WO2024234020A2

WO2024234020A2 - Denova synthetic 5' untranslated regions (utr)

Info

Publication number: WO2024234020A2
Application number: PCT/US2024/038246
Authority: WO
Inventors: Nicholas Valiante; Shiteshu SHRIMAL
Original assignee: Innovac Therapeutics
Priority date: 2024-03-27
Filing date: 2024-07-16
Publication date: 2024-11-14
Also published as: WO2024234020A3

Abstract

The invention relates to a polynucleotide, comprising at least one 5' untranslated region (5'UTR) and at least one open reading frame (ORF). It has improvd mRNA stability, protein expression and can induce improved immune response.

Description

DESCRIPTION

Denova Synthetic 5’ Untranslated Regions (UTR)

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application No. 63/570,338, filed on March 27, 2024. The entire contents of the above application is incorporated herein by reference.

TECHNICAL FIELD

The invention relates to the field of mRNA and the 5’ untranslated regions (UTR) of a mRNA.

BACKGROUND OF THE INVENTION

The invention relates to designing new synthetic 5’UTRs. The 5 ’UTR is the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon and is a primary determinant of translation efficiency. The eukaryotic 5 ’UTR contains the Kozak consensus sequence (GCCA/GCCAUGG), which contains the initiation codon (AUG). The eukaryotic 5’ UTR also contains cis-acting regulatory elements called upstream open reading frames (uORFs) and upstream AUGs (uAUGs) and termination codons, which have a great impact on the regulation of translation. Translation initiation and elongation are also affected by RNA secondary structure that forms within 5’UTRs and coding sequence (CDS), with the strongest structures showing the most negative effect on translation. Natural 5 ’UTR has higher GC content and presence of secondary structures which delays scanning by ribosomal machinery and decreases translational efficiency. Other ribosomal and regulatory protein might bind and effect the expression of the protein of interest. There is a need for a 5’UTR that increases the translational efficiency.

SUMMARY OF THE INVENTION

The invention relates to 5’ untranslated region (5’UTR) that can promote mRNA stability, transaltional efficiency and the mRNA having the 5’UTR which can induce a stronger immune response in a subject in need thereof.

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

In one aspect, the invention provides a polynucleotide, comprising the 5’UTR comprises or consists of the polynucleotide sequence having 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-8.

In one aspect, the invention provides a polynucleotide comprising at least one open reading frame (ORF) encoding N7N gE protein or variants thereof. In some embodiments, the variant of gE is N7N gE del569, gE del575 Y569A, gE del575 Y569K or gE del574 Y569A.

In one aspect, the invention provides a vector comprising the polynucleotide of the invention.

In one aspect, the invention provides a cell comprising the polynucleotide of the invention or the vector of the invention.

In one aspect, the invention provides a composition comprising the polynucleotide of the invention, the vector of the invention or the cell of the invention, and pharmaceutically acceptable excipients.

In one aspect, the invention provides method for preventing or treating a disease, comprising administering the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention.

In one aspect, the invention provides the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention for use in preventing or treating a disease a subject in need thereof.

In one aspect, the invention provides the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention for use as a vaccine or for use in gene therapy.

In one aspect, the invention provides the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention for use in regulating the expression of one or more genes (e.g., downregulating a disease-causing gene) or modifying one or more genes (e.g., replacing a disease-causing gene with a healthy copy of the gene).

In one aspect, the invention provides the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention for veterinary use.

In one aspect, the invention provides use of the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention in the manufacture of a medicament for preventing or treating a disease.

In one aspect, the invention provides a kit comprising the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention, and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are presented for the purpose of illustration only, and are not intended to be limiting.

Figure 1: Schematic representation of the vector.

Figure 2: mRNA constructs with different 5’UTRs were in vitro translated in RRL (Rabbit reticulocyte lysate) and generated product was run on SDS-PAGE and immunoblotted with anti-gE N7N antibody.

Figure 3: mRNA constructs with different 5’UTRs were transiently transfected in HeLa cells at three different amounts ( 0.5. 1 and 2 ug) by using lipofectamine messenger max (ThermoFisher). After 20 hours, cell lysates were collected and analyzed by immunoblotting using anti-VZV gE antibody and data was quantified.

