1 liter (L) to 50L or more (for example, 5L, 10L, 15L, 20L, 25L, 30L, 35L, 40L, 45L, 50L or more). In some embodiments, the method may be performed in a volume of 5L to 10L, 5L to 15L, 5L to 20L, 5L to 25L, 5L to 30L, 5L to 35L, 5L to 40L, 5L to 45L, 10L to 15L, 10L to 20L, 10L to 25L, 20L to 30L, 10L to 35L, 10L to 40L, 10L to 45L, 10L to 50L, 15L to 20L, 15L to 25L, 15L to 30L, 15L to 35L, 15L to 40L, 15L to 45L, or 15 to 50L.

In some embodiments, the bioreactor can produce at least 1g of circular RNA. In some embodiments, the bioreactor can produce 1-200g of circular RNA (e.g., 1-10g, 1-20g, 1-50g, 10-50g, 10-100g, 50-100g, 50-200g of circular RNA). In some embodiments, the amount produced is measured per liter (e.g., 1-200g/liter), per batch or reaction (e.g., 1-200g/batch or reaction), or per unit time (e.g., 1-200g/hour or/day).

In some embodiments, more than one bioreactor can be used in series to increase production capacity (eg, one, two, three, four, five, six, seven, eight, or nine bioreactors can be used in series).

How to use

In some embodiments, cyclic polyribonucleotides encoding anti-fusion polypeptides (e.g., polypeptides of Table 1) are used to treat or prevent viral infections (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).

In some embodiments, circular polynucleotides encoding anti-fusion polypeptides (eg, polypeptides of Table 1) are used to reduce viral entry.

In some embodiments, circular polynucleotides encoding anti-fusion polypeptides (e.g., polypeptides of Table 1) can be administered to a subject to reduce the risk of viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).

For example, a cyclic polyribonucleotide as described herein (e.g., in a pharmaceutical composition) can be administered to a subject. In certain embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human mammal. In an embodiment, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, ape), an ungulate (e.g., a cattle, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In an embodiment, the subject is a bird, such as a member of the avian taxonomic group Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathus (e.g., ostrich, emu), Columbiformes (e.g., pigeon, pigeon), or Psittaciformes (e.g., parrot). In embodiments, the subject is an invertebrate, such as an arthropod (eg, an insect, arachnid, crustacean), a nematode, an annelid, a worm, or a mollusk.

In some embodiments, the present disclosure provides a method of changing a subject by providing a composition or formulation as described herein to the subject. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or RNA molecule as described herein), and a polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes a eukaryotic or prokaryotic cell comprising a nucleic acid as described herein.

In certain embodiments, the present disclosure provides a method for treating a viral infection of a subject by providing a composition or preparation as described herein to a subject in need. In certain embodiments, the composition or preparation is or includes a nucleic acid molecule (e.g., a DNA molecule or RNA molecule as described herein), and provides polynucleotides to a eukaryotic subject. In certain embodiments, the composition or preparation is or includes a eukaryotic or prokaryotic cell comprising nucleic acid as described herein. In certain embodiments, the amount of polyribonucleotides provided and the duration are sufficient to treat a viral infection in a subject such as needing treatment.

In some embodiments, the method can be used to treat or prevent HIV. For example, in some embodiments, the cyclic polyribonucleotide encodes an anti-fusion polypeptide targeting HIV, and the composition can be used to treat or prevent HIV.

In some embodiments, the method can be used to treat or prevent SARS-CoV-2. For example, in some embodiments, the cyclic polyribonucleotide encodes an anti-fusion polypeptide targeting SARS-CoV-2, and the composition can be used to treat or prevent SARS-CoV-2.

In some embodiments, the method can be used to treat or prevent HCV. For example, in some embodiments, the cyclic polyribonucleotide encodes an anti-fusion polypeptide targeting HCV, and the composition can be used to treat or prevent HCV.

In some embodiments, the method can be used for treating or preventing RSV.For example, in some embodiments, the anti-fusion polypeptide targeting RSV of cyclic polyribonucleotide encoding, and the composition can be used for treating or preventing RSV.

Dosage

Disclosed herein is a method of administering a certain level of cyclic polyribonucleotides encoding anti-fusion polypeptides (e.g., polypeptides of Table 1) or expressing a certain level of anti-fusion polypeptides (e.g., polypeptides of Table 1) in cells after providing at least two doses or compositions of cyclic polyribonucleotides to cells.Disclosed herein is a method of administering a certain level of cyclic polyribonucleotides or expressing a certain level of anti-fusion polypeptides (e.g., polypeptides of Table 1, such as polypeptides of any one of Tables 2-4) in subjects (e.g., mammals, such as humans) after providing at least two doses or compositions of cyclic polyribonucleotides to subjects (e.g., administering).Described compositions include cyclic polyribonucleotides encoding anti-fusion polypeptides as described herein.Methods of administering may include administering two or more doses of cyclic polyribonucleotide compositions, such as in a short period of time or in an extended period of time.In certain embodiments, compositions containing cyclic polyribonucleotides further include a pharmaceutically acceptable carrier or excipient.Cyclic polyribonucleotides encode anti-fusion polypeptides, which can be expressed in cells, such as after administration.

The methods described herein may include administering a dose sufficient to produce 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) in a subject. /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).

In some embodiments, the method may further comprise administering a second dose of the pharmaceutical composition. The method may further comprise administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more dose of the pharmaceutical composition. In some embodiments, subsequent doses help maintain a serum concentration of the anti-fusion polypeptide in the subject 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). In some embodiments, the subsequent dose is administered before the serum concentration of the anti-fusion polypeptide in the subject drops below 500 ng/mL.

In some embodiments, multiple doses are provided to produce a certain level of the composition or express a certain level of the anti-fusion polypeptide in a cell, tissue, or subject. In some embodiments, multiple doses are provided to produce or maintain a certain level of the composition or produce or maintain a certain level of the anti-fusion polypeptide in a cell, tissue, or subject over a period of time, for example, 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, ten months, eleven months, one year, or more) after the first dose of the pharmaceutical composition.

In some embodiments, the second dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, such as 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, such as one day to one week, such as two days, three days, four days, five days, six days, or one week, such as one week to one month, such as two weeks, three weeks or one month, such as one month to one year, such as 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 1 to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days, or 90 days to 180 days).

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, ten months, eleven months, one year, or more) after the second dose of the pharmaceutical composition.

In some embodiments, the third dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, such as 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, such as one day to one week, such as two days, three days, four days, five days, six days, or one week, such as one week to one month, such as two weeks, three weeks or one month, such as one month to one year, such as 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 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days, or 90 days to 180 days).

In some embodiments, the second dose is administered before the serum concentration of the anti-fusion polypeptide in the serum of the subject is less than about 500 ng/mL.

In some embodiments, the methods maintain 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, ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL or more), for example, 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, 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, ten months, eleven months, one year or more).

A method for administering a composition of a nucleic acid molecule described herein (e.g., a cyclic polyribonucleotide) of multiple doses includes providing two or more compositions to a cell, tissue, or subject (e.g., a mammal) over a period of time. According to certain embodiments, a composition of a nucleic acid molecule described herein of multiple doses can be administered to a subject over a determined time course. The method according to this aspect of the invention includes sequentially administering a composition of a nucleic acid molecule described herein (e.g., a cyclic polyribonucleotide, a linear polyribonucleotide, a cyclic polydeoxyribonucleotide, a linear polydeoxyribonucleotide) of multiple doses to a subject (e.g., in a pharmaceutical or veterinary composition). As used herein, "sequential administration" means that at different time points (e.g., on different days) separated by a predetermined interval (e.g., by hour, day, week, or month), each dose of a composition of a nucleic acid molecule described herein is administered to a subject. In some embodiments, the method provided by the present invention includes sequentially administering a single initial dose of a composition of a nucleic acid molecule described herein to a subject, followed by one or more second doses of the composition, optionally followed by one or more third doses of the composition.

The terms "initial dose", "secondary dose" and "tertiary dose" refer to the temporal order in which the composition of nucleic acid molecules described herein is administered. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen; the "secondary dose" is the dose administered after the initial dose; the "tertiary dose" is the dose administered after the second dose. The initial dose, the second dose and the third dose can all contain the same amount of the composition of nucleic acid molecules described herein, and in certain embodiments, can differ from each other in terms of the frequency of administration. In certain embodiments, during the course of treatment, the amount of the composition of nucleic acid molecules described herein contained in the initial, second and/or third doses differs from each other (e.g., appropriately up-regulated or down-regulated). In certain embodiments, one or more (e.g., 2, 3, 4 or 5) doses are administered as a "loading dose" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered at a less frequent rate.

In certain embodiments, each second and/or third dosage is used after the previous dosage immediately preceding. As used herein, phrase "previous dosage immediately preceding" means in the order of multiple administrations, the composition of the nucleic acid molecules described herein of the dosage is administered to the subject before the dosage immediately following in the administration order, without intermediate dosage. In certain embodiments, each second and/or third dosage is used every day, every 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after the previous dosage immediately preceding. In certain embodiments, each second and/or third dosage is used every 0.5 week, 1 week, 2 weeks, 3 weeks or 4 weeks after the previous dosage immediately preceding.

The method according to this aspect of the invention may include administering to a subject any number of second and/or third doses of a composition of nucleic acid molecules described herein. For example, in certain embodiments, only a single second dose is administered to a subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to a subject. Similarly, in certain embodiments, only a single third dose is administered to a subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to a subject.

In certain embodiments, the frequency with which the second and/or third dose is administered to a subject may vary during 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 a cell, 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, a fourth composition, a fifth composition, a sixth composition, a seventh composition, an eighth composition, a ninth composition, a tenth composition, or more compositions. In some embodiments, additional compositions are provided over the duration of the cell lifespan. In some embodiments, additional compositions are provided (e.g., administered) when a cell, tissue, or subject benefits from the composition.

In some embodiments, the first composition in the multiple dosing regimen includes a first amount of nucleic acid molecules disclosed herein (e.g., cyclic polyribonucleotides). In some embodiments, the second composition in the multiple dosing regimen includes a second amount of nucleic acid molecules disclosed herein (e.g., cyclic polyribonucleotides). In some embodiments, the third composition, the fourth composition, the fifth composition, the sixth composition, the seventh composition, the eighth composition, the ninth composition, the tenth composition or more compositions in the multiple dosing regimen include the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth amount or more of nucleic acid molecules disclosed herein (e.g., cyclic polyribonucleotides). In some embodiments, the second amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) is the same as the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) is the same as the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth amount or more of nucleic acid molecules (e.g., cyclic polyribonucleotides) is the same as the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the second amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) is less than the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) is less than the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth amount or more of nucleic acid molecules (e.g., cyclic polyribonucleotides) is less than the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the second amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) is greater than the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) is greater than the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth amount or more of nucleic acid molecules (e.g., cyclic polyribonucleotides) is greater than the first amount of nucleic acid molecules (e.g., cyclic polyribonucleotides). In some embodiments, the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the second composition changes no more than 1%, 5%, 10%, 15%, 20% or 25% of the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the first composition. In some embodiments, the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the second composition is less than 1%, 5%, 10%, 15%, 20% or 25% of the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the first composition. In some embodiments, the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the second composition is 0.1 times to 1000 times higher than the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the first composition. In some embodiments, the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the second composition is 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times higher than the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the first composition. In certain embodiments, the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the subsequent composition (e.g., the composition used after the first composition) is higher than the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the first composition by 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times. In certain embodiments, the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the second composition is lower than the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the first composition by 0.1 times to 1000 times. In certain embodiments, the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the second composition is lower than the amount of the nucleic acid molecule (e.g., cyclic polyribonucleotide) of the first composition by 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of subsequent compositions (e.g., compositions used after the first composition) is 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times lower than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of subsequent compositions (e.g., after a certain amount of nucleic acid molecules (e.g., cyclic polyribonucleotides)) is 0.1 times to 1000 times higher or lower than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of subsequent compositions (e.g., after a certain amount of nucleic acid molecules (e.g., cyclic polyribonucleotides)) is 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times higher or lower than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the first composition. For example, the first composition comprises 1 times of nucleic acid molecules (for example, cyclic polyribonucleotides), the second composition comprises 5 times of nucleic acid molecules (for example, cyclic polyribonucleotides) compared with the first composition, and the third composition comprises 0.2 times of nucleic acid molecules (for example, cyclic polyribonucleotides) compared with the first composition. In certain embodiments, compared with the amount of the nucleic acid molecules (for example, cyclic polyribonucleotides) of the first composition, the second composition comprises at least 5 times of nucleic acid molecules (for example, cyclic polyribonucleotides).

