Antiviral therapeutics

Abstract

The invention provides an hSAND protein and a CYB5R3 protein for use in the prevention or treatment of a viral infection in a subject. The invention also provides one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein for use in the prevention or treatment of a viral infection in a subject, a fusion protein, a polynucleotide encoding the fusion protein, a vector, a host cell, an immunogenic composition, and an antiviral vaccine.

Description

[0001] Our Reference: P608081PC00

[0002] Antiviral therapeutics

[0003] Field of Invention

[0004] Embodiments of the present invention described herein relate to the combination of an hSAND protein and a CYB5R3 protein for use in the prevention or treatment of a viral infection in a subject. In particular, fusion proteins of hSAND and a CYB5R3 are described, as well as polynucleotides encoding the individual proteins and fusion protein. Also described are immunogenic compositions and antiviral vaccines.

[0005] Background to the Invention

[0006] The burden of viral infections on the global economy and public health is unprecedented. This burden is increasing due to globalisation and the rise in the emergence and re-emergence of new viruses.

[0007] Traditionally, small-molecule catalysts or reagents (nonenzymatic catalysts) are used in antiviral drug discovery and synthesis. A prominent example of antivirals produced this way is broad-spectrum antiviral nucleoside triphosphate analogues (ANTAs) (Figure 12b). ANTAs have spectrum activity against several viruses and are rapidly repurposed to treat emerging viruses. For example, antiviral remdesivir was initially developed for Ebola and was the first drug approved to treat COVID-19. Hence, they are one of the largest groups of antiviral medications (>25 FDA-approved drugs) and are on the World Health Organization's List of Essential Medicines.

[0008] As a drug, ANTAs are synthesised as a nucleoside analogue prodrug (NAP). These prodrugs are delivered to cells, after which they are phosphorylated by the cellular or viral kinases leading to formation of multiple species including monophosphate, diphosphate, and triphosphate. Only the triphosphate form is active, and the monophosphate and diphosphate can inhibit other enzymes causing toxicity and affecting cellular function (Figure 12b). This process of conversion of NAP to ANTA is not efficient and has toxicity. Approval of a NAP is very costly because of all the optimisation and investments required to identify a suitable NAP that can be converted to its corresponding ANTA inside a cell with minimum toxicity and highest efficiency. There remains a need to develop broad spectrum antiviral therapeutics that can be produced in an efficient manner and with minimum toxicity.

[0009] Summary of the Invention

[0010] Members of the radical-S-adenosylmethionine (SAM) superfamily of enzymes are widespread in all Kingdoms of life.10-12They are commonly identified by a highly conserved [4Fe-4S] cluster coordinated to three cysteine residues, enabling SAM to coordinate to the fourth iron atom in the cluster (Fig. la).10'11The oxidized [4Fe-4S]2+cluster receives electrons to reductively cleave SAM and generate 5 '- deoxyadenosyl radical (5 '-dA*).13'14This radical intermediate performs hydrogen atom transfer (HAT) reactions, converting a substrate to its product. At least eight radical-SAM enzymes exist in humans,7affecting different aspects of human health and disease. For example, hSAND (human SAM-dependent nucleotide dehydratase) (also known as RSAD2 or viperin)15'16is known to perform vital biochemical reactions regulating the immune response, oncometabolism, or oncogene expression.8'9The nature of the partner protein providing electrons to hSAND, i.e. the electron transfer partner (ETP), has remained elusive.

[0011] During viral infection, the expression of hSAND is induced by interferons,16and uses two electrons20(Fig. lb) to catalyse the transformation of the nucleoside triphosphate, cytidine triphosphate (CTP), to its antiviral and immunoregulatory analogue 3 '-deoxy-3 ',4 '-didehydro-CTP (ddhCTP).21This nucleoside triphosphate analogue can chain terminate viral replication and boost the immune system to fight a wide range of viruses.

[0012] The inventors have identified CYB5R3, a member of the Cytochrome-bs reductases (CYB5R) family, as the ETP of hSAND. Members of CYB5R are known to reduce Cytochrome b5 (Cyt-b5), a known electron transfer partner of Cyt-P450 or Cyt-c.22

[0013] The inventors have demonstrated that the antiviral nature of hSAND can be harnessed by providing hSAND and CYB5R3 together as a therapeutic agent to treat or prevent viral infection. In particular, hSAND and CYB5R3 can be provided prior to a viral infection to facilitate generation of cellular ddhCTP without a virus being present, thus acting as a prophylactic to prevent or block early entry of a virus into the cell. Furthermore, hSAND and CYB5R3 can be provided during a viral infection to boost the generation of cellular ddhCTP to treat the viral infection. A particular advantage of the invention is that the hSAND- and CYB5R3- based therapeutic agents can be used to broadly and non-specifically treat and prevent viral infections, without requiring prior knowledge of the viral antigens.

