De novo synthesis of 5 '-untranslated regions (utr)

Abstract

The present invention relates to a polynucleotide comprising at least one 5'untranslated region (5 'UTR) and at least one open reading frame (ORF), which improves mRNA stability, protein expression and is capable of inducing an improved immune response.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Patent Application No. 63 / 570,338, filed March 27, 2024. The entire contents of the above application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of mRNA and its 5' untranslated region (UTR). Background Technology

[0003] This invention relates to the design and de novo synthesis of the 5'UTR. The 5'UTR is the direct region upstream of the start codon in messenger RNA (mRNA) and is a major determinant of translation efficiency. The eukaryotic 5'UTR contains a Kozak concordant sequence (GCCA / GCCAUGG) containing the start codon (AUG). The eukaryotic 5'UTR also contains cis-regulatory elements called upstream open reading frames (uORFs) and upstream AUGs (uAUGs), as well as a stop codon, which have a significant impact on translation regulation. Translation initiation and elongation are also influenced by RNA secondary structures formed within the 5'UTR and coding sequence (CDS), with the strongest structures exhibiting the most negative impact on translation. The native 5'UTR has a high GC content and high secondary structure, which delays the scanning of ribosomal mechanisms and reduces translation efficiency. Other ribosomes and regulatory proteins may bind and affect the expression of the target protein.

[0004] A 5'UTR is needed to improve translation efficiency. Summary of the Invention

[0005] This invention relates to a 5' untranslated region (5'UTR) that can promote mRNA stability and translation efficiency, and to mRNAs containing the 5'UTR, which can induce a stronger immune response in subjects in need.

[0006] In one aspect, the present invention provides a polynucleotide comprising at least one 5' untranslated region (5'UTR) and at least one open reading frame (ORF).

[0007] In one aspect, the present invention provides a polynucleotide, wherein the 5'UTR comprises or is composed of a polynucleotide sequence having 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any polynucleotide sequence selected from SEQ ID NO:1-8.

[0008] In one aspect, the present invention provides a polynucleotide comprising at least one open reading frame (ORF) encoding a VZV gE protein or a variant thereof. In some embodiments, the variant of gE is VZV gE del569, gE del575Y569A, gE del575 Y569K, or gE del574 Y569A.

[0009] In one aspect, the present invention provides a carrier comprising the polynucleotide of the present invention.

[0010] In one aspect, the present invention provides a cell comprising the polynucleotide of the present invention or the carrier of the present invention.

[0011] In one aspect, the present invention provides a composition comprising the polynucleotide of the present invention, the carrier of the present invention or the cell of the present invention, and a pharmaceutically acceptable excipient.

[0012] In one aspect, the present invention provides a method for preventing or treating a disease, comprising administering the polynucleotide of the present invention, the carrier of the present invention, the cell of the present invention, or the composition of the present invention.

[0013] In one aspect, the present invention provides the polynucleotide of the present invention, the carrier of the present invention, the cell of the present invention, or the composition of the present invention for the prevention or treatment of a disease in a subject in need.

[0014] In one aspect, the present invention provides the polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, or the composition of the present invention, which are used as vaccines or for gene therapy.

[0015] In one aspect, the present invention provides the polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, or the composition of the present invention for regulating the expression of one or more genes (e.g., downregulating a pathogenic gene) or modifying one or more genes (e.g., replacing a pathogenic gene with a copy of a healthy gene).

[0016] In one aspect, the present invention provides the polynucleotide of the present invention, the carrier of the present invention, the cell of the present invention, or the composition of the present invention for use in veterinary medicine.

[0017] In one aspect, the present invention provides the use of the polynucleotides of the present invention, the carriers of the present invention, the cells of the present invention, or the compositions of the present invention in the preparation of medicaments for the prevention or treatment of diseases.

[0018] In one aspect, the present invention provides a kit comprising the polynucleotide of the present invention, the vector of the present invention, the cells of the present invention or the composition of the present invention, and instructions for use. Attached Figure Description

[0019] The following figures are for illustrative purposes only and are not intended to be limiting.

[0020] Figure 1 : Schematic diagram of the carrier.

[0021] Figure 2 mRNA constructs with different 5'UTRs were translated in vitro in RRL (rabbit reticulocyte lysate), and the resulting products were analyzed by SDS-PAGE electrophoresis and immunoblotting with anti-gE VZV antibody.

[0022] Figure 3 mRNA constructs with different 5'UTRs were transiently transfected into HeLa cells using lipofectamine messenger max (ThermoFisher) at three different amounts (0.5, 1, and 2 μg). Twenty hours later, cell lysates were collected and analyzed by Western blotting using an anti-VZV gE antibody, and the data were quantified.

[0023] Figure 4 Anti-VZV gE antibody titers induced by mRNAs with different 5'UTRs.