In certain embodiments, the first composition comprises a higher amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) than the second composition. In certain embodiments, the first composition comprises a higher amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) than the 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th composition.

In some embodiments, a plurality of (e.g., two or more) compositions of nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding anti-fusion polypeptides administered with a multiple dosing regimen as described herein are the same composition. In some embodiments, a plurality of (e.g., two or more) compositions of nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding anti-fusion polypeptides administered with a multiple dosing regimen as described herein are different compositions. In some embodiments, the same composition comprises nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding the same anti-fusion polypeptides. In some embodiments, different compositions comprise nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding different anti-fusion polypeptides, or a combination thereof.

In certain embodiments, in multiple dosing regimens, the method for administering nucleic acid molecules (e.g., cyclic polyribonucleotides) provided herein includes administering nucleic acid molecules to a subject in need multiple times (multiple doses), for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 100, 150, 200 or 500 administrations, with an interval of 1 day to 56 days (e.g., about 49 days, 42 days, 35 days, 28 days, 21 days, 14 days or 7 days). In certain embodiments, in multiple dosing regimens, the method provided herein includes administering nucleic acid molecules to a subject in need at least 3 times, with an interval of about 7 days. In some embodiments, in subjects who receive administration of multiple doses of a nucleic acid molecule provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), the level of anti-fusion polypeptide (e.g., plasma anti-fusion polypeptide) remains at a level that changes by less than 50%, 40%, 30%, 20%, or 10% over a period of more 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 subjects who receive administration of multiple doses of a nucleic acid molecule provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), the level of the anti-fusion polypeptide (e.g., plasma anti-fusion polypeptide level) is maintained at a first level for a period of more 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, eighth, or last dose, wherein the first level is higher than the level of the anti-fusion polypeptide measured shortly after the first dose (e.g., the level measured about 12, 24, 36, or 48 hours after the first dose). In some embodiments, in subjects who receive administration of multiple doses of a nucleic acid molecule provided herein (e.g., at least 3 doses, about 7 days apart), the level of the anti-fusion polypeptide (e.g., plasma anti-fusion polypeptide level) is maintained at a first level for a period of more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eighth or last dose, wherein the first level is higher than the level of the anti-fusion polypeptide measured shortly after the first dose (e.g., the level measured about 12, 24, 36 or 48 hours after the first dose).

Delivery Method

The cyclic polyribonucleotide encoding the anti-fusion polypeptide described herein (eg, the polypeptide of Table 1) can be included in a pharmaceutical composition with or without a carrier.

The pharmaceutical compositions described herein can be formulated, for example, containing a carrier (such as a drug carrier and/or a polymer carrier, such as a liposome), and delivered to a subject in need (e.g., a human or non-human agricultural animal or livestock, such as cattle, dogs, cats, horses, poultry) by known methods. Such methods include, but are not limited to, transfection (e.g., lipid-mediated cationic polymers, calcium phosphate, dendrimers); electroporation or other membrane-destroying methods (e.g., nuclear transfection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, microparticle bombardment ("gene gun"), fugene, direct acoustic loading, cell squeezing, phototransfection, protoplast fusion, piercing infection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Delivery methods are also described, for example, in Gori et al., Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy. 2015 Jul, 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 certain embodiments, cyclic polyribonucleotides can be delivered in "naked" delivery formulations. Naked delivery formulations deliver cyclic polyribonucleotides to cells without the aid of carriers and without covalent modification of cyclic polyribonucleotides or without partial or complete encapsulation of cyclic polyribonucleotides.

Naked delivery preparation is the preparation without carrier, and wherein cyclic polyribonucleotide is not combined with the covalent modification of the part that helps to be delivered to cell and cyclic polyribonucleotide is not partially or completely encapsulated.In certain embodiments, the cyclic polyribonucleotide that is not combined with the covalent modification of the part that helps to be delivered to cell can be the polyribonucleotide that is not combined with the part (such as protein, micromolecule, particle, polymer or biopolymer) that helps to be delivered to cell covalently.In certain embodiments, cyclic polyribonucleotide can be delivered in delivery preparation together with protamine or protamine salt (for example protamine sulfate).

The polyribonucleotide that does not have the covalent modification that helps to be delivered to the part bonded of cell can not contain modified phosphate group.For example, the polyribonucleotide that does not have the covalent modification that helps to be delivered to the part bonded of cell can not contain thiophosphate, selenophosphate, borophosphate, borophosphate, hydrogen phosphate, phosphoramidate, phosphorodiamidate, alkyl or aryl phosphonate or phosphotriester.

In some embodiments, the naked delivery formulation may be free of any or all of the following: transfection agents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, the naked delivery formulation can be free of phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydride-modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethylenimine), poly(tetramethylenimine), polypropyleneimine, aminoglycoside-polyamines, 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-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propylammonium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamido glycylidene spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin.

Naked delivery formulations can include non-carrier excipients. In certain embodiments, non-carrier excipients can include inactive ingredients that do not show active cell penetration. In certain embodiments, non-carrier excipients can include buffers, such as PBS. In certain embodiments, non-carrier excipients can be solvents, non-aqueous solvents, diluents, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulators, disintegrants, adhesives, buffers, lubricants, or oils.

In certain embodiments, the naked delivery preparation may include a diluent, such as a parenteral acceptable diluent. The diluent (e.g., a parenteral acceptable diluent) may be a liquid diluent or a solid diluent. In certain embodiments, the diluent (e.g., a parenteral acceptable diluent) may be an RNA solubilizing agent, a buffer or an isotonic agent. The example of an RNA solubilizing agent includes water, ethanol, methanol, acetone, formamide and 2-propanol. Examples of buffers include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-acetylamino)-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 isotonic agents include glycerol, mannitol, polyethylene glycol, propylene glycol, trehalose or sucrose.

In some embodiments, preparation includes cell penetrating agent.In some embodiments, preparation is a topical preparation, and includes cell penetrating agent.Cell penetrating agent can include organic compound, such as alcohol with one or more hydroxyl functional groups.In some cases, cell penetrating agent includes alcohol, such as but not limited to monohydric alcohol, polyol, unsaturated aliphatic alcohol and alicyclic alcohol.Cell penetrating agent can include one or more of the following: methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenylethyl alcohol, butanol, amyl alcohol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl alcohol (particularly denatured alcohol), glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycol (PEG) -400, ethoxylated fatty acid or hydroxyethyl cellulose.In certain embodiments, cell penetrating agent includes ethanol.Cell penetrating agent can include any amount as described in WO 2020/180751 or WO 2020/180752 or any cell penetrating agent in any preparation, which is hereby incorporated by reference in its entirety.

In some embodiments, a pharmaceutical preparation as disclosed herein, a pharmaceutical composition as disclosed herein, a drug substance as disclosed herein, or a finished drug as disclosed herein is in a parenteral nucleic acid delivery system. A parenteral nucleic acid delivery system may include a pharmaceutical preparation as disclosed herein, a pharmaceutical composition as disclosed herein, a disclosed drug substance, or a finished drug as disclosed herein and a parenteral acceptable diluent. In some embodiments, a pharmaceutical preparation as disclosed herein, a pharmaceutical composition as disclosed herein, a drug substance as disclosed herein, or a finished drug as disclosed herein in a parenteral nucleic acid delivery system does not contain any carrier.

The disclosure further relates to a host or host cell comprising cyclic polyribonucleotides as described herein. In certain embodiments, the host or host cell is a vertebrate, a mammal (such as a human) or other organisms or cells.

In certain embodiments, compared with the response caused by reference compound (for example, corresponding to the linear polynucleotide of described cyclic polyribonucleotide), cyclic polyribonucleotide has the undesirable response of the host immune system of reduction or can not produce described undesirable response.In an embodiment, cyclic polyribonucleotide is non-immunogenic in host.Some immune responses include but are not limited to humoral immune response (for example, the generation of immunogen-specific antibody) and cell-mediated immune response (for example, lymphocyte proliferation).

In certain embodiments, host or host cell are contacted with (for example, delivered to or applied to) cyclic polyribonucleotide.In certain embodiments, host is mammal, such as mankind.Cyclic polyribonucleotide or linear polyribonucleotide, expression product or both amounts can be measured in host at any time after administration.In certain embodiments, the time course of host growth in culture is measured.If growth increases or decreases in the presence of cyclic polyribonucleotide or linear polyribonucleotide, then cyclic polyribonucleotide or expression product or both are considered to be effective in increasing or reducing the growth of host.

The method of delivering a cyclic polyribonucleotide molecule as described herein to a cell, tissue or subject comprises administering to the cell, tissue or subject a pharmaceutical composition, a pharmaceutical raw material or a pharmaceutical finished product as described herein.

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 connective tissue, muscle tissue, neural tissue, or epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.).

In certain embodiments, the delivery method is an in vivo method. For example, the delivery method of cyclic polyribonucleotides as described herein includes parenteral administration to a subject in need, and a pharmaceutical composition as described herein, a drug bulk drug, or a finished drug product is administered parenterally to a subject in need. For another example, the method of delivering cyclic polyribonucleotides to the cells or tissues of a subject includes administering a pharmaceutical composition as described herein, a drug bulk drug, or a finished drug product parenterally to the cells or tissues. In certain embodiments, the amount of cyclic polyribonucleotides effectively triggers a biological response in a subject. In certain embodiments, the amount of cyclic polyribonucleotides effectively has a biological effect on the cells or tissues of a subject. In certain embodiments, a pharmaceutical composition as described herein, a drug bulk drug, or a finished drug product includes a carrier. In certain embodiments, a pharmaceutical composition as described herein, a drug bulk drug, or a finished drug product includes a diluent without any carrier.

In some embodiments, the pharmaceutical composition, drug substance, or finished drug product is administered parenterally. In some embodiments, the pharmaceutical composition, drug substance, or finished drug product is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphatically, subcutaneously, or intramuscularly. In some embodiments, parenteral administration is intravenous, intramuscular, ocular, subcutaneous, intradermal, or topical.

In some embodiments, the pharmaceutical composition, drug substance, or finished drug product as described herein is administered intramuscularly. In some embodiments, the pharmaceutical composition, drug substance, or finished drug product as described herein is administered subcutaneously. In some embodiments, the pharmaceutical composition, drug substance, or finished drug product as described herein is administered topically. In some embodiments, the pharmaceutical composition, drug substance, or finished drug product as described herein is administered intratracheally.