[0014] Furthermore, the inventors have demonstrated that this system can be used to generate ddhCTP in vitro, to allow large-scale production of this antiviral compound.

[0015] To this end, the inventors have generated hSAND and CYB5R3 polypeptides and fusion proteins, as well as nucleotides encoding said polypeptides and fusion proteins. A particular aspect developed is an antiviral mRNA-based vaccine encoding an hSAND protein and a CYB5R3 protein. It is envisaged that this invention can replace the multistep and inefficient processes of making an ANTA and delivering it to a target cell. Instead, the mRNA-based vaccine will be delivered to a target cell (e.g., using liposomes or other vehicles) which will lead to expression of hSAND and CYB5R3. hSAND then converts CTP directly to ddhCTP, which directly exerts its antiviral activity.

[0016] In a first aspect, there is provided an hSAND protein and a CYB5R3 protein for use in the prevention or treatment of a viral infection in a subject, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity.

[0017] Advantageously, the hSAND protein and the CYB5R3 protein can be used together to produce the antiviral compound ddhCTP to treat an existing viral infection or to act as a prophylactic agent. As discussed above, the CYB5R3 protein functions as the electron transfer partner of the hSAND protein, which uses two electrons to catalyse the transformation of CTP to ddhCTP.

[0018] The term "hSAND protein", as used herein, refers to the human radical-S- adenosylmethionine (SAM) dependent nucleotide dehydratase (hSAND) and encompasses naturally-occurring hSAND, artificially synthesised hSAND, as well as fragments and variants of hSAND, provided that the hSAND protein has functional nucleotide dehydratase activity (i.e., the hSAND protein can catalyse the dehydration of nucleoside triphosphates (NTPs) to their ddh analogues in the presence of an electron transfer partner). hSAND is described in Ji Y, Wei L, Da A, Stark H, Hagedoorn PL, Ciofi-Baffoni S, Cowley SA, Louro RO, Todorovic S, Mroginski MA, Nicolet Y, Roessler MM, Le Brun NE, Piccioli M, James WS, Hagen WR, Ebrahimi KH ("Radical-SAM dependent nucleotide dehydratase (SAND), rectification of the names of an ancient iron-sulfur enzyme using NC-IUBMB recommendations." Front Mol Biosci. 2022 Oct 21;9: 1032220. doi: 10.3389 / fmolb.2022.1032220. PMID: 36387278). The DNA sequence of hSAND can be identified by GenBank accession number AC017076.14 and is provided as SEQ ID NO: 1. The mRNA sequence of hSAND can be found by GenBank accession number AF442151.1. The amino acid sequence of hSAND is provided as SEQ ID NO: 2.

[0019] In typical embodiments, the hSAND protein comprises the Q195GKK surface loop of hSAND.

[0020] In some embodiments, the hSAND protein is a truncated variant of hSAND lacking the hydrophobic domain. The amino acid sequence of the hydrophobic domain of hSAND is provided as SEQ ID NO: 3. In some embodiments, the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 (modified hSAND, wherein the hydrophobic domain has been removed and replaced with a methionine residue) or an amino acid sequence having at least 80% identity to SEQ ID NO: 4.

[0021] In some embodiments, the hSAND protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 91% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 92% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 93% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 94% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 96% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 97% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 98% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having at least 99% identity to SEQ ID NO: 4. In some embodiments, the hSAND protein has an amino acid sequence having 100% identity to SEQ ID NO: 4.

[0022] The term "CYB5R3 protein", as used herein, refers to the NADH-cytochrome b5 reductase 3 protein and encompasses naturally-occurring CYB5R3, artificially synthesised CYB5R3, as well as fragments and variants of CYB5R3, provided that the CYB5R3 protein has functional electron transfer partner activity (i.e., the CYB5R3 protein can act as the electron transfer partner of the hSAND protein in the nucleotide dehydratase mechanism). The DNA sequence of CYB5R3 can be found by GenBank accession number AY341030.1 and is provided as SEQ ID NO: 5. The mRNA sequence of CYB5R3 can be found by GenBank accession number BT009821.1. The amino acid sequence of CYB5R3 is provided as SEQ ID NO: 6.