[0024] Figure 5 T cell-mediated immune responses generated in mice immunized with mRNA containing different 5'UTRs and formulated with SM102. Detailed Implementation

[0025] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The term "technique" as used herein is intended to refer to techniques commonly understood in the art, including variations or equivalent substitutions that would be obvious to one of ordinary skill in the art. While it is believed that the following terms are fully understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0026] As used herein, the terms “including,” “comprising,” “having,” “containing,” or other variations thereof are inclusive or open-ended and do not exclude other elements or method steps not listed.

[0027] As used herein, the terms “implementation,” “disclosed herein,” or “this disclosure” are not intended to be limiting but are generally applicable to any embodiment as defined in the claims or described herein. These terms are used interchangeably herein.

[0028] As used herein, the terms “treatment,” “under treatment,” “therapy,” etc., refer to the elimination, reduction, or improvement of a disease or condition, and / or its associated symptoms. While not excluding the possibility that treatment of a disease or condition requires the complete elimination of said disease, condition, or its associated symptoms, the term “treatment” and its synonyms contemplate the administration of a therapeutically effective amount of the disclosed peptides or compositions to a subject requiring such treatment. The treatment may be symptomatic, such as suppressing symptoms. It may be administered short-term, medium-term directed, or long-term, such as within the context of maintenance therapy.

[0029] Throughout this disclosure, the term “an” or “a” entity refers to one or more of the entity; for example, “a polynucleotide” should be understood to represent one or more polynucleotides. Therefore, the terms “an (or a)”, “one or more”, and “at least one” are used interchangeably herein.

[0030] The term “composition” or “pharmaceutical composition” refers to a composition comprising the polynucleotide described herein and, for example, a pharmaceutically acceptable carrier, excipient, or diluent for administration to a subject in need of treatment.

[0031] The term "pharmaceutically acceptable" means, within the limits of reasonable medical judgment, a composition suitable for contact with tissues in humans and animals without excessive toxicity or other complications commensurate with a reasonable benefit / risk ratio.

[0032] "Effective dose" refers to the amount of polynucleotide, carrier, or composition disclosed herein that is effective for treatment when administered to a subject in a single dose or as part of a series of administrations. For example, in relation to varicella-zoster virus (VZV) infection, the dose is effective when administration results in one or more of the following: prevention of infection, relief of symptoms, elimination or reduction of pathogen, etc. The dose may be a fixed dose applicable to all treated subjects, or it may vary depending on the weight, health status and physical condition of the subject to be treated, the degree of prevention, relief or reduction desired, the formulation of the polynucleotide (e.g., mRNA) or composition disclosed herein, a professional assessment of the medical condition, and other relevant factors.

[0033] The term "subject" refers to any subject who requires treatment with the polynucleotides or compositions provided herein, particularly mammalian subjects. Mammal subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, dairy cows, apes, monkeys, orangutans, chimpanzees, etc. In one embodiment, the subject is a human subject.

[0034] Polynucleotides The present invention provides a polynucleotide comprising at least one 5' untranslated region (5'UTR) and at least one open reading frame (ORF).

[0035] The term "polynucleotide," in its broadest sense, includes any compound and / or substance comprising a nucleotide polymer. Exemplary nucleic acids or polynucleotides of the present invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization) or hybrids thereof. In a preferred embodiment, the polynucleotide is messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a target polypeptide and is capable of being translated in vitro, in vivo, in situ, or ex vivo to produce the encoded target polypeptide.

[0036] The term "polynucleotide" refers to modified and unmodified polynucleotides, and can also refer to DNA, RNA (e.g., mRNA), or hybrid molecules containing both DNA and RNA portions.

[0037] The term "variant" refers to a molecule whose amino acid or nucleotide sequence differs from the natural or reference sequence. Compared to the natural or reference sequence, the amino acid or nucleotide sequence variant may have substitutions, deletions, and / or insertions at certain positions within the amino acid or nucleotide sequence.

[0038] DNA: DNA is the common abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, a polymer composed of nucleotides. These nucleotides are typically monomers of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate. These monomers themselves consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and are polymerized through a characteristic backbone structure. This backbone structure is typically formed by a phosphodiester bond between the sugar moiety (deoxyribose) of the first nucleotide and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers, that is, the sequence of bases attached to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, for example, through A / T base pairing and G / C base pairing.