In some embodiments, the pharmaceutical composition, drug substance or drug product is administered by injection. Administration can be systemic or local. In some embodiments, any delivery method as described herein is carried out with a carrier. In some embodiments, any delivery method as described herein can be carried out without the aid of a carrier or a cell penetrating agent.

In certain embodiments, after applying step, at least 1 day, at least 2 days, at least 3 days, at least 4 days or at least 5 days in cell, tissue or experimenter, detected cyclic polyribonucleotide or the product translated from described cyclic polyribonucleotide.In certain embodiments, before applying step, evaluate cyclic polyribonucleotide in cell, tissue or experimenter or the existence of the product translated from described cyclic polyribonucleotide.In certain embodiments, after applying step, evaluate cyclic polyribonucleotide in cell, tissue or experimenter or the existence of the product translated from described cyclic polyribonucleotide.

Preparations

In some embodiments of the present disclosure, cyclic polyribonucleotide as herein described can be formulated in compositions, for example, for being delivered to the compositions of cell, plant, invertebrate, non-human vertebrate or human experimenter, for example agriculture, veterinary medicine or pharmaceutical composition.In certain embodiments, cyclic polyribonucleotide is formulated in pharmaceutical composition.In certain embodiments, composition comprises cyclic polyribonucleotide and diluent, carrier, adjuvant or their combination.In specific embodiments, composition comprises cyclic polyribonucleotide as herein described and carrier or does not contain the diluent of any carrier.In certain embodiments, the composition comprising cyclic polyribonucleotide and the diluent not containing any carrier is used for naked delivery of cyclic polyribonucleotide to experimenter.

The pharmaceutical composition may optionally include one or more additional active substances, such as therapeutic and/or preventive active substances. The pharmaceutical composition may optionally include an inactive substance serving as a medium or medium of a composition as described herein (e.g., a composition comprising cyclic polyribonucleotides), such as any inactive ingredient approved by the U.S. Food and Drug Administration (FDA) and listed in the inactive ingredient data. The pharmaceutical composition of the present invention may be sterile and/or pyrogen-free. The overall considerations in the preparation and/or production of medicaments can be found in the following documents: for example, Remington: The Science and Practice of Pharmacy [Remington: Pharmaceutical Science and Practice] 21st edition, Lippincott Williams & Wilkins [Lippincott Williams and Wilkins Publishing Company], 2005 (incorporated herein by reference). Non-limiting examples of inactive substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonicity agents, thickeners, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof.

Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals are well known, and ordinary veterinary pharmacists can design and/or perform such modifications with only ordinary experimentation (if any). We envision that subjects to whom the pharmaceutical compositions are administered include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

The formulation of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed later. Generally, such preparation methods include the steps of combining the active ingredient with an excipient and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, dividing, shaping and/or packaging the product.

In some embodiments, the reference standard for the amount of cyclic polyribonucleotide molecules present in the formulation is at least 30% (w/w), 40% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w) of the total ribonucleotide molecules in the pharmaceutical formulation. , 95% (w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.1% (w/w), 99.2% (w/w), 99.3% (w/w), 99.4% (w/w), 99.5% (w/w), 99.6% (w/w), 99.7% (w/w) ), 99.8% (w/w), 99.9% (w/w), or 100% (w/w) of the molecule.

In some embodiments, the reference standard for the amount of linear polyribonucleotide molecules present in the formulation is the presence of no more than 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, The linear polyribonucleotide molecule of 1 μg/ml, 2 μg/ml, 3 μg/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 1 μg/ml, 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 g/ml, 300 μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml, 1 mg/ml, 1.5 mg/ml or 2 mg/ml.

In certain embodiments, the reference standard about the amount of the linear polyribonucleotide molecules present in the preparation is that the total ribonucleotide molecules in the pharmaceutical preparation are no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) of the linear polyribonucleotide molecules.

In certain embodiments, the reference standard for the amount of the nicked polyribonucleotide molecules present in the preparation is that the total ribonucleotide molecules in the pharmaceutical preparation are no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w) or 15% (w/w) of the nicked polyribonucleotide molecules.

In certain embodiments, the reference standard of the amount of the nicked and linear polyribonucleotide molecules of the combination present in the preparation is to account for the total ribonucleotide molecules in the pharmaceutical preparation and be no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) of the combination of nicked and linear polyribonucleotide molecules. In certain embodiments, pharmaceutical preparation is the intermediate pharmaceutical preparation of final cyclic polyribonucleotide finished drug. In certain embodiments, pharmaceutical preparation is a bulk drug or active pharmaceutical ingredient (API). In certain embodiments, pharmaceutical preparation is a finished drug for application to a subject.

In some embodiments, the preparation of cyclic polyribonucleotides (before, during or after reducing linear RNA) is further processed to substantially remove DNA, protein contaminants (e.g., cellular proteins (such as host cell proteins) or protein process impurities), endotoxins, mononucleotide molecules, and/or process-related impurities.

In some embodiments, the pharmaceutical formulation disclosed herein may include: (i) a compound disclosed herein (e.g., a cyclic polyribonucleotide); (ii) a buffer; (iii) a nonionic 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).

preservative

The compositions or pharmaceutical compositions provided herein may include materials for single administration, or may include materials for multiple administration (e.g., "multiple dose" kits). Polyribonucleotides may exist in linear or cyclic forms. The compositions or pharmaceutical compositions may include one or more preservatives, such as thimerosal or 2-phenoxyethanol. Preservatives may be used to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, Onamer M or other agents known to those skilled in the art. In ophthalmic products, for example, such preservatives may be used in an amount of 0.004% to 0.02%. In compositions described herein, preservatives (e.g., benzalkonium chloride) may be used in an amount of 0.001% to less than 0.01%, such as 0.001% to 0.008%, preferably about 0.005% by weight.

Polyribonucleotides can be susceptible to the influence of RNA enzymes that may be abundant in the surrounding environment. Compositions provided herein can include reagents that inhibit RNA enzyme activity, thereby preventing polyribonucleotide degradation. In some cases, the composition or pharmaceutical composition include any RNA enzyme inhibitors known to those skilled in the art. Alternatively or in addition, polyribonucleotides and cell penetrants and/or pharmaceutically acceptable diluents or carriers, vehicles, excipients or other reagents in the composition provided herein can be prepared in an environment without RNA enzymes. The composition can be prepared in an environment without RNA enzymes.

In some cases, the compositions provided herein can be sterile. The compositions can be formulated as sterile solutions or suspensions in suitable vehicles known in the art. The compositions can be sterilized by conventional known sterilization techniques, for example, the compositions can be sterile filtered.

Salt

In some cases, the compositions or pharmaceutical compositions provided herein include one or more salts. In order to control tonicity, the compositions provided herein may include physiological salts such as sodium salts. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and/or magnesium chloride, etc. In some cases, the compositions are formulated with one or more pharmaceutically acceptable salts. The one or more pharmaceutically acceptable salts may include inorganic ions, such as those of sodium, potassium, calcium, magnesium ions, etc. Such salts may include 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. Polyribonucleotides may exist in linear or cyclic form.

Buffer/pH

The compositions or pharmaceutical compositions provided herein may include one or more buffers, such as Tris buffers; borate buffers; succinate buffers; histidine buffers (e.g., containing aluminum hydroxide adjuvants); or citrate buffers. In some cases, a buffer in the range of 5-20 mM is included.

The compositions or pharmaceutical compositions provided herein can have a pH of about 5.0 to about 8.5, about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.8. The compositions or pharmaceutical compositions can have a pH of about 7. The polyribonucleotides can exist in linear or cyclic form.

Detergents/Surfactants

Depending on the intended route of administration, the compositions or pharmaceutical compositions provided herein may include one or more detergents and/or surfactants, such as polyoxyethylene sorbitan ester surfactants (commonly referred to as "Tweens"), such as polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO) and/or butylene oxide (BO), such as linear EO/PO block copolymers, sold under the DOWFAX ^™ trademark; octylphenol polyethers in which the number of repeating ethoxy(oxy-1,2-ethanediyl) groups can vary, such as octylphenol polyether-9 (Triton X-100 or tert-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol polyoxyethylene ethers, such as Tergitol ^™ NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol (called Brij surfactants), such as triethylene glycol monolauryl ether (Brij30); and sorbitan esters (commonly referred to as "SPAN"), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, octylphenol polyethers (such as octylphenol polyether 9 (Triton X-100) or tert-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB") or sodium deoxycholate. The one or more detergents and/or surfactants may be present only in trace amounts. In some cases, the composition may contain less than 1 mg/ml of each octylphenol polyether-10 and polysorbate 80. Nonionic surfactants can be used herein. Surfactants can be classified by their "HLB" (hydrophilic/lipophilic balance value). In some cases, the HLB of the surfactant is at least 10, at least 15 and/or at least 16. Polyribonucleotides can exist in linear or circular form.

Thinner

In some embodiments, the compositions of the present disclosure comprise cyclic polyribonucleotides and a diluent. In some embodiments, the compositions of the present disclosure comprise linear polyribonucleotides and a diluent.

Diluent can be non-carrier excipient.Non-carrier excipient is used as the vehicle or medium of composition (such as, cyclic polyribonucleotide as described herein).Non-carrier excipient is used as the vehicle or medium of composition (such as, linear polyribonucleotide as described herein).Non-limiting examples of non-carrier excipient include solvent, aqueous solvent, non-aqueous solvent, dispersion medium, diluent, dispersant, suspending agent, surfactant, isotonic agent, thickening agent, emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, dispersant, granulating agent, disintegrant, adhesive, buffer (for example, phosphate buffered saline (PBS)), lubricant, oil and mixture thereof.Non-carrier excipient can be any inactive ingredient that does not show cell penetration effect by approval of U.S. Food and Drug Administration (FDA) and is listed in the inactive ingredient database.Non-carrier excipient can be any inactive ingredient suitable for being applied to non-human animals (for example, suitable for veterinary use). Modification of compositions suitable for administration to humans in order to render them suitable for administration to various animals is well understood, and a veterinary pharmacist of ordinary skill can design and/or perform such modifications with no more than ordinary experimentation, if any.

In certain embodiments, cyclic polyribonucleotide can be delivered in the form of naked delivery preparation, such as comprising diluent. Naked delivery preparation delivers cyclic polyribonucleotide to cell, without the need to be modified or partially or completely encapsulated by means of carrier and without the need to cyclic polyribonucleotide, capped polyribonucleotide or its complex.

Naked delivery preparation is the preparation without carrier and wherein cyclic polyribonucleotide is not in conjunction with the covalent modification of the part that helps to be delivered to cell, or there is not part or complete encapsulation of cyclic polyribonucleotide.In certain embodiments, not in conjunction with the covalently modified cyclic polyribonucleotide of the part that helps to be delivered to cell is the polyribonucleotide that is not covalently bound to protein, micromolecule, particle, polymer or biopolymer.Not in conjunction with the covalently modified cyclic polyribonucleotide of the part that helps to be delivered to cell does not contain modified phosphate group.For example, not in conjunction with the covalently modified cyclic polyribonucleotide of the part that helps to be delivered to cell does not contain thiophosphate, selenophosphate, borophosphate, borophosphate, hydrogen phosphate, phosphoramidate, diaminophosphoroester, alkyl or aryl phosphonate or phosphotriester.