[0023] In some embodiments, the CYB5R3 protein is a truncated variant of CYB5R3 lacking the hydrophobic domain. The amino acid sequence of the hydrophobic domain of CYB5R3 is provided as SEQ ID NO: 7. In some embodiments, the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 (modified CYB5R3, wherein the hydrophobic domain has been removed and replaced with a methionine residue) or an amino acid sequence having at least 80% identity to SEQ ID NO: 8.

[0024] In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 91% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 92% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 93% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 94% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 96% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 97% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having at least 99% identity to SEQ ID NO: 8. In some embodiments, the CYB5R3 protein has an amino acid sequence having 100% identity to SEQ ID NO: 8.

[0025] The term "prevention or treatment of a viral infection" as used herein refers to precluding, reducing the risk of developing and delaying the onset of a viral infection or one or more symptoms or complications associated with the viral infection, alleviating, ameliorating or reducing the severity or frequency of, inhibiting the progress of, reversing or abrogating a viral infection or one or more symptoms or complications associated with the viral infection, and alleviating, ameliorating or eradicating one or more causes of the viral infection. Treating the viral infection includes ameliorating at least one symptom of the viral infection, even if the underlying pathophysiology is not affected. In particular embodiments, the products of the invention are for use in the prevention of a viral infection.

[0026] In some embodiments, the viral infection is selected from: an infection caused by a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-sense ssRNA virus, a negative-sense ssRNA virus, an RNA virus that reverse transcribes, or a DNA virus that reverse transcribes.

[0027] In some embodiments, the viral infection is an infection caused by a virus from a family selected from: Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Bornaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picobirnaviridae, Picobirna, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Togaviridae, or Delta.

[0028] In some embodiments, the viral infection is an infection caused a virus from the Coronaviridae family. In some embodiments, the viral infection is caused by a severe acute respiratory syndrome (SARS) coronavirus or a MERS coronavirus. In some embodiments, the viral infection is caused by SARS coronavirus 2 (SARS- CoV-2).

[0029] In some embodiments, the viral infection is a respiratory viral infection. In some embodiments, the respiratory viral infection is caused by a rhinovirus, a respiratory syncytial virus (RSV), an adenovirus, a bocavirus, a coronavirus, a metapneumovirus, or a parainfluenza virus.

[0030] The subject may be any living multi-cellular vertebrate organism, including human and non-human mammals, such as non-human primates (e.g., chimpanzees and other apes and monkey species), pigs, camels, bats, sheep, cows, dogs, cats, rodents (e.g., mice, rats and guinea pigs), birds and the like. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0031] In a second aspect, there is provided one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein for use in the prevention or treatment of a viral infection in a subject, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity.

[0032] The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect. Further, the medical use (e.g., the viral infection, the subject, etc.) is as defined above with respect to the first aspect.

[0033] In some embodiments, the one or more polynucleotides comprises or consists of one polynucleotide (i.e., both the hSAND protein and the CYB5R3 protein are encoded by a single polynucleotide). In some embodiments, the one or more polynucleotides comprises or consists of two polynucleotides (i.e., a first polynucleotide encodes the hSAND protein and a second polynucleotide encodes the CYB5R3 protein).

[0034] In some embodiments, the one or more polynucleotides are DNA sequences. In some embodiments, the one or more polynucleotides are cDNA sequences. In some embodiments, the one or more polynucleotides are RNA sequences. In some embodiments, the one or more polynucleotides are mRNA sequences.

[0035] In some embodiments, the one or more polynucleotides comprises the nucleotide sequence set out in SEQ ID NO: 10 (modified hSAND; codon-optimised for expression in humans) and the nucleotide sequence set out in SEQ ID NO: 13 (modified CYB5R3; codon-optimised for expression in humans). In some embodiments, the one or more polynucleotides comprises a first polynucleotide having the nucleotide sequence set out in SEQ ID NO: 10 (modified hSAND) and a second polynucleotide having the nucleotide sequence set out in SEQ ID NO: 13 (modified CYB5R3). In some embodiments, the one or more polynucleotides comprises one polynucleotide having a first nucleotide sequence set out in SEQ ID NO: 10 (modified hSAND) and a second nucleotide sequence set out in SEQ ID NO: 13 (modified CYB5R3).

[0036] In some embodiments, the one or more polynucleotides comprises the nucleotide sequence set out in SEQ ID NO: 9 (modified hSAND; codon-optimised for expression in E. coli) and the nucleotide sequence set out in SEQ ID NO: 12 (modified CYB5R3; codon-optimised for expression in E. coli).