Figure 4: Anti-VZV gE antibody titer induced by mRNA with different 5’UTRs.

Figure 5: T-cell-mediated immune responses from mice immunized with mRNA differing in 5’UTRs and formulated with SM102.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined below, all technical and scientific terms used herein have the same meanings as commonly understood by an ordinary skilled person in the art. References to techniques used herein are intended to refer to techniques that are generally understood in the art, including those obvious changes or equivalent replacements of the techniques for those skilled in the art. While it is believed that the following terms are well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

As used herein, the terms “including”, “comprising”, “having”, “containing” or “comprising”, and other variants thereof, are inclusive or open, and do not exclude other unlisted elements or method steps.

As used herein, the terms “embodiment”, “disclosed herein” or “disclosure” are not meant to be limiting, but applies generally to any of the embodiments defined in the claims or described herein. These terms are used interchangeably herein.

As used herein, the terms “treat”, “treating”, “treatment” and the like refer to eliminating, reducing, or ameliorating a disease or condition, and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. The term “treat” and synonyms contemplate administering a therapeutically effective amount of the polypeptide or the composition disclosed herein to a subject in need of such treatment. The treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a longterm treatment, for example within the context of a maintenance therapy.

Throughout this disclosure, the terms “a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

The terms “composition” or “pharmaceutical composition” refer to compositions comprising the polynucleotide described herein, along with e.g., pharmaceutically acceptable carriers, excipients, or diluents for administration to a subject in need of treatment.

The term “pharmaceutically acceptable” refers to compositions that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity or other complications commensurate with a reasonable benefit/risk ratio.

An “effective amount” is that amount of a polynucleotide, a vector or a composition provided herein, the administration of which to a subject, either in a single dose or as part of a series, is effective for treatment. For example, with respect to Varicella zoster virus (VZV) infection, an amount is effective, for example, when its administration results in one or more of prevention of the infection, mitigation of the symptoms, elimination or reduction of the pathogens and the like. This amount can be a fixed dose for all subjects being treated, or can vary depending upon the weight, health, and physical condition of the subject to be treated, the extent of prevention, mitigation or reduction is desired, the formulation of the polynucleotide (e.g., mRNA) or the composition disclosed herein, a professional assessment of the medical situation, and other relevant factors.

The term “subject” is meant any subject, particularly a mammalian subject, in need of treatment with the polynucleotide or the composition provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on. In one embodiment, the subject is a human subject.

Polynucleotide

The invention provides a polynucleotide, comprising at least one 5’ untranslated region (5’UTR) and at least one open reading frame (ORF).

The term “polynucleotide,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L- ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2’-amino functionalization, and 2’-amino-a-LNA having a 2’ -amino functionalization) or hybrids thereof. In a preferred embodiment, the polynucleotide is a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

The term “polynucleotide,” refers to both modified and un-modified polynucleotide, and also can refer to a DNA, a RNA (e.g., mRNA) or a hybridmolecule comprising DNA and RNA portions.

The term “variant” refers to molecules which differ in their amino acid sequence or nucleotide sequence from a native or reference sequence. The amino acid sequence or nucleotide sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence or nucleotide sequence, as compared to a native or reference sequence. DNA: DNA is the usual abbreviation for deoxy-ribonucleic-acid. It is a nucleic acid molecule, i.e., a polymer consisting of nucleotides. These nucleotides are usually deoxy- adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine- monophosphate and deoxy-cytidine-monophosphate monomers which are - by themselves - composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerise by a characteristic backbone structure. The backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the DNA-sequence. DNA may 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, e.g. by A T-base-pairing and G/C-base-pairing.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic-acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually adenosine- monophosphate, uridine-monophosphate, guanosinemonophosphate and cytidine- monophosphate monomers which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific succession of the monomers is called the RNA-sequence. Usually RNA may be obtainable by transcription of a DNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in the so-called premature RNA which has to be processed into so- called messenger-RNA, usually abbreviated as mRNA. Processing of the premature RNA, e.g. in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications such as splicing, 5'- capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5 ’-cap, a 5’UTR, an open reading frame, a 3’UTR and a poly(A) sequence. Aside from messenger RNA, several non-coding types of RNA exist which may be involved in regulation of transcription and/or translation. Sequence of a nucleic acid molecule: The sequence of a nucleic acid molecule is typically understood to be the particular and individual order, i.e. the succession of its nucleotides. The sequence of a protein or peptide is typically understood to be the order, i.e. the succession of its amino acids. Sequence identity: Two or more sequences are identical if they exhibit the same length and order of nucleotides or amino acids. The percentage of identity typically describes the extent to which two sequences are identical, i.e. it typically describes the percentage of nucleotides that correspond in their sequence position with identical nucleotides of a reference-sequence. For determination of the degree of identity, the sequences to be compared are considered to exhibit the same length, i.e. the length of the longest sequence of the sequences to be compared. This means that a first sequence consisting of 8 nucleotides is 80% identical to a second sequence consisting of 10 nucleotides comprising the first sequence. In other words, in the context of the present invention, identity of sequences preferably relates to the percentage of nucleotides of a sequence which have the same position in two or more sequences having the same length. Gaps are usually regarded as nonidentical positions, irrespective of their actual position in an alignment.