In some embodiments, the naked delivery formulation is free of any or all of the following: transfection agents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. In some embodiments, the naked delivery formulation does not contain phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydride-modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethylenimine), poly(tetramethylenimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationic gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)- Imidazolium chloride (DOTIM), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamido glycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL) or globulin.

In certain embodiments, the naked delivery formulation includes non-carrier excipients. In certain embodiments, non-carrier excipients include inactive ingredients that do not show cell penetration. In certain embodiments, non-carrier excipients include buffers, such as PBS. In certain embodiments, non-carrier excipients are solvents, non-aqueous solvents, diluents, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulators, disintegrants, binders, buffers, lubricants or oils.

In some embodiments, naked delivery preparation includes diluent.Diluent can be liquid diluent or solid diluent.In some embodiments, diluent is RNA solubilizing agent, buffer or isotonic agent.The example of RNA solubilizing agent includes water, ethanol, methanol, acetone, formamide and 2-propanol.The example of buffer includes 2-(N-morpholino) ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl) amino] acetic acid (ADA), N-(2-acetylamino)-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 isotonic agents include glycerol, mannitol, polyethylene glycol, propylene glycol, trehalose or sucrose.

Carrier

In some embodiments, the compositions of the present disclosure comprise cyclic polyribonucleotides and a carrier. In some embodiments, the compositions of the present disclosure comprise linear polyribonucleotides and a carrier.

In certain embodiments, the composition comprises a cyclic polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In certain embodiments, the composition comprises a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.

In other embodiments, compositions comprises cyclic polyribonucleotide in cell, vesicle or other carriers based on film or via cell, vesicle or other carriers based on film.In other embodiments, compositions comprises linear polyribonucleotide in cell, vesicle or other carriers based on film or via cell, vesicle or other carriers based on film.In one embodiment, compositions comprises cyclic polyribonucleotide in liposome or other similar vesicles.In one embodiment, compositions comprises linear polyribonucleotide in liposome or other similar vesicles.Liposome is spherical vesicle structure, and described spherical vesicle structure is made up of single layer or multilayer lipid bilayer around internal aqueous compartment and relatively impermeable external lipophilic phospholipid bilayer.Liposome can be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their cargo across biological membranes and the blood-brain barrier (BBB) (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, Vol. 2011, Article ID 469679, p. 12, 2011. doi:10.1155/2011/469679).

Vesicles can be made of several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, which relates to the teaching of multilamellar vesicle lipid preparation and is incorporated herein by reference). Although vesicle formation can be spontaneous when lipid film is mixed with aqueous solution, vesicle formation can also be accelerated by applying force in the form of oscillation using a homogenizer, sonicator or extrusion equipment (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery [Drug Delivery Magazine], Vol. 2011, Article ID 469679, p. 12, 2011.doi:10.1155/2011/469679). Extruded lipids can be prepared by extrusion through a filter of decreasing size as described in Templeton et al., Nature Biotech, 15:647-652, 1997, which is incorporated herein by reference for its teachings on the preparation of extruded lipids.

In certain embodiments, the compositions disclosed herein include cyclic polyribonucleotides and lipid nanoparticles, such as lipid nanoparticles as described herein. In certain embodiments, the compositions disclosed herein include linear polyribonucleotides and lipid nanoparticles. Lipid nanoparticles are another example of carriers that provide biocompatibility and biodegradable delivery systems for cyclic polyribonucleotide molecules as described herein. Lipid nanoparticles are another example of carriers that provide biocompatibility and biodegradable delivery systems for linear polyribonucleotide molecules as described herein. Nanostructured lipid carriers (NLC) are modified solid lipid nanoparticles (SLN), and the modified solid lipid nanoparticles retain the characteristics of SLN, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNP) are an important part of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled release of drugs. Lipid-polymer nanoparticles (PLN) can also be used, i.e., a novel carrier that combines liposomes and polymers. These nanoparticles have the complementary advantages of PNP and liposomes. PLNs consist of a core-shell structure; the polymer core provides a stable structure, while the phospholipid shell provides good biocompatibility. Therefore, these two components increase drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs. For review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi: 10.3390/nano7060122.

Other non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-based materials), protein carriers (e.g., proteins covalently linked to cyclic polyribonucleotides or proteins covalently linked to linear polyribonucleotides), or cationic carriers (e.g., cationic lipid polymers or transfection reagents). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin. Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethylenimine), poly(tetramethylenimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationic gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)- ) imidazolium chloride (DOTIM), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propylammonium (DOSPA), 3B-[N-(N,N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamido glycylidene spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE) and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Non-limiting examples of protein carriers include human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL) or globulin.

Exosomes can also be used as drug delivery vehicles for circular RNA compositions or preparations described herein. Exosomes can be used as drug delivery vehicles for linear polyribonucleotide compositions or preparations described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B [Acta Pharmaceutica Sinica B] Vol. 6, No. 4, pp. 287-296; doi.org/10.1016/j.apsb.2016.02.001. In vitro differentiated erythrocytes can also be used as carriers for circular RNA compositions or preparations described herein. In vitro differentiated erythrocytes can also be used as carriers for linear polyribonucleotide compositions or preparations described herein. See, e.g., International Patent Publication Nos. WO 2015/073587; WO 2017/123646; WO 2017/123644; WO 2018/102740; WO 2016/183482; WO 2015/153102; WO 2018/151829; WO 2018/009838; Shi et al. 2014. Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America]. 111(28):10131-10136; U.S. Patent 9,644,180; Huang et al. 2017. Nature Communications [Nature-Communications] 8:423; Shi et al. 2014. Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America]. 111(28):10131-10136. For example, the fusion compositions described in International Patent Publication No. WO 2018/208728 can also be used as a carrier to deliver circular polyribonucleotide molecules described herein. For example, the fusion compositions described in WO 2018/208728 can also be used as a carrier to deliver linear polyribonucleotide molecules described herein.

Virosome and virus-like particle (VLP) also can be used as carrier and circular polyribonucleotide molecule as herein described is delivered to the cell of targeting. Virosome and virus-like particle (VLP) also can be used as carrier and linear polyribonucleotide molecule as herein described is delivered to the cell of targeting.

Plant nanovesicles and plant messenger bags (PMPs) such as International Patent Publication Nos. WO 2011/097480, WO 2013/070324, WO 2017/004526 or WO2020/041784 can also be used as carriers to deliver circular RNA compositions or preparations described herein. Plant nanovesicles and plant messenger bags (PMPs) can also be used as carriers to deliver linear polyribonucleotide compositions or preparations described herein.

Microbubbles can also be used as carriers to deliver cyclic polyribonucleotide molecules described herein. Microbubbles can also be used as carriers to deliver linear polyribonucleotide molecules described herein. See, for example, US 7115583; Beeri, R. et al., Circulation. [Circulation] October 1, 2002; 106 (14): 1756-1759; Bez, M. et al., Nat Protoc. [Natural Experiment Manual] April 2019; 14 (4): 1015-1026; Hernot, S. et al., Adv Drug Deliv Rev. [Advanced Drug Delivery Review] June 30, 2008; 60 (10): 1153-1166; Rychak, J.J. et al., Adv Drug Deliv Rev. [Advanced Drug Delivery Review] June 2014; 72: 82-93. In some embodiments, microbubbles are albumin-coated perfluorocarbon microbubbles.

The carrier comprising cyclic polyribonucleotide as herein described can comprise multiple particles.Described particle can have the median particle size (for example 30 to 50,50 to 100,100 to 200,200 to 300,300 to 400,400 to 500,500 to 600,600 to 700,100 to 500,50 to 500 or 200 to 700 nanometers) of 30 to 700 nanometers.The size of particle can be optimized to be deposited in the cell with the useful load that is conducive to including cyclic polyribonucleotide.Cyclic polyribonucleotide is deposited in some cell type and can be conducive to different particle sizes.For example, particle size can be optimized so that cyclic polyribonucleotide is deposited in antigen presenting cells.Particle size can be optimized so that cyclic polyribonucleotide is deposited in dendritic cells.In addition, particle size can be optimized so that cyclic polyribonucleotide is deposited in drainage lymph node cells.

Lipid Nanoparticles

The compositions, methods and delivery systems provided by the present disclosure can adopt any suitable carrier or delivery form as described herein, including lipid nanoparticles (LNP) in certain embodiments. In certain embodiments, lipid nanoparticles include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic or zwitterionic lipids); one or more conjugated lipids (such as PEG conjugated lipids described in Table 5 of WO 2019217941 or conjugated to a polymer; it is incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol).

Lipids that can be used for nanoparticle formation (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO 2019217941, which is incorporated herein by reference—for example, the lipid-containing nanoparticles may include one or more lipids in Table 4 of WO 2019217941. The lipid nanoparticles may include additional elements, such as polymers, such as the polymers described in Table 5 of WO 2019217941, which is incorporated by reference.

In some embodiments, the conjugated lipid, when present, may include one or more of: PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019051289 (incorporated by reference), and combinations of the foregoing.

In some embodiments, the sterol that can be incorporated into the lipid nanoparticles includes one or more of cholesterol or cholesterol derivatives, such as those in WO 2009/127060 or US 2010/0130588, which are incorporated by reference. Additional exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, which are incorporated by reference herein.

In some embodiments, lipid particles include ionizable lipids, non-cationic lipids, conjugated lipids and sterols that inhibit particle aggregation. The amount of these components can be changed independently to obtain desired properties. For example, in some embodiments, lipid nanoparticles include ionizable lipids, non-cationic lipids, conjugated lipids and sterols, and the amount of the ionizable lipids is about 20mol% to about 90mol% of total lipids (in other embodiments, the ionizable lipids can be 20%-70% (mol), 30%-60% (mol) or 40%-50% (mol) of the total lipids present in the lipid nanoparticles; about 50mol% to about 90mol%); the amount of the non-cationic lipids is about 5mol% to about 30mol% of total lipids; the amount of the conjugated lipids is about 0.5mol% to about 20mol% of total lipids, and the amount of the sterols is about 20mol% to about 50mol% of total lipids. The ratio of total lipids to nucleic acids can be changed as needed. For example, the ratio of total lipids to nucleic acids (mass or weight) can be about 10: 1 to about 30: 1.

In some embodiments, the ratio of lipid to nucleic acid (mass/mass ratio; w/w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and nucleic acid can be adjusted to provide a desired N/P ratio, such as 3, 4, 5, 6, 7, 8, 9, 10 or higher N/P ratios. Typically, the total lipid content of the lipid nanoparticle formulation can be in the range of about 5 mg/ml to about 30 mg/mL.

Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivering the compositions described herein, e.g., nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) include:

In some embodiments, LNPs comprising formula (i) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising formula (ii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising formula (iii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising formula (v) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising formula (vi) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising formula (viii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising formula (ix) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

in

^X1 is O, ^NR1 or a direct bond, ^X2 is C2-5 alkylene, ^X3 is C(=O) or a direct bond, ^R1 is H or Me, ^R3 is C1-3 alkyl, ^R2 is C1-3 alkyl, or ^R2 together with the nitrogen atom to which it is attached and 1-3 carbon atoms of ^X2 form a 4-membered, 5-membered or 6-membered ring, or ^X1 is ^NR1 , ^R1 and ^R2 together with the nitrogen atom to which they are attached form a 5-membered or 6-membered ring, or ^R2 and ^R3 together with the nitrogen atom to which they are attached form a 5-membered, 6-membered or 7-membered ring, ^Y1 is C2-12 alkylene, and ^Y2 is selected from

n is 0 to 3, ^R4 is C1-15 alkyl, ^Z1 is C1-6 alkylene or a direct bond,

^Z2 is

(in either orientation) or absent, provided that if Z ¹ is a direct bond, then Z ² is absent;

^R5 is C5-9 alkyl or C6-10 alkoxy, ^R6 is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and ^R7 is H or Me, or a salt thereof, provided that if ^R3 and ^R2 are C2 alkyl, ^X1 is O, ^X2 is a straight-chain C3 alkylene, ^X3 is C(=0), ^Y1 is a straight-chain Ce alkylene, ( ^Y2 ) ^nR4 is

, ^R4 is a linear C5 alkyl, ^Z1 is a C2 alkylene, ^Z2 is absent, W is a methylene, and ^R7 is H, then ^R5 and ^R6 are not Cx alkoxy.

In some embodiments, LNPs comprising formula (xii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising formula (xi) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

in

In some embodiments, the LNP comprises a compound having formula (xiii) and a compound having formula (xiv).

In some embodiments, LNPs comprising formula (xv) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, LNPs comprising a formulation having formula (xvi) are used to deliver a polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

in

In some embodiments, lipid compounds used to form lipid nanoparticles for delivering compositions described herein, e.g., nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) are made by one of the following reactions:

In certain embodiments, the LNP including formula (xxi) is used for delivering polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions as described herein to cells. In certain embodiments, the LNP of formula (xxi) is the LNP described by WO 2021113777 (e.g., lipid of formula (1), such as lipid of Table 1 of WO 2021113777).

in

Each n is independently an integer from 2 to 15; _L1 and _L3 are each independently -OC(O)-* or -C(O)O-*, wherein "*" represents the point of attachment to _R1 or _R3 ;

_R1 and _R3 are each independently a linear or branched _C9 - _C20 alkyl group or a _C9 -C ₂₀ alkenyl, which is optionally substituted with one or more substituents selected from the group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonealkyl;

_R2 is selected from the group consisting of:

In certain embodiments, the LNP including formula (xxii) is used for delivering polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions as described herein to cells. In certain embodiments, the LNP of formula (xxii) is the LNP described in WO 2021113777 (e.g., lipids of formula (2), such as lipids of Table 2 of WO 2021113777).

in

Each n is independently an integer from 1 to 15;

_R1 and _R2 are each independently selected from the group consisting of:

_R3 is selected from the group consisting of:

In certain embodiments, the LNP including formula (xxiii) is used for delivering polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions as described herein to cells. In certain embodiments, the LNP of formula (xxiii) is the LNP described in WO 2021113777 (e.g., lipids of formula (3), such as lipids of Table 3 of WO 2021113777).

in

X is selected from -O-, -S- or -OC(O)-*, wherein * represents the point of attachment to R ₁ ;

_R1 is selected from the group consisting of:

and _R2 is selected from the group consisting of:

In some embodiments, the compositions described herein (e.g., nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs comprising ionizable lipids. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); for example, as described in Example 1 of US 9,867,888 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadecane-9,12-dienoate (LP01), for example, as synthesized in Example 13 of WO 2015/095340 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), for example, as synthesized in Examples 7, 8, or 9 of US 2012/0027803, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as synthesized in Examples 14 and 16 of WO 2010/053572, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is an imidazolyl cholesteryl ester (ICE) lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazol-4-yl)propanoate, such as structure (I) from WO2020/106946 (incorporated herein by reference in its entirety).

In some embodiments, the ionizable lipid can be a cationic lipid, an ionizable cationic lipid, such as a cationic lipid that can exist in a positively charged form or a neutral form according to pH, or an amine-containing lipid that can be easily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. Exemplary cationic lipids include one or more positively charged amine groups. In some embodiments, the lipid particles include cationic lipids prepared with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (such as sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments, the cationic lipid can be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein can have an effective pKa exceeding 6.0. In an embodiment, the lipid nanoparticle may include a second cationic lipid having an effective pKa different from the first cationic lipid (e.g., greater than the first effective pKa). The lipid nanoparticles may include 40 mol % to 60 mol % of cationic lipids, neutral lipids, steroids, polymer-conjugated lipids and therapeutic agents encapsulated in or associated with the lipid nanoparticles, such as nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)). In some embodiments, nucleic acids are co-formulated with cationic lipids. Nucleic acids can be adsorbed onto the surface of LNPs (e.g., LNPs comprising cationic lipids). In some embodiments, nucleic acids can be encapsulated in LNPs (e.g., LNPs comprising cationic lipids). In some embodiments, lipid nanoparticles may include targeting moieties, such as targeting moieties coated with targeting agents. In embodiments, LNP formulations are biodegradable. In some embodiments, lipid nanoparticles comprising one or more lipids described herein (e.g., formula (i), (ii), (vii), and/or (ix)) encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% of the RNA molecules.

Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of WO 2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulae: X of US 2016/0311759; I of US 20150376115 or US 2016/0376224; I, II, or III of US 20160151284; I, IA, II, or IIA of US 20170210967; I-c of US 20150140070; A of US 2013/0178541; I of US 2013/0303587 or US 2013/0123338; I of US 2015/0141678; II, III, IV, or V of US 2015/0239926; I of US 2017/0119904; I or II of WO 2017/117528; US A of US 2012/0149894; A of US 2015/0057373; A of WO 2013/116126; A of US 2013/0090372; A of US 2013/0274523; A of US 2013/0274504; A of US 2013/0053572; A of WO 2013/016058; A of WO 2012/162210; I of US 2008/042973; I, II, III or IV of US 2012/01287670; I or II of US 2014/0200257; I, II or III of US 2015/0203446; I or III of US 2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID or III-XXIV of US 2014/0308304; US 2013/0338210; I, II, III or IV of WO 2009/132131; A of US 2012/01011478; I or XXXV of US 2012/0027796; XIV or XVII of US 2012/0058144; US 2013/0323269; I of US 2011/0117125; I, II or III of US 2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV or XVI of US 2011/0076335; I or II of US 2006/008378; I of US 2013/0123338; I or X-A-Y-Z of US 2015/0064242; XVI, XVII or XVIII of US 2013/0022649; I, II or III of US 2013/0116307; I, II or III of US 2013/0116307; I or II of US 2010/0062967; I-X of US 2013/0189351; I of US 2014/0039032; V of 2018/0028664; I of US 2016/0317458; I of US 2013/0195920; 5, 6 or 10 of US10,221,127; III-3 of WO 2018/081480; I-5 or I-8 of WO 2020/081938; 18 or 25 of US 9,867,888; A of US 2019/0136231; II of WO 2020/219876; 1 of US 2012/0027803; OF-02 of US 2019/0240349; 23 of US 10,086,013; cKK-E12/A6 of Miao et al. (2020); WO C12-200 of 2010/053572; 7C1 of Dahlman et al. (2017); 304-013 or 503-013 of Whitehead et al.; TS-P4C2 of US 9,708,628; I of WO 2020/106946; I of WO 2020/106946; and (1), (2), (3) or (4) of WO 2021/113777. Exemplary lipids further include lipids of any one of Tables 1-16 of WO 2021/113777.

In some embodiments, the ionizable lipid is MC3 (6Z, 9Z, 28Z, 31Z) - heptatriacontane-6, 9, 28, 31-tetraen-19-yl-4- (dimethylamino) butyrate (DLin-MC3-DMA or MC3), for example, as described in Example 9 of WO 2019051289A9 (incorporated by reference in its entirety). In some embodiments, the ionizable lipid is lipid ATX-002, for example, as described in Example 10 of WO 2019051289A9 (incorporated by reference in its entirety). In some embodiments, the ionizable lipid is (13Z, 16Z) -A, A-dimethyl-3-nonyldios-13, 16-dien-1-amine (compound 32), for example, as described in Example 11 of WO 2019051289A9 (incorporated by reference in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO 2019051289A9 (incorporated by reference in its entirety).

Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16- O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucylphosphatidylcholine (DMPC), dierucylphosphatidylglycerol (DMPG), dierucylphosphatidylcholine (DMPG), dierucylphosphatidylcholine (DMPC), dierucylphosphatidylcholine ... The phospholipids of the present invention are preferably phosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), di-re-oleoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, bis-hexadecyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine or its mixture. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably an acyl group derived from a fatty acid with a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearyl or oleoyl. In certain embodiments, additional exemplary lipids include, but are not limited to, those described by Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, which is incorporated herein by reference. In some embodiments, such lipids include plant lipids (e.g., DGTS) found to improve liver transfection with mRNA.

Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids, such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, ricinoleic acid glyceride, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO 2017/099823 or U.S. Patent Publication US 2018/0028664, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II or IV of US 2018/0028664, which is incorporated by reference in its entirety. The non-cationic lipid may account for 0%-30% (mole) of the total lipid present in, for example, a lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5%-20% (mole) or 10%-15% (mole) of the total lipid present in the lipid nanoparticle. In an embodiment, the molar ratio of ionizable lipid to neutral lipid is about 2: 1 to about 8: 1 (e.g., about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1 or 8: 1).

In some embodiments, the lipid nanoparticles do not include any phospholipids.

In some aspects, the lipid nanoparticles may further include components such as sterols to provide membrane integrity. An exemplary sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2,-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether and 6-ketocholestanol; non-polar analogs such as 5a-cholestanes, cholesterenone, 5a-cholestanone, 5p-cholestanone and cholesterol decanoate; and mixtures thereof. In certain embodiments, cholesterol derivatives are polar analogs, for example, cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO 2009/127060 and U.S. Patent Publication US 2010/0130588, each of which is incorporated herein by reference in its entirety.

In some embodiments, components that provide membrane integrity, such as sterols, can comprise 0%-50% (mol) of the total lipid present in the lipid nanoparticle (e.g., 0%-10%, 10%-20%, 20%-30%, 30%-40%, or 40%-50%). In some embodiments, such components are 20%-50% (mol), 30%-40% (mol) of the total lipid content of the lipid nanoparticle.

In certain embodiments, lipid nanoparticles may include polyethylene glycol (PEG) or conjugated lipid molecules. Typically, these are used to suppress the aggregation of lipid nanoparticles and/or provide spatial stabilization. Exemplary conjugated lipids include but are not limited to PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates and mixtures thereof. In certain embodiments, conjugated lipid molecules are PEG-lipid conjugates, for example (methoxy polyethylene glycol) conjugated lipids.

Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described in, for example, US 5,885,613, US 6,287,591, US In some embodiments, the PEG-lipid is a compound of formula III, III-a-1, III-a-2, III-b-1, III-b-2 or V of US 2018/0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid has US20150376115 or US 2016/0376224, both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-distearylglycerol, PEG-dilaurylglyceramide, PEG- Dimyristylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from the group consisting of:

In some embodiments, lipids conjugated to molecules other than PEG can also be used to replace PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates) and cationic polymer lipid (GPL) conjugates can be used to replace PEG-lipids or used together with PEG-lipids.

Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO 2019051289A9, the entire contents of which are incorporated herein by reference in their entirety.