[0037] In some embodiments, the one or more polynucleotides comprises the RNA sequence set out in SEQ ID NO: 11 (modified hSAND) and the RNA sequence set out in SEQ ID NO: 14 (modified CYB5R3). In some embodiments, the one or more polynucleotides comprises a first polynucleotide having the RNA sequence set out in SEQ ID NO: 11 (modified hSAND) and a second polynucleotide having the RNA sequence set out in SEQ ID NO: 14 (modified CYB5R3). In some embodiments, the one or more polynucleotides comprises one polynucleotide having a first RNA sequence set out in SEQ ID NO: 11 (modified hSAND) and a second RNA sequence set out in SEQ ID NO: 14 (modified CYB5R3).

[0038] The one or more polynucleotides may be codon optimised to enhance expression in particular host cells. Codon optimisation methods are known in the art and refer to modifying the nucleotide sequence in order to enhance protein expression in a host cell of interest by replacing one or more codons of the native sequence with codons that are more frequently used in the genes of that host cell or in the genes of the host the cell was derived from. Various species exhibit particular bias for certain codons of a particular amino acid. In certain embodiments, the one or more polynucleotides are codon optimised to enhance expression in a host cell selected from bacteria, yeast, fungi, plant, mammalian and / or insect cells. In some embodiments, the one or more polynucleotides are codon-optimised to enhance expression in humans.

[0039] In a third aspect, there is provided a fusion protein comprising an hSAND protein and a CYB5R3 protein fused together, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity. The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect.

[0040] For example, in some embodiments, the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 (modified hSAND) or an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 (modified CYB5R3) or an amino acid sequence having at least 80% identity to SEQ ID NO: 8. As discussed above, the hSAND protein may have an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4. Similarly, as discussed above, the CYB5R3 protein may have an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 8.

[0041] The term "fusion protein", as used herein, refers to a chimeric protein having two or more polypeptides which are not usually contiguous joined together. The term "fusion polypeptide" can be used interchangeably with the term "fusion protein". In a more particular sense, the term "fusion protein" may also refer to a polypeptide comprising an hSAND protein and a CYB5R3 protein covalently linked, either directly or via an amino acid or a linker. The polypeptides forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C- terminus, N-terminus to N-terminus, or N- terminus to C-terminus.

[0042] Techniques for producing fusion polypeptides are known in the art and include linking the coding sequences encoding the polypeptides such that they are in structure and that expression of the fused polypeptide is under the control of the same promoter(s) and terminator. Fusion proteins can also be constructed using intein technology in which post-translational fusions are created (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0043] In some embodiments, the hSAND protein and the CYB5R3 protein are fused together via a linker. The term "linker" as used herein refers to an entity that connects the hSAND protein and the CYB5R3 protein to form a multi-element agent. Any suitable linker can be used, provided the linker does not affect the nucleotide dehydratase activity of the hSAND protein and the electron transfer partner activity of the CYB5R3 protein within the fusion protein. A variety of different linker elements that can appropriately be used when engineering fusion proteins are known in the art (Holliger et al, 1993; Poljak, 1994). In some embodiments, the linker is an amino acid linker. The amino acid linker may also be referred to as a peptide linker. In some embodiments, the linker comprises the amino acid sequence set out in SEQ ID NO: 18 (linker sequence). In some embodiments, the linker consists of the amino acid sequence set out in SEQ ID NO: 18 (linker sequence).

[0044] In some embodiments, the linker is 1-30 amino acids in length. In some embodiments, the linker is 2-25 amino acids in length. In some embodiments, the linker is 3-20 amino acids in length. In some embodiments, the linker is 4-15 amino acids in length. In some embodiments, the linker is 5-10 amino acids in length. In some embodiments, the linker is at least 1, 2, 3, 4, 5, 8, or 10 amino acids in length. In some embodiments, the linker is no more than 30, 25, 20, 15, 10, 9, 8, 7, 6 or 5 amino acids in length.

[0045] The hSAND protein and the CYB5R3 protein may be in any orientation. For example, the hSAND protein may be N-terminal to the CYB5R3 protein, or the hSAND protein may be C-terminal to the CYB5R3 protein. Typically, the C- terminal end of the hSAND protein is connected to the N-terminal end of the CYB5R3 protein, optionally via a linker.

[0046] In some embodiments, the fusion protein comprises the amino acid sequence set out in SEQ ID NO: 17 (fusion protein) or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein consists of the amino acid sequence set out in SEQ ID NO: 17 (fusion protein) or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17.

[0047] In some embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion protein comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 17.