[0039] RNA, mRNA: RNA is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, a polymer composed of nucleotides. These nucleotides are typically monomers of adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate, linked together along a so-called backbone. This backbone is formed by phosphodiester bonds between the sugar (ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. The specific sequential sequence of monomers is called the RNA sequence. RNA is typically obtained through transcription of a DNA sequence, for example, within a cell. In eukaryotic cells, transcription usually takes place in the nucleus or mitochondria. In vivo, transcription of DNA typically produces so-called immature RNA, which must be processed into so-called messenger RNA, commonly abbreviated as mRNA. The processing of immature RNA, for example in eukaryotes, involves a variety of different post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. The sum of these processes is also called RNA maturation. Mature messenger RNA typically provides a nucleotide sequence that provides the amino acid sequence that can be translated into a specific peptide or protein. Typically, mature mRNA contains a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a poly(A) sequence. Besides messenger RNA, several non-coding RNA types exist that may be involved in the regulation of transcription and / or translation. The sequence of a nucleic acid molecule is generally understood as its specific and unique sequence, i.e., a continuous sequence of its nucleotides. The sequence of a protein or peptide is generally understood as its amino acid sequence, i.e., a continuous sequence of its amino acids. Sequence identity: If two or more sequences exhibit the same length and nucleotide or amino acid sequence, they are identical. The percentage of identity typically describes the degree to which two sequences are identical; that is, it typically describes the percentage of nucleotides that correspond to the same nucleotides in a reference sequence at a sequence position. To determine the degree of identity, the sequences to be compared are considered to have the same length, i.e., the length of the longest sequence among the sequences to be compared. This means that a first sequence consisting of 8 nucleotides has 80% identity with a second sequence consisting of 10 nucleotides containing that first sequence. In other words, in the context of this invention, sequence identity preferably refers to the percentage of sequence nucleotides at the same position in two or more sequences of the same length. Gap positions are generally considered to be non-identical positions, regardless of their actual position in the alignment.

[0040] In some embodiments, the mRNA or RNA disclosed herein is either a non-replicating mRNA or a non-replicating RNA. In some embodiments, the mRNA or RNA disclosed herein is either a self-amplifying mRNA (SAM) or a self-amplifying RNA (saRNA).

[0041] Typically, the basic components of an mRNA molecule include at least one coding region, a 5' UTR, a 3' UTR, a 5' cap, and a poly-A tail.

[0042] 5' Cap: A 5' cap is an entity, typically a modified nucleotide entity, that is usually "capped" at the 5' end of mature mRNA. 5' caps are typically formed from modified nucleotides, particularly derivatives of guanine nucleotides. Preferably, the 5' cap is linked to the 5' end via a 5'-5'-triphosphate linker bond. The 5' cap can be methylated, for example, m7GpppN, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5' end of RNA. Other examples of 5' cap structures include glycerol groups, inverted deoxygenated abase residues (partially), 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-dehydrated hexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threopentafuranosyl nucleotides, acyclic 3',4'-open nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abase residue, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abase residue, 1,4-butanediol phosphate, 3'-phosphoramide, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-thiophosphate, dithiophosphate, or bridged or non-bridged methylphosphonate moiety.

[0043] 5' Untranslated Region (5'UTR): The 5'UTR is generally understood as a specific portion of messenger RNA (mRNA). It is located at the 5' end of the open reading frame (OPF) of the mRNA. Typically, the 5'UTR begins at the transcription start site and ends one nucleotide before the start codon of the ORF. The 5'UTR may contain elements that control gene expression, also known as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites or 5' terminal oligopyrimidine segments. The 5'UTR can be post-transcriptionally modified, for example by adding a 5' cap. In the context of this invention, the 5'UTR corresponds to the mature mRNA sequence located between the 5' cap and the start codon. Preferably, the 5'UTR corresponds to the sequence extending from the nucleotide immediately following the 3' end of the 5' cap to the nucleotide immediately following the 5' end of the start codon in the protein-coding region. The nucleotide immediately following the 5' cap in the mature mRNA typically corresponds to the transcription start site. The term "corresponds to" means that the 5'UTR sequence can be an RNA sequence, such as a sequence in an mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to that RNA sequence. In the context of this invention, the term "5'UTR of a gene" refers to the sequence corresponding to the 5'UTR of the mature mRNA of that gene, i.e., the 5'UTR of the mRNA obtained by transcribing the gene and processing the immature mRNA. The term "5'UTR of a gene" encompasses both the DNA and RNA sequences of that 5'UTR.

[0044] Open Reading Frame (ORF): In the context of this invention, an open reading frame (ORF) is typically a sequence of several nucleotide triplets that can be translated into a peptide or protein. An ORF preferably contains a start codon at its 5' end, which is a combination of three consecutive nucleotides typically encoding the amino acid methionine (ATG or AUG), followed by a region typically of multiples of length 3. The ORF is preferably terminated by a stop codon (e.g., TAA, TAG, TGA). Typically, this is the only stop codon for the ORF. Therefore, in the context of this invention, an ORF is preferably a nucleotide sequence consisting of multiple nucleotides divisible by 3, beginning with a start codon (e.g., ATG or AUG) and preferably ending with a stop codon (e.g., TAA, TGA, or TAG, or UAA, UGA, UAG, respectively). The ORF can be isolated or integrated into a longer nucleic acid sequence, such as in a vector or mRNA. An ORF may also be referred to as a "protein-coding region" or "coding region."