In some embodiments, PEG or conjugated lipids may include 0%-20% (moles) of the total lipids present in the lipid nanoparticles. In some embodiments, the content of PEG or conjugated lipids is 0.5%-10% or 2%-5% (moles) of the total lipids present in the lipid nanoparticles. The molar ratio of ionizable lipids, non-cationic lipids, sterols and PEG conjugated lipids can be changed as needed. For example, lipid particles may include 30%-70% ionizable lipids by mole or total weight of the composition, 0%-60% cholesterol by mole or total weight of the composition, 0%-30% non-cationic lipids by mole or total weight of the composition and 1%-10% conjugated lipids by mole or total weight of the composition. Preferably, the composition includes 30%-40% ionizable lipids by mole or total weight of the composition, 40%-50% cholesterol by mole or total weight of the composition, and 10%-20% non-cationic lipids by mole or total weight of the composition. In some other embodiments, the composition is 50%-75% ionizable lipids by mole or total weight of the composition, 20%-40% cholesterol by mole or total weight of the composition, and 5%-10% non-cationic lipids by mole or total weight of the composition, and 1%-10% conjugated lipids by mole or total weight of the composition. The composition may contain 60%-70% ionizable lipids by mole or total weight of the composition, 25%-35% cholesterol by mole or total weight of the composition, and 5%-10% non-cationic lipids by mole or total weight of the composition. The composition may also contain up to 90% ionizable lipids by mole or total weight of the composition and 2% to 15% non-cationic lipids by mole or total weight of the composition. The formulation can also be a lipid nanoparticle formulation, for example comprising 8%-30% ionizable lipid by mole or total weight of the composition, 5%-30% non-cationic lipid by mole or total weight of the composition, and 0%-20% cholesterol by mole or total weight of the composition; 4%-25% ionizable lipid by mole or total weight of the composition, 4%-25% non-cationic lipid by mole or total weight of the composition, 2% to 25% cholesterol by mole or total weight of the composition, 10% to 35% conjugated lipid by mole or total weight of the composition, and 0%-20% cholesterol by mole or total weight of the composition; 5% cholesterol; or 2%-30% ionizable lipids by mole or total weight of the composition, 2%-30% non-cationic lipids by mole or total weight of the composition, 1% to 15% cholesterol by mole or total weight of the composition, 2% to 35% conjugated lipids by mole or total weight of the composition, and 1%-20% cholesterol by mole or total weight of the composition; or even up to 90% ionizable lipids by mole or total weight of the composition and 2%-10% non-cationic lipids by mole or total weight of the composition, or even 100% cationic lipids by mole or total weight of the composition. In some embodiments, the lipid particle formulation includes ionizable lipids, phospholipids, cholesterol and PEGylated lipids in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation includes ionizable lipids, cholesterol and PEGylated lipids in a molar ratio of 60:38.5:1.5.

In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, wherein for the ionizable lipid, the molar ratio of the lipid is in the range of 20 to 70 mole percent, with a target of 40-60, the mole percent of the non-cationic lipid is in the range of 0 to 30, with a target of 0 to 15, the mole percent of the sterol is in the range of 20 to 70, with a target of 30 to 50, and the mole percent of the PEGylated lipid is in the range of 1 to 6, with a target of 2 to 5.

In some embodiments, the lipid particle comprises an ionizable lipid/non-cationic lipid/sterol/conjugated lipid in a molar ratio of 50:10:38.5:1.5.

In one aspect, the present disclosure provides lipid nanoparticle formulations comprising phospholipids, phosphatidylcholine, phosphatidylcholine, and phosphatidylethanolamine.

In certain embodiments, one or more additional compounds may also be included. Those compounds may be administered alone, or additional compounds may be included in the lipid nanoparticles of the present invention. In other words, except nucleic acid or at least the second nucleic acid, the lipid nanoparticles may contain other compounds that are different from the first nucleic acid. In a non-limiting manner, other additional compounds may be selected from the group consisting of: small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptide mimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biomaterials, or any combination thereof.

In certain embodiments, LNP includes biodegradable, ionizable lipids. In certain embodiments, LNP includes (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecane-9,12-dienoate, also referred to as 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadecane-9,12-dienoate) or another ionizable lipid. See, e.g., WO 2019/067992, WO/2017/173054, WO 2015/095340, and WO2014/136086, and the lipids of the references provided therein. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, for example, where the ionizable lipid is cationic depending on pH.

In some embodiments, the average LNP diameter of the LNP formulation can be between tens of nm and hundreds of nm, for example, as measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of LNP formulations may be from about 50nm to about 100nm, from about 50nm to about 90nm, from about 50nm to about 80nm, from about 50nm to about 70nm, from about 50nm to about 60nm, from about 60nm to about 100nm, from about 60nm to about 90nm, from about 60nm to about 80nm, from about 60nm to about 70nm, from about 70nm to about 100nm, from about 70nm to about 90nm, from about 70nm to about 80nm, from about 80nm to about 100nm, from about 80nm to about 90nm, or from about 90nm to about 100nm. In some embodiments, the average LNP diameter of LNP formulations may be from about 70nm to about 100nm. In a specific embodiment, the average LNP diameter of LNP formulations may be about 80nm. In some embodiments, the average LNP diameter of LNP formulations may be about 100nm. In some embodiments, the LNP formulation has an average LNP diameter ranging from about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

In some cases, LNP can be relatively homogeneous.Polydispersity index can be used for indicating the homogeneity of LNP, for example the size distribution of lipid nanoparticles.Small (for example, less than 0.3) polydispersity index usually indicates narrow size distribution.The polydispersity index of LNP can be about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25.In certain embodiments, the polydispersity index of LNP can be about 0.10 to about 0.20.

The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. In certain embodiments, the zeta potential can describe the surface charge of LNP. Lipid nanoparticles with relatively low charge (positive or negative charge) are generally desirable because higher charged materials may not interact with cells, tissues and other elements in the body undesirably. In some embodiments, the zeta potential of the LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

The encapsulation efficiency of protein and/or nucleic acid describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with LNP after preparation relative to the initial amount provided. Encapsulation efficiency is ideally higher (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after crushing lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For lipid nanoparticles as described herein, the encapsulation efficiency of protein and/or nucleic acid can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

The LNP may optionally include one or more layers of coating. In some embodiments, the LNP may be formulated in a capsule, film or tablet having a coating. The capsule, film or tablet comprising the composition described herein may have any useful size, tensile strength, hardness or density.

WO 2020/061457 and WO 2021/113777 (each of which is incorporated herein by reference in its entirety) teach additional exemplary lipids, formulations, methods and characterizations of LNPs. Other exemplary lipids, formulations, methods and LNP characterizations are taught by Hou et al. Lipid nanoparticles for mRNA delivery [lipid nanoparticles for mRNA delivery]. Nat Rev Mater [Natural Review Materials] (2021). doi.org/10.1038/s41578-021-00358-0, which is incorporated herein by reference in its entirety (see, e.g., the exemplary lipids and lipid derivatives of Fig. 2 of Hou et al.).

In some embodiments, in vitro or ex vivo cell lipid transfection is performed using Lipofectamine MessengerMax (ThermoFisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNPs are prepared using GenVoy_ILM ionizable lipid mixtures (Precision NanoSystems). In certain embodiments, LNPs are prepared using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the preparation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl [German Applied Chemistry] 51 (34): 8529-8533 (2012), which is incorporated herein by reference in its entirety.

LNP formulations optimized for delivery of CRISPR-Cas systems (e.g., Cas9-gRNA RNPs, gRNA, Cas9 mRNA) are described in WO 2019067992 and WO 2019067910, both of which are incorporated by reference, and can be used to deliver the cyclic polyribonucleotides and linear polyribonucleotides described herein.

Additional specific LNP formulations useful for delivery of nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) are described in US 8,158,601 and US 8,168,775, both of which are incorporated by reference, including the formulation sold under the name ONPATTRO used in patisiran.

In an embodiment, the polyribonucleotide (for example, cyclic polyribonucleotide, linear polyribonucleotide) of at least part (for example, antigenic part) of encoding protein as herein described or polypeptide is formulated in LNP, wherein: (a) LNP comprises cationic lipid, neutral lipid, cholesterol and PEG lipid, (b) the mean particle size of LNP is between 80nm and 160nm, and (c) polyribonucleotide.In an embodiment, the polyribonucleotide (for example, cyclic polyribonucleotide, linear polyribonucleotide) prepared in LNP is a vaccine.

The exemplary administration of polyribonucleotide (for example, cyclic polyribonucleotide, linear polyribonucleotide) LNP can comprise about 0.1,0.25,0.3,0.5,1,2,3,4,5,6,8,10 or 100mg/kg (RNA).In certain embodiments, the dosage of the compositions of polyribonucleotide (for example, cyclic polyribonucleotide, linear polyribonucleotide) antigen as described herein is between 30-200mcg, for example 30mcg, 50mcg, 75mcg, 100mcg, 150mcg or 200mcg.

Reagent test kit

In some respects, present disclosure provides test kit.In certain embodiments, test kit includes (a) cyclic polyribonucleotides or pharmaceutical compositions as described herein encoding anti-fusion polypeptides (e.g., polypeptides of Table 1), and optionally (b) informational materials.Informational materials can be descriptive, instructive, marketing or other materials related to the purposes of the methods described herein and/or pharmaceutical compositions or cyclic polyribonucleotides for the methods described herein.Pharmaceutical compositions or cyclic polyribonucleotides can include materials for single administration (e.g., single dose form), or can include materials for multiple administrations (e.g., "multiple dose" test kits).

The form of the information material of test kit is not limited. In one embodiment, the information material may include information about the production of pharmaceutical composition, pharmaceutical raw material or finished drug, molecular weight, concentration, expiration date, batch or production site information of pharmaceutical composition, pharmaceutical raw material or finished drug, etc. In one embodiment, the information material relates to a method for applying a pharmaceutical composition dosage form. In one embodiment, the information material relates to a method for applying a cyclic polyribonucleotide dosage form.

In addition to the dosage form of pharmaceutical compositions and cyclic polyribonucleotides as described herein, the kit may also include other ingredients, such as solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter antagonists or sweeteners), spices, dyes or coloring agents (e.g., for coloring or dyeing one or more components in the kit), or other cosmetic ingredients, and/or the second medicament for treating illness or disorder described herein. Alternatively, other ingredients may be included in the kit, but in a composition or container different from pharmaceutical compositions or cyclic polyribonucleotides as described herein. In such embodiments, the kit may include pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) as described herein mixed with other ingredients, or pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) as described herein used together with other ingredients.

In some embodiments, the components of the kit are stored under inert conditions (e.g., under nitrogen or another inert gas such as argon). In some embodiments, the components of the kit are stored under anhydrous conditions (e.g., with a desiccant). In some embodiments, the components are stored in a light-proof container, such as an amber vial.

The dosage form of pharmaceutical composition as herein described or nucleic acid molecules (for example, cyclic polyribonucleotide) can be in any form, for example, liquid, dry or lyophilized form is provided.Preferred pharmaceutical composition as herein described or nucleic acid molecules (for example, cyclic polyribonucleotide) are substantially pure and/or sterile.When providing pharmaceutical composition as herein described or nucleic acid molecules (for example, cyclic polyribonucleotide) in liquid solution, liquid solution is preferably aqueous solution, wherein preferably sterile aqueous solution.When pharmaceutical composition as herein described or nucleic acid molecules (for example, cyclic polyribonucleotide) are provided in dry form, usually by adding suitable solvent to reconstruct.Solvent, for example sterile water or buffer can be optionally provided in test kit.