[0048] In some embodiments, the fusion protein further comprises a label, a purification tag, a peptide that enhances the biocompatibility, solubility, secretion, or half-life of the fusion protein, and any combination thereof. The label may be any suitable label for identifying the fusion protein. For example, the label may be a fluorescent label, such as GFP. Various labels and fluorescent labels are known to the skilled person. The purification tag may be any suitable tag that aids in the purification of the fusion polypeptide. Various purification tags are known to the skilled person, including polyhistidine tags (e.g., HisX6), GST peptide, FLAG peptide, streptavidin binding peptide, V5 epitope peptide, Myc peptide or HA peptide. The peptide that enhances the biocompatibility, solubility, secretion, or half-life of the fusion protein may be any suitable peptide known to the skilled person, such as a PAS polypeptide (a polypeptide consisting of proline, alanine and serine residues) or an albumin binding domain.

[0049] In some embodiments, the fusion protein comprising the following consecutive amino acids in N- to C-terminus orientation: (i) SEQ ID NO: 4 or a variant of SEQ ID NO:4; (ii) a linker of 1-30 amino acids linking SEQ ID NO:4 and SEQ ID NO:8 or the variants thereof; and (iii) SEQ ID NO:8 or a variant of SEQ ID NO:8. In some embodiments, the variants of SEQ ID NO: 4 or 8 have at least about 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 4 or 8. In certain embodiments, the variants of SEQ ID NO: 4 or 8 have conservative substitutions. In some embodiments, the linker has SEQ ID NO: 18 or a variant of SEQ ID NO: 18 having about 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 18.

[0050] In a fourth aspect, there is provided a polynucleotide encoding the fusion protein as described herein above and elsewhere.

[0051] The fusion protein is as defined above with respect to the third aspect.

[0052] In some embodiments, the polynucleotide has the nucleotide sequence set out in SEQ ID NO: 15 (fusion protein). In some embodiments, the polynucleotide has the RNA sequence set out in SEQ ID NO: 16 (fusion protein).

[0053] In some embodiments, the polynucleotide is codon optimised for expression in a particular host. In some embodiments, the polynucleotide is codon optimised for expression in humans.

[0054] In a fifth aspect, there is provided a vector comprising the polynucleotide as described herein above and elsewhere.

[0055] The polynucleotide is as defined above with respect to fourth aspect.

[0056] A vector may be any of a number of polynucleotides into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. In some embodiments, the vector comprises a promoter sequence. A promoter may include an untranslated polynucleotide usually located upstream of the coding region that contains the site for initiating transcription of the polynucleotide. The promoter region may also include other elements that act as regulators of gene expression. In further embodiments of the invention, the vector contains an additional region to aid in selection of cells that have the vector incorporated. The promoter sequence is often bounded (inclusively) at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RIMA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes.

[0057] In some embodiments, vector comprises one or more marker sequences suitable for use in the identification and selection of cells which have been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., p-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

[0058] An expression vector is one into which a desired nucleic acid may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Expression refers to the transcription and / or translation of an endogenous gene, transgene or coding region in a cell.

[0059] A coding sequence and regulatory sequences are operably joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RIMA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

[0060] The vector may be any suitable vector, such as a prokaryotic vector, a eukaryotic vector or a viral vector. The prokaryotic vector may be a plasmid vector, a phage vector, a phagemid vector, a cosmid vector or a bacterial artificial chromosome vector. The eukaryotic vector may be a yeast vector, an insect cell vector, a mammalian vector, or a plant cell vector. The viral vector may be an adenovirus vector, a lentivirus vector, or a retrovirus vector. In particular embodiments, the vector is a plasmid vector. In particular embodiments, the vector is a viral vector.

[0061] In some embodiments, the vector is a non-viral vector, i.e., a delivery system that transports the desired sequence(s) into cells without using a virus. Suitable non-viral vectors are known in the art and include, for example, lipid nanoparticles, N-acetylgalactosamine (GalNAc), polymer nanoparticles, liposome nanoparticles, and / or inorganic nanoparticles. See, for example, Geng et al. (Viral and non-viral vectors in gene therapy: current state and clinical perspectives, eBioMedicine, Volume 118, 2025, 105834, ISSN 2352-3964, https : / / doi;org / 10, 1016 / LebjonT2025;105834) and Wang et al. (Emerging non- viral vectors for gene delivery. J Nanobiotechnol 21, 272 (2023). https: / / doi.org / 10.1186 / sl2951-023-02044-5).

[0062] In a sixth aspect, there is provided a vector comprising one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein thereof has electron transfer partner activity.

[0063] The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect. Features of the vectors described in detail with respect to the fifth aspect are also applicable to this aspect.