[0045] 3' Untranslated Region (3'UTR): The 3'UTR is typically the portion of mRNA located between the protein-coding region (i.e., the open reading frame) and the poly(A) sequence of the mRNA. The 3'UTR of mRNA is not translated into an amino acid sequence. The 3'UTR sequence is usually encoded by a gene transcribed into the corresponding mRNA during gene expression. The genome sequence is first transcribed into immature mRNA, which contains optional introns. Then, the immature mRNA is further processed into mature mRNA during maturation. This maturation process includes 5' capping, splicing of the immature mRNA to remove optional introns, and 3' end modifications (e.g., 3' end polyadenylation of the immature mRNA and optional endonuclease or exonuclease cleavage, etc.). In the context of this invention, the 3'UTR corresponds to the mature mRNA sequence located on the 3' side of the protein-coding region stop codon (preferably immediately adjacent to the protein-coding region stop codon) and extending to the 5' side of the poly(A) sequence (preferably extending to the nucleotide immediately adjacent to the 5' side of the poly(A) sequence). The term "corresponds to" means that the 3'UTR sequence can be an RNA sequence, such as a sequence in an mRNA sequence used to define the 3'UTR sequence, or a DNA sequence corresponding to that RNA sequence. In the context of this invention, the term "gene's 3'UTR" refers to the sequence corresponding to the 3'UTR of the mature mRNA of the gene, i.e., the 3'UTR of the mRNA obtained by transcribing the gene and processing the immature mRNA. The term "gene's 3'UTR" encompasses both the DNA and RNA sequences of that 3'UTR.

[0046] Poly(A) sequence: A poly(A) sequence, also known as a poly(A) tail or 3'-poly(A) tail, is generally understood to be an adenine nucleotide sequence, for example, up to about 400 adenine nucleotides, for example from about 20 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, and most preferably from about 60 to about 250 adenine nucleotides. The poly(A) sequence is typically located at the 3' end of mRNA. In the context of this invention, the poly(A) sequence can be located within mRNA or any other nucleic acid molecule, for example in a vector, such as in a vector that serves as a template for RNA (preferably mRNA) generation, for example by transcription of that vector.

[0047] Polyadenylation: Polyadenylation is generally understood as the addition of a poly(A) sequence to a nucleic acid molecule (e.g., an RNA molecule, such as immature mRNA). Polyadenylation can be induced by a so-called polyadenylation signal. This signal is preferably located within a nucleotide segment at the 3' end of the nucleic acid molecule (e.g., the RNA molecule) to be polyadenylated. The polyadenylation signal typically contains a hexamer composed of adenine and uracil / thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences, preferably hexamer sequences, are also conceivable. Polyadenylation typically occurs during the processing of immature mRNA (also known as pre-mRNA). RNA maturation (from immature mRNA to mature mRNA) typically includes a polyadenylation step.

[0048] The sequence of a polynucleotide is generally understood as the specific and unique sequence of its constituent nucleotides, that is, the continuous arrangement of its nucleotides. The sequence of a protein or peptide is generally understood as the sequence of its constituent amino acids, that is, the continuous arrangement of its amino acids.

[0049] Sequence identity: Two or more sequences are considered identical if they exhibit the same length and nucleotide or amino acid sequence. The percentage of identity typically describes the degree to which two sequences are identical; that is, it typically describes the percentage of nucleotides at a sequence position that correspond to the same nucleotide in a reference sequence. To determine the degree of identity, the sequences to be compared are considered to have the same length, i.e., the length of the longest sequence among the sequences to be compared. This means that a first sequence consisting of 8 nucleotides has 80% identity with a second sequence consisting of 10 nucleotides containing that first sequence. In other words, in the context of this invention, sequence identity preferably refers to the percentage of nucleotides at the same position in two or more sequences of the same length. Gap positions are generally considered to be non-identical positions, regardless of their actual position in the alignment.

[0050] Transfection: The term "transfection" refers to the introduction of nucleic acid molecules or polynucleotides, such as DNA or RNA (e.g., mRNA) into cells, preferably eukaryotic cells. In the context of this invention, the term "transfection" encompasses any method known to those skilled in the art for introducing nucleic acid molecules into cells (preferably eukaryotic cells, such as mammalian cells). Such methods include, for example, electroporation, liposome transfection (e.g., transfection based on cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers (such as DEAE-glucan or polyethyleneimine). Preferably, the introduction is non-viral.

[0051] Vaccines: Vaccines are generally understood as prophylactic or therapeutic materials that provide at least one antigen (preferably an immunogen). The antigen or immunogen can be derived from any material suitable for vaccination. For example, the antigen or immunogen can be derived from pathogens, such as bacterial or viral particles, or from tumor or cancerous tissue. The antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response.