In some embodiments, the mRNA or RNA disclosed herein is nonreplicating mRNA or non-replicating RNA, respectively. In some embodiments, the mRNA or RNA disclosed herein is self-amplifying mRNA (SAM) or selfamplifying RNA (saRNA), respectively.

Typically, the basic components of an mRNA molecule include at least a coding region, a 5’UTR, a 3’UTR, a 5’ cap and a poly-A tail. 5 ’-cap: A 5 ’-cap is an entity, typically a modified nucleotide entity, which generally ‘caps’ the 5 ’-end of a mature mRNA. A 5’ -cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. Preferably, the 5 ’-cap is linked to the 5 ’-terminus via a 5’ -5’ -triphosphate linkage. A 5 ’-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5’ nucleotide of the nucleic acid carrying the 5 ’-cap, typically the 5 ’-end of an RNA. Further examples of 5 ’cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4’, 5’ methylene nucleotide, l-(beta-D- erythrofuranosyl) nucleotide, 4’-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3 ’,4’ -seco nucleotide, acyclic 3,4- dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3 ’-3’- inverted nucleotide moiety, 3 ’-3 ’-inverted abasic moiety, 3’ -2’ -inverted nucleotide moiety, 3 ’-2’ -inverted abasic moiety, 1,4-butanediol phosphate, 3’- phosphoramidate, hexylphosphate, aminohexyl phosphate, 3 ’-phosphate, 3’phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety.

5 ’-untranslated region (5’UTR): A 5’UTR is typically understood to be a particular section of messenger RNA (mRNA). It is located 5’ of the open reading frame of the mRNA. Typically, the 5’UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5’UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5 ’-Terminal Oligopyrimidine Tract. The 5’UTR may be posttranscriptionally modified, for example by addition of a 5 ’-cap. In the context of the present invention, a 5 ’UTR corresponds to the sequence of a mature mRNA which is located between the 5 ’cap and the start codon. Preferably, the 5’UTR corresponds to the sequence which extends from a nucleotide located 3’ to the 5’- cap, preferably from the nucleotide located immediately 3’ to the 5 ’cap, to a nucleotide located 5 ’ to the start codon of the protein coding region, preferably to the nucleotide located immediately 5’ to the start codon of the protein coding region. The nucleotide located immediately 3’ to the 5 ’cap of a mature mRNA typically corresponds to the transcriptional start site. The term “corresponds to” means that the 5’UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5’UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 5’UTR of a gene”, is the sequence which corresponds to the 5’UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “5’UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 5’UTR.

Open reading frame: An open reading frame (ORF) in the context of the invention may typically be a sequence of several nucleotide triplets which may be translated into a peptide or protein. An open reading frame preferably contains a start codon, i.e. a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG or AUG), at its 5 ’-end and a subsequent region which usually exhibits a length which is a multiple of 3 nucleotides. An ORF is preferably terminated by a stop-codon (e.g., TAA, TAG, TGA). Typically, this is the only stop-codon of the open reading frame. Thus, an open reading frame in the context of the present invention is preferably a nucleotide sequence, consisting of a number of nucleotides that may be divided by three, which starts with a start codon (e.g. ATG or AUG) and which preferably terminates with a stop codon (e.g., TAA, TGA, or TAG or UAA, UGA, UAG, respectively). The open reading frame may be isolated or it may be incorporated in a longer nucleic acid sequence, for example in a vector or an mRNA. An open reading frame may also be termed ‘protein coding region’ or ‘coding region’.