[0064] In a seventh aspect, there is provided a host cell comprising: (i) the fusion protein as described herein above and elsewhere;

[0065] (ii) the polynucleotide as described herein above and elsewhere; and / or

[0066] (iii) the vector as described herein above and elsewhere.

[0067] The fusion protein is as defined above with respect to the third aspect. The polynucleotide is as defined above with respect to the fourth aspect. The vector is as defined above with respect to the fifth and sixth aspects.

[0068] The host cell may be any suitable host cell, such as a prokaryotic cell or a eukaryotic cell. As used herein, the term "host cell" refers to a cell which harbours a vector of the invention, as well as a cell that is suitable for use in expressing a recombinant gene or protein. It is not intended that the present invention be limited to any particular type of cell. Indeed, it is contemplated that any suitable cell will find use in the present invention as a host cell. A host cell according to the invention may permit the expression of a polynucleotide of the invention. Thus, the host cell may be, for example, a bacterial, a yeast, a fungal, a plant, an insect or a mammalian cell. In some embodiment, the host cell is a bacterial, a yeast, or a mammalian cell. In some embodiments, the host cell is a human cell.

[0069] In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is an antigen presenting cell. In some embodiments, the host cell is a dendritic cell or a macrophage.

[0070] In an eighth aspect, there is provided an immunogenic composition comprising:

[0071] (i) an hSAND protein having nucleotide dehydratase activity and a CYB5R3 protein having electron transfer partner activity; or

[0072] (ii) the fusion protein as described herein above and elsewhere.

[0073] The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect. The fusion protein is as described above with respect to the third aspect.

[0074] For example, in some embodiments, the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 (modified hSAND) or an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 (modified CYB5R3) or an amino acid sequence having at least 80% identity to SEQ ID NO: 8.

[0075] In a ninth aspect, there is provided an immunogenic composition comprising:

[0076] (i) one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity;

[0077] (iii) the polynucleotide encoding the fusion protein as described herein above and elsewhere; or

[0078] (iv) the vector as described herein above and elsewhere.

[0079] The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect. The one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein are as defined above with respect to the second aspect. The polynucleotide encoding the fusion protein is as described above with respect to the fourth aspect. The vector is as defined above with respect to the fifth and sixth aspects.

[0080] For example, in some embodiments, the one or more polynucleotides comprises the nucleotide sequence set out in SEQ ID NO: 10 (modified hSAND) and the nucleotide sequence set out in SEQ ID NO: 13 (modified CYB5R3). As a further example, the polynucleotide encoding the fusion protein has the nucleotide sequence set out in SEQ ID NO: 15.

[0081] In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" is employed herein to refer to those materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject extract from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; sterile distilled water; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa. : Mack Publishing Co., 1995), which discloses typical carriers and conventional methods of preparing pharmaceutical formulations.

[0082] Diluents, wetting agents, fillers, extenders, binders, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, perfuming agents, sweeteners, preservatives and antioxidants can also be present in the compositions.

[0083] The immunogenic composition may be a liquid, gel, or gel-like formulation (e.g., suspensions, solutions, emulsions, aqueous solutions).

[0084] As will be appreciated by one skilled in the art, the formulation may vary depending on the type of therapeutic agent, for example whether a polynucleotide or a polypeptide is being administered. The skilled person will be able to select the appropriate formulation.

[0085] In a tenth aspect, there is provided an antiviral vaccine comprising at least one ribonucleic (RNA) polynucleotide having an open reading frame encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity. The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect. The at least one ribonucleic (RNA) polynucleotide is as defined above with respect to the second aspect.

[0086] For example, in some embodiments, the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 (modified hSAND) or an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 (modified CYB5R3) or an amino acid sequence having at least 80% identity to SEQ ID NO: 8.

[0087] The term "antiviral vaccine", as used herein, refers to a therapeutic agent that can be administered as a prophylactic agent to prevent viral infection. The skilled person will, however, recognise that the antiviral vaccine may also be administered during a viral infection to treat the viral infection.

[0088] In some embodiments, the at least one RNA polynucleotide is a messenger RNA (mRNA). In some embodiments, the at least one RNA polynucleotide is an mRNA comprising a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly(A) tail. In some embodiments, the 5' cap is a 5' terminal cap 7mG(5')ppp(5')NlmpNp.

[0089] An "open reading frame" is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.

[0090] The antiviral vaccine may be formulated in any suitable manner as known by one skilled in the art (see, for example, Zeng C, Zhang C, Walker PG, Dong Y. Formulation and Delivery Technologies for mRNA Vaccines. Curr Top Microbiol Immunol. 2022;440:71-110. doi: 10.1007 / 82_2020_217. PMID: 32483657; PMCID: PMC8195316).