[0052] Vector: The term "vector" refers to a nucleic acid molecule or polynucleotide. In the context of this invention, a vector is suitable for integrating or carrying a desired nucleic acid sequence, such as a nucleic acid sequence containing an open reading frame. Such vectors can be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that allows convenient storage of nucleic acid molecules (e.g., mRNA molecules). Therefore, the vector may contain a sequence corresponding to, for example, a desired mRNA sequence or a portion thereof, such as a sequence corresponding to the open reading frame and 3'UTR of the mRNA. An expression vector can be used to produce expression products, such as RNA (e.g., mRNA), or peptides, polypeptides, or proteins. For example, an expression vector may contain a sequence required for transcribing a vector sequence fragment, such as a promoter sequence, for example, an RNA promoter sequence. A cloning vector is typically a vector containing a cloning site that can be used to integrate a nucleic acid sequence into the vector. A cloning vector can be, for example, a plasmid vector or a phage vector. A transfer vector can be a vector suitable for introducing nucleic acid molecules into cells or organisms (e.g., a viral vector). In the context of this invention, a vector can be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. Preferably, the vector in the context of this application comprises a cloning site, a selection marker (e.g., an antibiotic resistance factor), and a sequence suitable for vector proliferation (e.g., an origin of replication). Preferably, the vector in the context of this application is a plasmid vector.

[0053] Peptide: A peptide, or polypeptide, is typically a polymer of amino acid monomers linked together by peptide bonds. It usually contains fewer than 50 monomeric units. However, the term peptide does not exclude molecules with more than 50 monomeric units. Long peptides, also known as polypeptides, typically contain 50 to 600 monomeric units.

[0054] The polynucleotides used in this invention can be prepared using any existing technology, including but not limited to chemical synthesis, enzymatic synthesis (commonly referred to as in vitro transcription (IVT)), or by enzymatic or chemical cleavage of longer precursors. Methods for synthesizing RNA are known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: apractical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: HumanaPress, 2005; both incorporated herein by reference). The process of designing and synthesizing the primary constructs of this invention typically includes gene construction, mRNA production (with or without modification), and purification steps. In enzymatic synthesis methods, a target polynucleotide sequence encoding a target polypeptide is first selected for integration into a vector to be amplified to produce a cDNA template. Optionally, the target polynucleotide sequence and / or any flanking sequences may be codon-optimized. Then, mRNA is produced via in vitro transcription (IVT) using the cDNA template. After production, the mRNA can be purified and cleaned.

[0055] In one embodiment, the polynucleotide is RNA, preferably mRNA. In one embodiment, the polynucleotide is DNA. In one embodiment, the polynucleotide is DNA. In one embodiment, the polynucleotide includes a 3' untranslated region (3'UTR). In one embodiment, the polynucleotide includes a poly A tail.

[0056] In one embodiment, the 5'UTR comprises or is composed of a polynucleotide sequence having 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any polynucleotide sequence selected from SEQ ID NO: 1-8.

[0057] In one embodiment, the 5'UTR comprises or consists of any polynucleotide sequence selected from or composed of SEQ ID NO: 1-8.

[0058] In one embodiment, the ORF comprises a polynucleotide sequence encoding an amino acid sequence of a VZV antigen or a variant thereof. In some embodiments, the VZV antigen is a VZV glycoprotein. In some embodiments, the VZV glycoprotein is selected from gE, gI, gB, gH, gD, gK, gC, gN, gM, and variants thereof. In some embodiments, the VZV glycoprotein is a gE protein or a variant thereof.

[0059] In one embodiment, the amino acid sequence encoded by the ORF comprises or consists of an amino acid sequence of a truncated VZV antigen sequence.

[0060] Carriers, cells and compositions Vector: The term "vector" refers to a nucleic acid molecule, preferably an artificial nucleic acid molecule. In the context of this invention, a vector is suitable for integrating or carrying a desired nucleic acid sequence, such as a nucleic acid sequence containing an open reading frame. Such vectors can be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that allows convenient storage of nucleic acid molecules (e.g., mRNA molecules). Therefore, the vector may contain a sequence corresponding to, for example, a desired mRNA sequence or a portion thereof, such as a sequence corresponding to the 5'UTR and open reading frame of the mRNA. An expression vector can be used to produce expression products, such as RNA (e.g., mRNA), or peptides, polypeptides, or proteins. For example, an expression vector may contain a sequence required for transcribing a vector sequence fragment, such as a promoter sequence, for example, an RNA promoter sequence. A cloning vector is typically a vector containing a cloning site that can be used to integrate a nucleic acid sequence into the vector. A cloning vector can be, for example, a plasmid vector or a phage vector. A transfer vector can be a vector suitable for introducing nucleic acid molecules into cells or organisms (e.g., a viral vector). In the context of this invention, a vector can be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. Preferably, the vector in the context of this application comprises a cloning site, a selection marker (e.g., an antibiotic resistance factor), and a sequence suitable for vector proliferation (e.g., an origin of replication). Preferably, the vector in the context of this application is a plasmid vector.

[0061] In one aspect, the present invention provides a vector comprising the polynucleotide of the present invention. In one embodiment, the vector comprises a T7 promoter.