3 ’-untranslated region (3’UTR): A 3’UTR is typically the part of an mRNA which is located between the protein coding region (i.e., the open reading frame) and the poly(A) sequence of the mRNA. A 3’UTR of the mRNA is not translated into an amino acid sequence. The 3’UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns. The pre-mature mRNA is then further processed into mature mRNA in a maturation process. This maturation process comprises the steps of 5 ’capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3 ’-end, such as polyadenylation of the 3 ’-end of the premature mRNA and optional endo- or exonuclease cleavages etc. In the context of the present invention, a 3 ’UTR corresponds to the sequence of a mature mRNA which is located 3’ to the stop codon of the protein coding region, preferably immediately 3 ’ to the stop codon of the protein coding region, and which extends to the 5 ’-side of the poly(A) sequence, preferably to the nucleotide immediately 5’ to the poly(A) sequence. The term “corresponds to” means that the 3’UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3’UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 3’UTR of a gene”, is the sequence which corresponds to the 3’UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “3’UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 3’UTR.

Poly(A) sequence: A poly(A) sequence, also called poly(A) tail or 3’-poly(A) tail, is typically understood to be a sequence of adenine nucleotides, e.g., of up to about 400 adenine nucleotides, e.g. 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, most preferably from about 60 to about 250 adenine nucleotides. A poly(A) sequence is typically located at the 3 ’end of an mRNA. In the context of the present invention, a poly(A) sequence may be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector.

Polyadenylation: Polyadenylation is typically understood to be the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, e.g. to a premature mRNA. Polyadenylation may be induced by a so called polyadenylation signal. This signal is preferably located within a stretch of nucleotides at the 3 ’-end of a nucleic acid molecule, such as an RNA molecule, to be polyadenylated. A polyadenylation signal typically comprises a hexamer consisting of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences, preferably hexamer sequences, are also conceivable. Polyadenylation typically occurs during processing of a pre-mRNA (also called premature-mRNA). Typically, RNA maturation (from pre-mRNA to mature mRNA) comprises the step of poly adenylation.

Sequence of a polynucleotide: The sequence of a polynucleotide is typically understood to be the particular and individual order, i.e., the succession of its nucleotides. The sequence of a protein or peptide is typically understood to be the order, i.e., the succession of its amino acids.

Sequence identity: Two or more sequences are identical if they exhibit the same length and order of nucleotides or amino acids. The percentage of identity typically describes the extent to which two sequences are identical, i.e., it typically describes the percentage of nucleotides that correspond in their sequence position with identical nucleotides of a reference-sequence. For determination of the degree of identity, the sequences to be compared are considered to exhibit the same length, i.e., the length of the longest sequence of the sequences to be compared. This means that a first sequence consisting of 8 nucleotides is 80% identical to a second sequence consisting of 10 nucleotides comprising the first sequence. In other words, in the context of the present invention, identity of sequences preferably relates to the percentage of nucleotides of a sequence which have the same position in two or more sequences having the same length. Gaps are usually regarded as non-identical positions, irrespective of their actual position in an alignment.

Transfection: The term “transfection” refers to the introduction of nucleic acid molecules or polynucleotides, such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably into eukaryotic cells. In the context of the present invention, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, preferably into eukaryotic cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g., 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-dextran or polyethylenimine etc. Preferably, the introduction is non- viral.

Vaccine: A vaccine is typically understood to be a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen. The antigen or immunogen may be derived from any material that is suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as from bacteria or virus particles etc., or from a tumor or cancerous tissue. The antigen or immunogen stimulates the body’s adaptive immune system to provide an adaptive immune response.

Vector: The term ‘vector’ refers to a nucleic acid molecule or a polynucleotide. A vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molecule, for example, of an mRNA molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a desired mRNA sequence or a part thereof, such as a sequence corresponding to the open reading frame and the 3’UTR of an mRNA. An expression vector may be used for production of expression products such as RNA, e.g., mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a promoter sequence, e.g. an RNA promoter sequence. A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bacteriophage vector. A transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. A vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector. Preferably, a vector is a DNA molecule. Preferably, a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. Preferably, a vector in the context of the present application is a plasmid vector.