[0091] In some embodiments, the antiviral vaccine is formulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a noncationic lipid, a sterol, and a PEG-modified lipid; optionally wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid and the sterol is a cholesterol. n some embodiments, a lipid nanoparticle has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, a lipid nanoparticle has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the lipid nanoparticle has a polydispersity value of less than 0.4 (e.g., less than 0.3, 0.2 or 0.1). In some embodiments, the nanoparticle has a net neutral charge at a neutral pH value.

[0092] In some embodiments, the antiviral vaccine further comprises an adjuvant. Adjuvants, generally, are agents that enhance the immune response to a drug or vaccine. In some embodiments, the adjuvant is selected from the group consisting of aluminium-based adjuvants (such as aluminium phosphate, aluminium hydroxide, and phosphate aluminium hydroxide), MF59, AS03, TLR agonist molecule-based adjuvants (such as AS04 and CpG ODN 1018), AS01, virus-like particles, virosomes, PLA / PLGA, Matrix M, and flagellin. Suitable adjuvants are known to the skilled person (see, for example, Zhao, T., Cai, Y., Jiang, Y. et al. Vaccine adjuvants: mechanisms and platforms. Sig Transduct Target Ther 8, 283 (2023), and Facciola A, Visalli G, Lagana A, Di Pietro A. An Overview of Vaccine Adjuvants: Current Evidence and Future Perspectives. Vaccines (Basel). 2022 May 22;10(5):819).

[0093] In some embodiments, the antiviral vaccines described herein are chemically modified. In other embodiments, the antiviral vaccines are unmodified. The term "chemically modified" refers to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5'-terminal mRNA cap moieties.

[0094] In an eleventh aspect, there is provided an antiviral vaccine comprising at least one RNA polynucleotide having an open reading frame encoding the fusion protein as described herein above and elsewhere.

[0095] The fusion protein is as defined above with respect to the third aspect. The at least one ribonucleic (RNA) polynucleotide is as defined above with respect to the fourth aspect. Features of the antiviral vaccine as described in the tenth aspect are also applicable to this aspect. For example, in some embodiments, the at least one RIMA polynucleotide having an open reading frame encodes a fusion protein comprising the amino acid sequence set out in SEQ ID NO: 17 (fusion protein) or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17.

[0096] In a twelfth aspect, there is provided the fusion protein as described herein above and elsewhere (and particularly in the third aspect of the invention), or the polynucleotide as described herein above and elsewhere (and particularly in the fourth aspect of the invention), or the vector as described herein above and elsewhere (and particularly in the fifth and sixth aspects of the invention), or the cell as described herein above and elsewhere (and particularly in the seventh aspect of the invention), or the immunogenic composition as described herein above and elsewhere (and particularly in the eighth and ninth aspects of the invention), or the antiviral vaccine as described herein above and elsewhere (and particularly in the tenth and eleventh aspects of the invention) for use in the prevention or treatment of a viral infection in a subject.

[0097] In a thirteenth aspect, there is provided a ferritin nanocage presenting on its surface an hSAND protein having nucleotide dehydratase activity. In some embodiments, the ferritin nanocage comprises at least one monomer having the amino acid sequence set out in SEQ ID NO: 19.

[0098] Also provided is a ferritin nanocage presenting on its surface a CYB5R3 protein having electron transfer partner activity. In some embodiments, the ferritin nanocage comprises at least one monomer having the amino acid sequence set out in SEQ ID NO: 20.

[0099] Also provided is a ferritin nanocage presenting on its surface an hSAND protein having nucleotide dehydratase activity and a CYB5R3 protein having electron transfer partner activity. In some embodiments, the ferritin nanocage comprises at least one monomer having the amino acid sequence set out in SEQ ID NO: 19 and at least one monomer having the amino acid sequence set out in SEQ ID NO: 20.

[0100] Also provided is the ferritin nanocage as described herein for use in the prevention or treatment of a viral infection in a subject. Ferritin nanocages and methods for producing therapeutic ferritin nanocages are known to the skilled person (see Ebrahimi K. "Ferritin as a Platform for Creating Antiviral Mosaic Nanocages: Prospects for Treating COVID-19." Chembiochem. 2021 Apr 16;22(8): 1371-1378).

[0101] The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect. The medical use is as defined above with respect to the first aspect.

[0102] In a fourteenth aspect, there is provided a method of producing ddhCTP, wherein the method comprises: providing a cell in vitro with an hSAND protein having nucleotide dehydratase activity and a CYB5R3 protein having electron transfer partner activity, thereby producing ddhCTP in the cell.