[0062] Vaccines are generally understood to be prophylactic or therapeutic materials that provide at least one antigen (preferably an immunogen). The antigen or immunogen can be derived from any material suitable for vaccination. For example, the antigen or immunogen can be derived from pathogens, such as bacterial or viral particles, or from tumor or cancerous tissue. The antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response.

[0063] In one aspect, the present invention provides a cell comprising the polynucleotide of the present invention or the carrier of the present invention.

[0064] In one aspect, the present invention provides a composition comprising the polynucleotide of the present invention, the carrier of the present invention or the cell of the present invention, and a pharmaceutically acceptable excipient.

[0065] In some embodiments, the composition comprises the polynucleotide and lipid nanoparticles of the present invention.

[0066] Uses of polynucleotides, carriers, cells, and compositions In one aspect, the present invention provides a method for preventing or treating a disease, comprising administering an effective amount of the polynucleotide of the present invention, the carrier of the present invention, the cell of the present invention, or the composition of the present invention.

[0067] In one aspect, the present invention provides the polynucleotide of the present invention, the carrier of the present invention, the cell of the present invention, or the composition of the present invention for the prevention or treatment of a disease in a subject in need.

[0068] In one aspect, the present invention provides the polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, or the composition of the present invention, which are used as vaccines or for gene therapy.

[0069] In one aspect, the present invention provides the polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, or the composition of the present invention for regulating the expression of one or more genes (e.g., downregulating a pathogenic gene) or modifying one or more genes (e.g., replacing a pathogenic gene with a copy of a healthy gene).

[0070] In one aspect, the present invention provides the use of the polynucleotides of the present invention, the carriers of the present invention, the cells of the present invention, or the compositions of the present invention in the preparation of medicaments for the prevention or treatment of diseases.

[0071] In one aspect, the present invention provides a kit comprising the polynucleotide of the present invention, the vector of the present invention, the cells of the present invention or the composition of the present invention, and instructions for use.

[0072] In one aspect, the present invention provides the polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, or the composition of the present invention for veterinary use, for example, for treating animal diseases or for use as an animal vaccine.

[0073] In some embodiments, the composition comprises nanoparticles, such as lipid nanoparticles.

[0074] In some implementations, the disease is VZV infection.

[0075] In some embodiments, the polynucleotide is administered as a naked polynucleotide (e.g., naked mRNA) or as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. In a non-limiting example, the pharmaceutically acceptable excipient is polyethyleneimine (PEI) or lipid nanoparticles (LNP). Other examples of liposomes that can be used to administer the polynucleotide (e.g., mRNA) or the composition include protamine, cationic nanoemulsions, modified dendritic polymer nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid nanoparticles, cationic lipid-cholesterol nanoparticles, cationic lipid-cholesterol PEG nanoparticles, cationic lipid transfection reagents sold under the trademark LIPOFECTAMINE, non-liposomal transfection reagents sold under the trademark FUGENE, or any combination thereof, as the pharmaceutically acceptable excipient.

[0076] In some embodiments, the composition may optionally contain one or more additional active substances, such as therapeutic and / or preventative active substances.

[0077] In some embodiments, the regulation of expression of one or more genes or the modification of one or more genes is mediated by CRISPR (clustered regularly spaced short palindromic repeats). In some embodiments, the polynucleotides of the present invention, the vectors of the present invention, the cells of the present invention, or the compositions of the present invention are used in CRISPR; for example, the 5'UTR described herein can be used to express CRISPR Cas proteins, dCas proteins, or gRNAs (guide RNAs).

[0078] Example The present invention is further illustrated by the following embodiments, which should not be construed as limiting the invention. All references cited in this application are expressly incorporated herein by reference.

[0079] Methods and Materials plasmid construction Gene fragments encoding the 5'UTR (Mod, SEQ ID NO: 9), gE del574 Y569A, and α-globin 3'UTR (SEQ ID NO: 19) were purchased from Integrated DNA Technologies Inc. (IDT). The pUC19 vector (Thermo Scientific) and the gE del574 Y569A gene fragment were digested with EcoRI and BamHI restriction endonucleases, purified, ligated, and transformed in NEB® Stable Competent E. coli (NEB). Colonies were screened to obtain positive clones with the correct gE del574Y569A pUC19 plasmid sequence. The double-stranded Poly A primers (containing 121 Poly A regions followed by BbsI and SphI restriction endonuclease sites) and the gE del574 Y569A pUC19 plasmid were digested with BamHI and SphI restriction endonucleases, purified, ligated, and transformed in NEB® Stable Competent E. coli (NEB). Colonies were screened to obtain positive clones with the correct gE del574 Y569A Poly A pUC19 sequence.

[0080] The amino acid sequence of wild-type VZV gE is the NCBI reference sequence NP_040190.1 (https: / / www.ncbi.nlm.nih.gov / protein / NP_040190.1, the entire contents of which are incorporated herein by reference). The amino acid sequence of mutant VZV gE used in this article is based on the above-mentioned wild-type VZV gE (NP_040190.1).