Peptide: A peptide or polypeptide is typically a polymer of amino acid monomers, linked by peptide bonds. It typically contains less than 50 monomer units. Nevertheless, the term peptide is not a disclaimer for molecules having more than 50 monomer units. Long peptides are also called polypeptides, typically having between 50 and 600 monomeric units.

Polynucleotides for use in accordance with the invention may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT) or enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference). The process of design and synthesis of the primary constructs of the invention generally includes the steps of gene construction, mRNA production (either with or without modifications) and purification. In the enzymatic synthesis method, a target polynucleotide sequence encoding the polypeptide of interest is first selected for incorporation into a vector which will be amplified to produce a cDNA template. Optionally, the target polynucleotide sequence and/or any flanking sequences may be codon optimized. The cDNA template is then used to produce mRNA through in vitro transcription (IVT). After production, the mRNA may undergo purification and clean-up processes.

In one embodiment, the polynucleotide is a RNA, preferably a mRNA. In one embodiment, the polynucleotide is a DNA. In one embodiment, the polynucleotide is a DNA. In one embodiment, the polynucleotide comprises a 3’ untranslated region (3’UTR). In one embodiment, the polynucleotide comprises a poly A tail.

In one embodiment, the 5’UTR comprises or consists of the polynucleotide sequence having 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-8.

In one embodiment, the 5’UTR comprises or consists of any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1- 8.

In one embodiment, the ORF comprises the polynucleotide sequence encoding the amino acid sequence of N7N antigen or variants thereof. In some embodiments, the N7N antigen is a N7N glycoprotein. In some embodiments, the N7N glycoproteins are selected from the group consisting of gE, gl, gB, gH, gD, gK, gC, gN, gM and variants thereof. In some embodiments, the N7N glycoprotein is gE protein or variants thereof.

In one embodiment, the amino acid sequence encoded by the ORF comprises or consist of amino acid sequence of truncated N7N antigen sequence. Vector, cell and composition

Vector: The term “vector” refers to a nucleic acid molecule, preferably to an artificial nucleic acid molecule. A vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molecule, for example, of an mRNA molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a desired mRNA sequence or a part thereof, such as a sequence corresponding to the 5’UTR and the open reading frame of an mRNA. An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a promoter sequence, e.g. an RNA promoter sequence. A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bacteriophage vector. A transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. A vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector. Preferably, a vector is a DNA molecule. Preferably, a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. Preferably, a vector in the context of the present application is a plasmid vector.

In one aspect, the invention provides a vector, comprising the polynucleotide of the invention. In an embodiment, the vector comprises T7 promoter.

A vaccine is typically understood to be a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen. The antigen or immunogen may be derived from any material that is suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as from bacteria or virus particles etc., or from a tumor or cancerous tissue. The antigen or immunogen stimulates the body’s adaptive immune system to provide an adaptive immune response.

In some embodiments, the composition comprises the polynucleotide of the invention and a lipid nanoparticle.

Use of the polynucleotide, vector, cell and composition

In one aspect, the invention provides a method for preventing or treating a disease, comprising administering an effective amount of the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention.

In one aspect, the invention provides the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention for use in regulating the expression of one or more genes (e.g., downregulating a disease-causing gene) or modifying one or more genes (e.g., replacing a disease-causing gene with a healthy copy of the gene). In one aspect, the invention provides use of the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention in the manufacture of a medicament for preventing or treating a disease.

In one aspect, the invention provides the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention for veterinary use, for example, for treating diseases in animals or for use as a vaccine in animals.

In some embodiments, the composition comprises nanoparticle, for example, lipid nanoparticle.

In some embodiments, the disease is N7N infection.

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

In some embodiments, the composition may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances.

In some embodiments, regulating the expression of one or more genes or modifying one or more genes is mediated by CRISPR (clustered regularly interspaced short palindromic repeats). In some embodiments, the polynucleotide of the invention, the vector of the invention, the cell of the invention or the composition of the invention is used in the CRISPR, for example, the 5’UTR described herein can be used for the expression of Cas proteins, dCas proteins or gRNA (guide RNA) of the CRISPR.

EXAMPLES

The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention. The contents of all references cited throughout this application are expressly incorporated herein by reference.