The kit may include one or more containers for compositions containing dosage forms described herein. In certain embodiments, the kit contains separate containers, separators or compartments for compositions and informational materials. For example, pharmaceutical compositions or cyclic polyribonucleotides may be contained in bottles, vials or syringes, and informational materials may be contained in plastic sleeves or bags. In other embodiments, the separate elements of the kit are contained in single, undivided containers. For example, the dosage forms of pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein are contained in bottles, vials or syringes, with informational materials in the form of labels attached thereto. In certain embodiments, the kit includes multiple (e.g., a pack) of separate containers, each containing one or more unit dosage forms of pharmaceutical compositions or cyclic polyribonucleotides described herein. For example, the kit includes multiple syringes, ampoules, foil packages or blister packs, each containing a single unit dosage of dosage forms described herein.

The container of the kit can be airtight, waterproof (eg, not affected by changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for use with the dosage form, such as a syringe, a pipette, forceps, a measuring spoon, a swab (eg, a cotton swab or a wooden swab), or any such device.

The kit of the present invention may include dosage forms of different strengths to provide a subject with a dosage suitable for one or more of the initiation regimen, induction regimen, or maintenance regimen described herein. Alternatively, the kit may include a scored tablet to allow the user to administer a split dose as desired.

Examples

The following examples are presented in order to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared, and evaluated, and are intended to be purely exemplary of the present disclosure and are not intended to limit the scope of what the inventors regard as their invention.

Example 1: Expression of anti-fusion polypeptides from RNA in mammalian cells

This example demonstrates the expression of one or more open reading frames (ORFs) encoding one or more OC43-HR2P and EK1 peptides in Huh-7 cells.

In this example, circular RNAs encoding one OC43-HR2P peptide (SEQ ID NO: 289), one EK1 peptide (SEQ ID NO: 288), multiple OC43-HR2P peptides, multiple EK1 peptides, and combinations of OC43-HR2P peptides, peptide analogs, and EK1 peptides were designed. The circular RNAs were designed to include an IRES, a secretion signal, a furin site, one or more OC43-HR2P peptides, analogs, and/or EK1 sequences, and a spacer element. The circular RNAs were transfected into Huh-7 cells using Lipofectamine MessengerMax (Invitrogen LMRNA001) according to the manufacturer's instructions.

In one study, peptide expression was monitored in vitro over a time course.

Example 2: Inhibition of MERS-CoV S protein-mediated cell-cell fusion

To determine the inhibition of MERS-CoV fusion in the case of 293T target cells, a MERS-CoV S protein-mediated cell-to-cell fusion assay was used. This example uses 293T cells transfected with plasmid pAAV-IRES-EGFP encoding EGFP (293T/EGFP) or pAAV-IRES-MERS-EGFP encoding MERS-CoV S protein and EGFP (293T/MERS/EGFP) and cultured in DMEM containing 10% FBS at 37°C for 48 hours. Huh-7 cells (5×10 ⁴ ) expressing the MERS-CoV receptor DPP4 prepared according to Example 1 were incubated in 96-well plates at 37°C for 5 hours, followed by the addition of 1×10 ⁴ 293T/EGFP or 293T/MERS/EGFP cells, respectively.

After 4 hours of co-culture at 37°C, 293T/MERS/EGFP cells that were fused or not fused with Huh-7 cells were counted under an inverted fluorescence microscope (Nikon Eclipse Ti-S) (293T/EGFP cells were used as negative controls). Fusion cells were considered to be cells that were 2 times or more larger than unfused cells, and the difference in fluorescence intensity in fused cells was compared with that in unfused cells. The percentage of inhibition of intercellular fusion can be calculated using the following formula: (1-(EN)/(PN))×100. 'E' represents the percentage of intercellular fusion in the experimental group. 'P' represents the percentage of intercellular fusion in the positive control group, in which no circRNA was added. 'N' is the percentage of intercellular fusion in the negative control group, in which 293T/MERS/EGFP cells were replaced by 293T/EGFP cells. The concentration of 50% inhibition (IC ₅₀ ) can be calculated using CalcuSyn software. Co-culture can be continued for 48 hours at 37°C, and the formation of syncytia, for example, can be measured. The intracellular S protein ELISA can be applied to measure antiviral activity two days after viral challenge.

Example 3: Inhibition of infection with pseudotyped SARS-CoV-2 and MERS-CoV

By co-transfection of 293T cells with plasmid pNL4-3.luc.RE (encoding HIV-1 expressing luciferase of Env defective type) and pcDNA3.1-MERS-CoV-S plasmid, SARS or MERS pseudovirus carrying SARS-CoV-2 or MERS-CoV S protein and defective HIV-1 genome expressing luciferase as reporter molecule are prepared. In order to detect the inhibitory activity of expressed peptides on SARS or MERS pseudovirus infection, 293T (293T/ACE2) cells and Huh-7 cells (10 ⁴ per well in 96-well plates) transfected with ACE2 transfected with circRNA of the present invention and not transfected therewith are infected with SARS or MERS-CoV pseudovirus respectively. After infection, the culture is re-fed with fresh culture medium 12 hours after infection and incubated for another 72 hours. The cells are washed with PBS and lysed using the lysis reagent contained in the luciferase kit (Promega). Aliquots of cell lysates were transferred to 96-well Costar flat-bottom luminometer plates (Corning Costar) followed by the addition of luciferase substrate (Promega). Relative light units were immediately determined in an Ultra 384 luminometer (Tecan US).

Example 4: Expression of SARS-CoV-2 anti-fusion peptides

This example demonstrates the expression of SARS-CoV-2 anti-fusion peptides from circular RNA.

Several SARS-CoV-2 anti-fusion polypeptides were designed based on the HR2 region shown in Figure 1 (Figures 2 and 3). The circular RNA was designed to include an IRES and a nucleotide sequence encoding a SARS-CoV-2 anti-fusion polypeptide. In this example, the DNA construct was designed to include a spacer element and a polynucleotide cargo. The construct was designed to include a combination of an IRES and an ORF as a polynucleotide cargo. The ORF was designed to include an IL-2 secretion signal sequence, a nucleotide sequence encoding a SARS-CoV-2 anti-fusion polypeptide, and a nucleotide sequence encoding a HiBiT tag with a GGGGS peptide linker. The IRES is EMCV.

The amino acid and nucleic acid sequences of all constructs used are shown below.

The N-terminal IL-2 secretion signal is shown in uppercase (20 AA or 60 nucleotides)

Bold = Furin

Bold and italic = HiBiT tag with G4S peptide linker

HR2 full length

HR2A

HR2C

HR2B

EK1

Circular RNA is produced by self-splicing using the method described herein.Unmodified linear RNA is synthesized from a DNA template including the motif listed above using T7 RNA polymerase by in vitro transcription in the presence of 7.5 mM NTP.The template DNA is removed by treating with DNase.The linear RNA synthesized is purified with RNA purification kit (New England Biolabs, T2050).Self-splicing occurs during transcription.Circular RNA encoding anti-fusion peptides is purified by urea polyacrylamide gel electrophoresis (urea-PAGE) or reverse phase column chromatography.

To measure the expression of SARS-CoV-2 anti-fusion polypeptides, 0.4 pmol of circular RNA was delivered to HEK293 cells using lipofectamine. Expression was measured 48 hours later. Various polypeptides were expressed in HEK293 cells. Total expression (ng/ml and nM) is shown in Figure 2.

Table 2: HR2 construct expression

Expression (ng/mL) Expression (nM) HR2 full length 69.2 8.4 HR2A 5 1.3 HR2C 1.4 0.4 HR2B 0 0 EK1 0.1 0

Example 5: Inhibition of pseudotyped SARS-CoV-2 infection

This example demonstrates the inhibition of pseudotyped SARS-CoV2-infection by anti-fusion polypeptides expressed by circular RNA.

The circular RNA is designed to include an internal ribosome entry site (IRES) and a nucleotide sequence encoding an anti-fusion polypeptide of SARS-CoV-2. In this example, the DNA construct is designed to include a spacer element, and a combination of EMCV IRES and ORF as a polynucleotide cargo. The ORF is designed to include an IL-2 secretion signal sequence and a nucleotide sequence encoding a full-length anti-fusion polypeptide of HR2, as well as a nucleotide sequence encoding a HiBiT peptide tag. The ORF is also designed to include an IL-2 secretion signal sequence and a nucleotide sequence encoding a full-length anti-fusion polypeptide of HR2, without a nucleotide sequence encoding a HiBiT peptide tag.

Circular RNA is produced by self-splicing using the methods described herein. In the presence of 7.5 mM NTP, unmodified linear RNA is synthesized from a DNA template including the motifs listed above using T7 RNA polymerase by in vitro transcription. Template DNA is removed by treatment with DNase. The synthesized linear RNA is purified with an RNA purification kit (New England Biolabs, T2050). Self-splicing occurs during transcription. Circular RNA encoding the full-length anti-fusion polypeptide of HR2 is purified by urea polyacrylamide gel electrophoresis (urea-PAGE) or reverse phase column chromatography.

In order to detect the inhibitory activity of the expressed polypeptide on SARS pseudovirus infection, 293T (293T/ACE2) cells ( ¹⁰ per well in 96-well plates) transfected with ACE2 that have been transfected with circular RNA and not transfected with it were infected with SARS-CoV-2 pseudovirus. A single transfection reagent (without circular RNA) was used as a control ("mock"). After infection, the culture was re-fed with fresh culture medium 12 hours after infection and incubated for another 72 hours. The cells were washed with PBS and lysed using the lysis reagent included in the luciferase kit (Promega). An aliquot of the cell lysate was transferred to a 96-well Costar flat-bottom luminometer plate (Corning Costa), followed by the addition of a luciferase substrate (Promega). Relative light units were immediately determined in an Ultra 384 luminometer (Tecan, USA).

Cyclic polyribonucleotides encoding anti-fusion polypeptides described herein were used to determine the in vitro inhibition of fusion efficacy when using Omicron and Delta pseudoviruses. HR2 full-length anti-fusion polypeptides (FIG. 4A) expressed in cells and HR2 full-length anti-fusion polypeptides and HR2 full-length anti-fusion polypeptides conjugated with HiBiT tags inhibited the fusion of Delta and Omicron strains (FIG. 4A). There was no change in cell viability (measured by cellTiter glo) (FIG. 4B), indicating that the reduction of luciferase signaling (FIG. 4A) was attributed to the inhibition of viral fusion.

To measure the expression of the peptides in vivo, 120 pmol of circRNA formulated into lipid nanoparticles were delivered to mice by intravenous injection. The expression was measured using HiBiT extracellular detection system (#N3030, Promega) with 10% serum. As shown in Table 3 and Figure 5, the anti-fusion polypeptide was highly expressed at 6 hours and significantly decreased at 24 hours.

Table 3: HR2 expression

6 hours (ng/mL) 24 hours (ng/mL) HR2 full-length HiBiT 24.8 0.2 No HiBiT 0.02 0.01

Example 6: Inhibition of pseudotyped SARS-CoV-2 infection

This example demonstrates pseudotyping of SARS-CoV2-infection using anti-fusion peptides.

The anti-fusion peptides of SARS-CoV-2 were established based on the HR2, HR2A, HR2B and HR2C regions of the SARS-CoV-2 spike peptide and the EK1 peptide ( Figure 2 ).

Functional assays were performed to measure in vitro pseudovirus neutralization by EK-1 and HR2A polypeptides. HR2A polypeptides showed efficacy against Wuhan and Omicron strains (Figures 6A and 6B).