[0081] Using homologous recombination, VZV gE del569, gE del575Y569A, gE del575 Y569K, and gE del574 Y569A, encoding different 5'UTR sequences, as well as a sequence using human α-globin as the 3'UTR, were cloned into the pUC57 Poly A vector. This vector contains 121 poly A regions, followed by the Sap1 / BspQ1 site for plasmid linearization.

[0082] All constructed constructs were confirmed by Sanger sequencing.

[0083] 5'UTR builder construction The gE del574 Y569A Poly A pUC19 plasmid was digested with EcoRI and NcoI restriction endonucleases to remove the Mod 5'UTR sequence. The resulting vector and duplexes containing different 5'UTRs (SEQ ID NOs: 10-18) (purchased from IDT) were incubated in NEB HiFi builder mixture at 50°C for 1 hour. The mixture was then transformed into NEB® StableCompetent E. coli (NEB) and colonies were allowed to grow. Multiple clones were screened to obtain positive clones of all 5'UTR constructs. All constructed constructs were confirmed by Sanger sequencing.

[0084] Carrier linearization The plasmid with the pUC19 backbone was digested with Bbs1 enzyme and the plasmid with the pUC57 backbone was digested with Sap1 / BspQ1 enzyme to linearize the vector. Plasmid linearization was confirmed by agarose gel electrophoresis and purified using a Qiagen purification column.

[0085] mRNA generation and purification Using a linearized vector as a template, mRNA was generated using the NEB HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040S) according to the manufacturer's protocol. In short, an in vitro transcription reaction (IVT) was performed at 37°C for 2 hours (NEB) using a linearized template containing transcription buffer, T7 RNA polymerase, ATP, GTP, CTP, UTP, or m1Ψ. A capping reagent (TriLink CleanCap Reagent AG, N-7113) was also added to the reaction to generate capped mRNA (Cap 1). The plasmid template was removed using DNase I (NEB), and the generated mRNA was purified using LiCl precipitation or the Mega Clear Kit (Thermo). The mRNA was quantified and electrophoresed to check the integrity and quality of the generated mRNA.

[0086] Rabbit reticulocyte system in vitro translation An in vitro translation reaction was set up using the Promega Rabbit Reticulocyte System (L4960) and Transcend tRNA (L5061) with the synthesized mRNA. The resulting proteins were electrophoresed and detected using Western blotting with streptavidin antibodies.

[0087] mammalian cell transfection and expression mRNA was transfected into mammalian cells (HeLa and / or HEK 293T cells) using lipofectamine messenger max according to the manufacturer's protocol (Thermo), and treated after 20 hours. RIPA lysis buffer was electrophoresed on SDS-PAGE and electroblotted onto a PVDF membrane, and VZV gE protein expression was monitored using an anti-VZV gE antibody (Virusys).

[0088] Example 1: Design and preparation of mRNAs with different 5'UTRs Numerous de novo 5' UTRs were designed, namely Anta 8 (SEQ ID NO: 1), Anta 24 (SEQ ID NO: 2), Anta30 (SEQ ID NO: 3), MC (SEQ ID NO: 4), NB (SEQ ID NO: 5), and LC (SEQ ID NO: 6). The regions surrounding the translation start sites of the human HBA1 and HBB1 5' UTRs were modified to obtain MHBA1 (SEQ ID NO: 7) and MHBB1 (SEQ ID NO: 8). Sequence 176 in Table 4 of patent US10881730B2 was also used as a reference for comparison and labeled Mod (SEQ ID NO: 9).

[0089] As shown in the diagram above ( Figure 1 As shown in the diagram, the 5'UTR was cloned upstream of the gE VZV coding sequence to obtain the plasmid. A modified T7 promoter sequence was added before the 5'UTR sequence so that the generated mRNA could be co-transcribed and capped. A human α-globin 3'UTR was added after the CDS region, followed by a Poly A tail region and a recognition site for linearizing the generated plasmid.

[0090] In vitro transcription was performed using a linearized plasmid (using N1-methylpseudouridine instead of uridine) to generate mRNA, followed by DNase1 treatment to digest the linearized plasmid. The mRNA underwent further processing and purification.

[0091] Example 2: 5'UTR improves mRNA stability and protein expression To analyze how the 5'UTR in mRNA constructs affects protein production, rabbit reticulocyte lysis buffer (RRL) was used. RRL is a cell-free mammalian system used to characterize protein products of RNA transcripts and to study transcriptional and translational regulation. mRNA constructs with different 5'UTRs were translated in vitro in RRL, and the resulting proteins were detected using an anti-gE VZV antibody. Since the 5'UTR was the only difference among all tested mRNA constructs, differences in gE VZV protein expression were directly correlated with the 5'UTR. The highest protein expression was observed in the Anta30 5'UTR, followed by LC and Anta24. Similar expression levels were observed for the Anta8, NB, MC, and Mod 5'UTRs, while MHBA1 showed the lowest expression in the RRL. Figure 2 ).