Methods and Materials

Plasmid generation

A gene block encoding 5’UTR (Mod, SEQ ID NO: 9), gE del574 Y569A and alpha globin 3’UTR (SEQ ID NO: 19) was ordered from Integrated DNA Technologies Inc. (IDT). The pUC19 vector (Thermo Scientific) and gE del 574 Y569A gene block was restriction enzyme digested with EcoRI and BamHI, purified, ligated, and transformed in NEB® Stable Competent E. coli (NEB). Colonies were screened and a positive clone with correct sequence of gE del574 Y569A pUC19 plasmid was obtained. A duplex Poly A primer (containing 121 Poly A region followed by a BbsI and SphI restriction enzyme site) and gE del574 Y569A pUC19 plasmid were restriction enzyme digested with BamHI and SphI, purified, ligated, and transformed in NEB® Stable Competent E. coli (NEB). Colonies were screened and a positive clone with correct sequence of gE del574 Y569A Poly A pUC19 was obtained.

The amino acid sequence of WT VZV gE is NP_040190.1 of the NCBI reference sequence (https ://www. ncbi.nlm.nih.gov/protein/NP_040190.1 , which is incorporated herein in its entirety). The amino acid sequence of the mutant VZV gE used herein is based on the WT VZV gE (NP_040190.1) as above.

A homologous recombination approach was used to clone sequences encoding VZV gE del569, gE del575 Y569A, gE del575 Y569K, gE del574 Y569A with different 5’UTR sequences and human alpha globin as 3’UTR in pUC57 Poly A vector. The vector has 121 poly A region followed by Sapl/BspQl site for plasmid linearization.

All generated constructs were confirmed with Sanger Sequencing.

5’UTR construct generation gE del574 Y569A Poly A pUC19 plasmid was restriction enzyme digested with EcoRI and Ncol to excise the Mod 5’UTR sequence. The generated vector was incubated with the duplexes containing different 5’UTRs (SEQ ID NOs: 10-18) (ordered from IDT) in NEB HiFi builder mix at 50°C for 1 hour and the mix was transformed into NEB® Stable Competent E. coli (NEB) and colonies were allowed to grow. Multiple clones were screened to obtain the positive clones for all the 5’UTR constructs. All generated constructs were confirmed with Sanger Sequencing.

Vector Linearization

Plasmids with pUC19 backbone were restriction enzyme digested with Bbsl enzyme while plasmids with pUC57 backbone were restriction enzyme digested with Sapl/BspQl enzyme to linearize the vector. The plasmid linearization was confirmed on agarose gel and was purified on Qiagen clean up column. mRNA generation and purification Linearized vector was used as template to generate the mRNA by using NEB HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040S) and following manufacturer protocol. Briefly An invitro transcription reaction (IVT) was set up with linearized templates containing transcription buffer, T7 RNA polymerase, ATP, GTP, CTP, UTP or ml'P for 2 hours at 37°C (NEB). A cap reagent (TriLink CleanCap Reagent AG, N-7113) was also added to the reactions to generate capped mRNA (Cap 1). DNase I (NEB) was used to remove the plasmid template and generated mRNA was cleaned up either using LiCl precipitation or Mega clear kit (Thermo). mRNA was quantified and electrophoresed to check the integrity and quality of generated mRNA.

InVitro -Translation in Rabbit Reticulocyte system

A invitro translation reaction was set up with synthesized mRNA using Promega Rabbit Reticulocyte System (L4960) with Transcend tRNA (L5061) and generated protein was electrophoresed and blotted with streptavidin antibody to detect the biotin tagged protein.

Transfection in mammalian cell and expression mRNAs were transfected in mammalian cells (HeLa and/or HEK 293T cells) using lipofectamine messenger max using manufacturer protocol (Thermo) and were processed after 20 hrs. RIPA lysates were electrophoresed on SDS PAGE and electro blotted to PVDF membrane and VZV gE protein expression was monitored with anti-VZV gE antibody (Virusys)

Example 1: Design and preparation of mRNA with different 5’UTRs

A large number of denovo 5’UTRs were designed namely Anta 8 (SEQ ID NO: 1), Anta 24 (SEQ ID NO: 2), Anta 30 (SEQ ID NO: 3), MC (SEQ ID NO: 4), NB (SEQ ID NO: 5) and LC (SEQ ID NO: 6). Region around translation initiation site were modified for human HBA1 and HBB1 5’UTRs which resulted into M HBA1 (SEQ ID NO: 7) and M HBB1 (SEQ ID NO: 8). The sequence 176 in Table 4 of the patent US10881730B2 is also used as reference to compare and is labelled as Mod (SEQ ID NO: 9).