Further peptide experiments were performed using HR2A-C and HR2 full-length peptides. For negative control, IPB19, an HIV peptide, was used; for positive control, ACE-Fc, an antibody that directly binds to the receptor, was used. Peptides were prepared using 4-fold serial dilutions (8 dilutions) and HEK293 ACE2 cells (starting dilution 10 μM). All peptides showed inhibition of Omicron and WT strains, and the IC50 values are shown in Table 4.

Table 4: IC50 values

Full-length HR2 Fc fusion was also tested and shown to maintain inhibitory activity against Omicron and Wuhan strains.

The HR2 full-length polypeptide ("HR2 full") was shown to successfully inhibit the fusion of Omicron BA.4/BA.5, SARS CoV-1, and Wuhan strains (Figures 7A, 7B, and 8A-8D).

Example 7: Expression of HIV anti-fusion polypeptide

This example demonstrates HIV anti-fusion polypeptide expression from circular RNA.

Several HIV anti-fusion polypeptides were designed (Figure 9). The circular RNA was designed to include an IRES and a nucleotide sequence encoding an HIV anti-fusion polypeptide. In this example, a DNA construct was designed to include a spacer element and a polynucleotide cargo (Figure 9). The construct was designed to include a combination of an IRES and an ORF as a polynucleotide cargo. The ORF was designed to include an IL-2 secretion signal sequence (SEQ ID NO: 332), a nucleotide sequence encoding an HIV anti-fusion polypeptide, and a nucleotide sequence encoding a HiBiT tag (having the sequence VSGWRLFKKIS (SEQ ID NO: 362)) with a GGS or GGGGS peptide linker. The IRES is EMCV or a modified CVB3.

Circular RNA is produced by self-splicing using the method described herein.Unmodified linear RNA is synthesized from a DNA template including the motif listed above using T7 RNA polymerase by in vitro transcription in the presence of 7.5mM NTP.The template DNA is removed by treating with DNase.The linear RNA synthesized is purified with RNA purification kit (New England Biolabs, T2050).Self-splicing occurs during transcription.Circular RNA encoding anti-fusion peptides is purified by urea polyacrylamide gel electrophoresis (urea-PAGE) or reverse phase column chromatography.

To measure the expression of HIV anti-fusion polypeptides, 0.4 pmol of circular RNA was delivered to HEK293 cells using lipofectamine. Expression was measured 48 hours later. As shown in Figures 10A and 10B, the polypeptides were expressed in HEK293 cells. As shown in Figures 11A and 11B, the expression between circular RNA and DNA plasmids was comparable. The total expression (ng/mL and nM) of various polypeptides is shown in Figure 12.

HIV polypeptide and nucleic acid sequences

T20

YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF(SEQ ID NO:313)

tatacttctctgatccactctctgatcgaggaatctcagaaccagcaggagaagaacgaacaggaactgctggaactggataagtgggcttctctgtggaactggttc(with EMCV IRES)(SEQ ID NO:363)

OR

tacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctggacaagtgggccagcctgtggaactggttc(with modified CVB3 IRES)(SEQ ID NO:364)

T1249

WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF(SEQ ID NO:318)

tggcaggagtgggaacagaagatcactgctctgctggaacaggctcagattcagcaggaaaagaacgaatacgaactgcagaagctggataagtgggcttctctgtggggagtggttc(with EMCV IRES)(SEQ ID NO:365)

OR

tggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaagaacgagtacgagctgcagaagctggacaagtgggccagcctgtggggagtggttc(with modified CVB3 IRES)(SEQ ID NO:366)

T1144

TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALREL(SEQ ID NO:316)

actacttgggaagcttgggatagagctatcgctgaatacgctgctagaattgaagctctgctgagagctctgcaggaacagcaggaaaagaacgaagctgctctgagagaactg(with EMCV IRES)(SEQ ID NO:367)

OR

accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaagaacgaggccgcctgcggggagctg(with modified CVB3 IRES)(SEQID NO:368)

1144-2-0

TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALRELDKWASLWNWF(SEQ ID NO:317)

accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaagaacgaggccgccctgcgggagctggacaagtgggccagcctgtggaactggttc(with modified CVB3 IRES) (SEQ ID NO: 369)

T_2410

MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL(SEQ ID NO:314)

atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctg(with modified CVB3 IRES)(SEQID NO:370)

T_2410_2.0

MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF(SEQ ID NO:315)

atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctggacaagtgggccagcctgtggaactggttc(with modified CVB3 IRES)(SEQ ID NO:371)

1249_2.0

TTWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF(SEQ ID NO:319)

accacctggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaagaacgagtacgagctgcagaagctggacaagtgggccagcctgtggggagtggttc(with modified CVB3IRES) (SEQ ID NO: 372)

290676

TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELREL(SEQ ID NO:323)

accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaagaacgaggccgagctgcggggagctg(with modified CVB3 IRES)(SEQID NO:373)

290676-2-0

TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELRELDKWASLWNWF(SEQ ID NO:324)

accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaagaacgaggccgagctgcgggagctggacaagtgggccagcctgtggaactggttc(with modified CVB3 IRES) (SEQ ID NO: 374)

2635

TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL(SEQ ID NO:320)

accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcggggagctg(with modified CVB3 IRES)(SEQID NO:375)

2635_2.0

TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF(SEQ ID NO:321)

accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcggagctggacaagtgggccagcctgtggaactggttc(with modified CVB3 IRES) (SEQ ID NO: 376)

2635_3.0

MTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF(SEQ ID NO:322)

atgacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcggaggctggacaagtgggccagcctgtggaactggttc (SEQ ID NO: 377)

Other embodiments

Although the invention has been described in conjunction with particular embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the invention that generally follow the principles of the invention and include such departures from the invention as come within known or customary practice in the art to which the invention pertains, and that may be applied to the basic features set forth hereinabove, and that follow within the scope of the claims. Other embodiments are in the claims.

Claims (36)

1. A cyclic poly-ribonucleotide, the cyclic polyribonucleotides comprise a polyribonucleotide cargo encoding an antifusogenic polypeptide.

2. The cyclic polyribonucleotide according to claim 1, wherein said polyribonucleotide cargo comprises an expression sequence encoding said antifusogenic polypeptide.

3. The circular polyribonucleotide of claim 1 or 2, wherein the circular polyribonucleotide comprises a splice point that links a5 'exon fragment and a 3' exon fragment.

4. The cyclic polyribonucleotide of claim 2 or 3, wherein said polyribonucleotide cargo comprises an IRES operably linked with said expression sequence encoding said antifusogenic polypeptide.

5. The circular polyribonucleotide of claim 4, wherein the circular polyribonucleotide further comprises a spacer region between the IRES and the 3 'exon fragment or the 5' exon fragment.

6. The cyclic polyribonucleotide according to claim 5, wherein the spacer region is at least 5 ribonucleotides in length.

7. The cyclic polyribonucleotide according to claim 6, wherein the spacer region is 5 to 500 ribonucleotides in length.

8. The cyclic polyribonucleotide according to any of claims 5-7, wherein the spacer region comprises a poly a, a poly a-C, a poly a-U or a poly a-G sequence.

9. The cyclic polyribonucleotide according to any of claims 1-8, wherein the cyclic polyribonucleotide is at least 500 ribonucleotides in length.

10. The cyclic polyribonucleotide according to claim 9, wherein the length of said cyclic polyribonucleotide is between 500 and 20,000 ribonucleotides.

11. A linear polyribonucleotide comprising, from 5' to 3', a 3' intron fragment of (a); (B) a 3' splice site; (C) 3' exon fragments; (D) A polyribonucleotide cargo encoding said antifusogenic polypeptide; (E) 5' exon fragments; (F) a 5' splice site; and (G) a 5' intron fragment.

12. The linear polyribonucleotide of claim 11, wherein said polyribonucleotide cargo comprises an expression sequence that encodes said antifusogenic polypeptide.

13. The linear polyribonucleotide of claim 12, wherein the polyribonucleotide cargo comprises an IRES operably linked to the expression sequence encoding the antifusogenic polypeptide.

14. The linear polyribonucleotide according to any one of claims 11-13, wherein the cyclic polyribonucleotide further comprises a spacer region between one or more of (a), (B), (C), (D), (E), (F) and (G).

15. The linear polyribonucleotide according to claim 14, wherein the spacer region is at least 5 ribonucleotides in length.

16. The linear polyribonucleotide according to claim 15, wherein the spacer region is 5 to 500 ribonucleotides in length.

17. The linear polyribonucleotide of any of claims 14-16, wherein the spacer region comprises a poly a, a poly a-C, a poly a-U, or a poly a-G sequence.

18. The linear polyribonucleotide according to any of claims 11-17, wherein the length of the linear polyribonucleotide is at least 500 ribonucleotides.

19. The linear polyribonucleotide of claim 18, wherein the length of the linear polyribonucleotide is between 500 and 20,000 ribonucleotides.

20. A DNA vector encoding the linear polyribonucleotide according to any of claims 11-19.

21. A method of expressing an antifusogenic polypeptide in a cell, the method comprising providing to the cell a circular polyribonucleotide according to any of claims 1-10, a linear polyribonucleotide according to any of claims 11-19, or a DNA vector according to claim 20 under conditions suitable for expression of the antifusogenic polypeptide.

22. A method of producing a circular polyribonucleotide from a linear polyribonucleotide according to any of claims 11-20, said method comprising providing said linear polyribonucleotide under conditions suitable for self-splicing of said linear polyribonucleotide to produce said circular polyribonucleotide.

23. A pharmaceutical composition comprising a cyclic polyribonucleotide according to any of claims 1-10, a linear polyribonucleotide according to any of claims 11-19 or a DNA carrier according to claim 20, and a diluent, carrier or excipient.

24. A method of expressing an anti-fusion polypeptide in a subject, the method comprising administering a first dose of the pharmaceutical composition of claim 23 in an amount sufficient to produce a serum concentration of at least 500ng/mL of the anti-fusion polypeptide in the subject.

25. The method of claim 24, further comprising administering a second dose of the pharmaceutical composition.

26. The method of claim 25, wherein the second dose is administered at least 1 day after the first dose of the pharmaceutical composition.

27. The method of claim 26, wherein the second dose is administered 1 to 90 days after the first dose of the pharmaceutical composition.

28. The method of any one of claims 25-27, wherein the second dose is administered before the serum concentration of the anti-fusion polypeptide in the serum of the subject is less than about 500 ng/mL.

29. The method of claim 28, wherein the method maintains a serum concentration of the anti-fusion polypeptide of at least 500ng/mL in the subject.

30. The method of any one of claims 24-29, wherein the method treats or prevents a viral infection in the subject.

31. The method of claim 30, wherein the pharmaceutical composition is administered to the subject in an amount and for a duration sufficient to treat or prevent the viral infection.

32. The method of any one of claims 24-31, wherein the method reduces viral entry.

33. A cyclic polyribonucleotide comprising a polyribonucleotide cargo that encodes a plurality of antifusogenic polypeptides.

34. The cyclic polyribonucleotide of claim 33, wherein said polyribonucleotide cargo comprises an expression sequence that encodes said antifusogenic polypeptide.

35. The cyclic-polyribonucleotide of claim 33 or 34, wherein said antifusogenic polypeptide is directed against the same virus.

36. The cyclic-polyribonucleotide of claim 33 or 34, wherein said antifusogenic polypeptide is directed against more than one virus.