[0092] Surprisingly, different patterns were observed when measuring protein expression in HeLa cells using mRNAs with different 5'UTRs. Almost identical protein expression levels were observed for mRNAs with Anta8, Anta24, Anta30, MHBA1, and Mod 5'UTRs, while a 25% reduction was observed for all other tested mRNAs. Figure 3 ).

[0093] Example 3: mRNA with 5'UTR enhances immune response to VZV To test the immunogenicity of the mRNA constructs, SM102 was used as an ionizable lipid to generate mRNA-lipid nanoparticles (mRNA-LNPs), and mice were immunized with 10 μg mRNA-LNPs administered 14 days apart. Mouse serum was collected, and antibody titers against gE VZV protein were measured. The antibody titer of gE VZV mRNA with Anta30 and MHBA1 5'UTRs was twice that of mRNAs with other 5'UTRs. Figure 4 ).

[0094] The structure of SM102 is as follows .

[0095] Th1-biased T cell responses are crucial for demonstrating that the expected vaccine will produce a good and durable immune response. Th1-biased T cell responses are essential for the prevention and control of VZV. VZV gE-specific T cell responses in mice immunized with various constructs were measured by in vitro restimulation of spleen cells with an overlapping peptide pool covering the full-length VZV gE. Secretion of IFNγ (a typical TH1 cytokine) was observed in spleen cells from mice immunized with all constructs, with the construct using Anta30 as the 5'UTR showing the highest response. Figure 5 ).

[0096] Various embodiments of this disclosure have been described above, and these descriptions are exemplary and not exhaustive, and are not limited to the embodiments of this disclosure. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the various embodiments described. The terminology chosen in this text is intended to best explain the principles and practical applications of the various embodiments, as well as the technical improvements made to the embodiments in the market, or to enable other persons of ordinary skill in the art to understand the embodiments of this disclosure.

[0097] sequence list

[0098] (EcoRI and NcoI sites are shown in bold.)

Claims

1. A polynucleotide comprising at least one 5’ untranslated region (5’UTR) and at least one open reading frame (ORF), wherein the 5’UTR comprises or consists of a polynucleotide sequence that is 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence selected from any one of SEQ ID NOs: 1-8.

2. The polynucleotide of claim 1, wherein the 5’UTR comprises or consists of a polynucleotide sequence selected from any one of SEQ ID NOs: 1-8.

3. The polynucleotide of claim 1 or 2, wherein the ORF comprises a polynucleotide sequence encoding an amino acid sequence of a VZV antigen or a variant thereof, optionally the VZV antigen is a VZV glycoprotein.

4. The polynucleotide of any one of claims 1-3, wherein the VZV glycoprotein is selected from gE, gI, gB, gH, gD, gK, gC, gN, gM and variants thereof, preferably the VZV glycoprotein is gE or a variant thereof, more preferably the variant of gE is VZV gE del569, gE del575 Y569A, gE del575 Y569K or gE del574 Y569A.

5. The polynucleotide of any one of claims 1-4, further comprising a 3’ untranslated region (3’UTR).

6. The polynucleotide of any one of claims 1-5, further comprising a poly A tail.

7. A polynucleotide comprising at least one open reading frame (ORF) encoding a VZV gE protein or a variant thereof, wherein the variant of gE is VZV gE del569, gE del575 Y569A, gE del575 Y569K or gE del574 Y569A.

8. A vector comprising the polynucleotide of any one of the preceding claims.

9. The vector of claim 8, further comprising a T7 promoter.

10. A cell comprising the polynucleotide of any one of claims 1-7 or the vector of claim 8 or 9.

11. The cell of claim 10, wherein the cell is a mammalian cell, preferably a human cell.

12. A composition comprising the polynucleotide of any one of claims 1-7, the vector of claim 8 or 9, or the cell of claim 10 or 11, and a pharmaceutically acceptable excipient.

13. A method of preventing or treating a disease, comprising administering to a subject in need thereof the polynucleotide of any one of claims 1-7, the vector of claim 8 or 9, the cell of claim 10 or 11, or the composition of claim 12.

14. The polynucleotide of any one of claims 1-7, the vector of claim 8 or 9, the cell of claim 10 or 11, or the composition of claim 12, for use in preventing or treating a disease in a subject in need thereof.

15. The polynucleotide of any one of claims 1-7, the vector of claim 8 or 9, the cell of claim 10 or 11, or the composition of claim 12, for use as a vaccine or in gene therapy.

16. Use of the polynucleotide of any one of claims 1-7, the vector of claim 8 or 9, the cell of claim 10 or 11, or the composition of claim 12, in the manufacture of a medicament for preventing or treating a disease.

17. The method or use of any one of claims 13-16, wherein the disease is varicella zoster virus infection.

18. A kit comprising the polynucleotide of any one of claims 1-7, the vector of claim 8 or 9, the cell of claim 10 or 11, or the composition of claim 12, and instructions for use.

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