The above 5’UTR were cloned as shown in the schematic (Figure 1), upstream to coding sequence of gE N7N resulting in a plasmid. A modified T7 promoter sequence was appended before the 5’UTR sequence, so that the generated mRNA could be co-transcriptionally capped. Human alpha globin 3’UTR was added after the CDS region followed by Poly A tail region and a recognition site to linearize the generated plasmid.

In vitro transcription was set up with linearized plasmid to generate mRNA (replacement of uridine with N1 methyl pseudouridine) followed by a DNasel treatment to digest the linearized plasmid. mRNA was further processed and purified.

Example 2: The 5’UTRs improved the mRNA stability and protein expression

To analyze how the 5’UTRs in mRNA constructs affect the protein production, rabbit reticulocyte lysate (RRL) was employed. RRL is a mammalian cell-free system which is used for characterizing protein products from RNA transcripts and investigating transcriptional and translational control. mRNA constructs with different 5’UTRs were in vitro translated in RRL and generated protein were detected with anti-gE VZV antibody. Differences in expression of gE VZV protein could be directly corelated to 5’UTR as the 5’UTR is the only difference in all the tested mRNA constructs.

Maximum protein expression was observed for Anta30 5’UTR followed by LC and Anta24. A similar expression level was observed for Anta8, NB, MC and Mod 5’UTRs while minimal expression in RRL was observed for MHBA1 (Figure 2).

Surprisingly, when mRNA with different 5’UTRs were measured for protein expression in HeLa cells a different pattern was observed. Almost similar protein expression levels were observed for mRNA’s with Anta8, Anta24, Anta30, M HBA1 and Mod 5’UTRs while a 25% decrease was observed for all other mRNAs tested (Figure 3).

Example 3: mRNAs with the 5’UTRs has enhanced immune response against VZV

To test the immunogenicity of mRNA constructs, mRNA-lipid nanoparticles (mRNA-LNP) were generated with SMI 02 as ionizable lipid, and mice were immunized with two doses of 10 ug mRNA-LNPs apart 14 days. Mice sera was collected, and antibody titers were measured against gE VZV protein. gE VZV mRNA with Anta30 and M HBA1 5’UTRs had antibody titers values twice as compared to mRNA’s with other 5’UTRs (Figure 4).

The structure

Thl-biased T cell response is essential to prove that intended vaccine can generate a good and lasting immune response. Thl-biased T cell response is critical for the prevention and control of VZV. VZV gE-specific T cell responses in mice immunized with various constructs were measured by ex- vivo restimulation of splenocytes with an overlapping peptide pool covering the full length VZV gE. Secretion of IFNy (a prototypic TH1 cytokine) was observed in splenocytes from mice immunized with all the constructs with highest response observed in mRNA construct with Anta30 as 5’UTR (Figure 5).

Various implementations of the present disclosure have been described above and the above description is only exemplary rather than exhaustive and is not limited to the implementations of the present disclosure. Many modifications and alterations, without deviating from the scope and spirit of the explained various implementations, are obvious for those skilled in the art. The selection of terms in the text aims to best explain principles and actual applications of each implementation and technical improvements made in the market by each embodiment, or enable others of ordinary skill in the art to understand implementations of the present disclosure.

Sequence Listing

*(The EcoRI and Ncol sites are 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 the polynucleotide sequence having 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-8.

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

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

4. The polynucleotide of any one of claims 1-3, wherein the VZV glycoproteins are selected from the group consisting of gE, gl, gB, gH, gD, gK, gC, gN, gM and variants thereof, preferably the N7N glycoprotein is gE or variants thereof, more preferably the variant of gE is N7N 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 VZV gE protein or variants 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 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 pharmaceutically acceptable excipients.

13. A method for preventing or treating a disease, comprising administering 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 to a subject in need thereof.

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 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 for use 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 anyone 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.