[0103] Any suitable cell may be used to produce the ddhCTP. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell or a mammalian cell. In some embodiments, the cell is a bacterial cell, such as an E. coli cell.

[0104] In some embodiments, the hSAND protein and the CYB5R3 protein are provided as a fusion protein. The description of the fusion protein in the above aspects is equally applicable to this aspect.

[0105] Any suitable method may be used to provide the cell with the hSAND protein and the CYB5R3 protein. In some embodiments, providing a cell in vitro with an hSAND protein and a CYB5R3 protein involves providing the cell with one or more polynucleotides encoding the hSAND protein and the CYB5R3 protein (as described herein above and elsewhere) or a vector comprises said one or more polynucleotides (as described herein above and elsewhere) such that the hSAND protein and the CYB5R3 protein are expressed in the cell. In some embodiments, providing a cell in vitro with an hSAND protein and a CYB5R3 protein involves providing the cell with a polynucleotide encoding a fusion protein (as described herein above and elsewhere) or a vector comprising said polynucleotide (as described herein above and elsewhere) such that the hSAND protein and the CYB5R3 protein are expressed in the cell. In certain embodiments, expression of the hSAND protein and the CYB5R3 protein is induced in the cell. In some embodiments, the method further comprises purifying the ddhCTP produced by the method. Any suitable method may be used to purify the ddhCTP from the cell. One suitable method is provided in Gizzi, A.S., Grove, T.L., Arnold, J. J. et al. ("A naturally occurring antiviral ribonucleotide encoded by the human genome." Nature 558, 610-614 (2018)).

[0106] The hSAND protein and the CYB5R3 protein are as defined above with respect to the first aspect. The one or more polynucleotides are as defined above with respet to the second aspect. The fusion protein is as defined above with respect to the third aspect. The polynucleotide encoding the fusion protein is as defined above with respect to the fourth aspect. The vector is as described above with respect to the fifth and sixth aspects. The host cell is as defined above with respect to the seventh aspect.

[0107] Methods of Treatment and Uses

[0108] The above description provides products and compositions for use in the prevention or treatment of a viral infection. It is also intended to provide methods of preventing or treating a viral infection by administering said products and compositions to a subject.

[0109] Accordingly, there is provided a method of preventing or treating a viral infection in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent comprises:

[0110] (i) an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity;

[0111] (ii) one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity;

[0112] (iii) the fusion protein as described herein above and elsewhere;

[0113] (iv) the polynucleotide encoding the fusion protein as described herein above and elsewhere;

[0114] (v) the vector as described herein above and elsewhere;

[0115] (vi) the cell as described herein above and elsewhere;

[0116] (vii) the immunogenic composition as described herein above and elsewhere; or (viii) the antiviral vaccine as described herein above and elsewhere; or (ix) the ferritin nanocage as described herein above and elsewhere.

[0117] The description above is equally applicable to these methods of treatment.

[0118] Administration is typically by the parenteral route. When administered parenterally, the therapeutic agent may be administered via various routes, including intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, transdermal absorption, etc.

[0119] The therapeutic agent may be administered as a single dose or in multiple doses. The therapeutic agent may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the subject. One skilled in the art will recognise that the dose can be appropriately adjusted according to the type of therapeutic agent being administered (i.e., polynucleotide, polypeptide, vector, etc).

[0120] The term "therapeutically effective amount" as used herein refers to an amount of the therapeutic agent that, when administered to a subject, is sufficient to prevent, reduce the risk of developing, delay the onset of, slow the progression of or cause regression of the viral infection, or to alleviate to some extent the viral infection or one or more symptoms or complications thereof, at least in some fraction of the subjects taking that agent. The term "therapeutically effective amount" also refers to an amount of the therapeutic agent that is sufficient to elicit the biological or medical response of a cell, tissue, organ, system, animal or human which is sought by a researcher, veterinarian, medical doctor or clinician. The therapeutically effective amount may vary depending on such factors as the viral infection being treated, the particular agent being administered, the size of the subject, or the severity of the viral infection. One skilled in the art may empirically determine the effective amount of a particular agent without necessitating undue experimentation.

[0121] In some embodiments, the method further comprises co-administering an additional therapeutic agent to the subject. The additional therapeutic agent may be administered by any route, such as oral or parenteral administration. The additional therapeutic agent may be administered sequentially or simultaneously with the therapeutic agent. In some embodiments, the additional therapeutic agent is a chemically-modified CTP, such as 2'-O-Methylcytidine-5'-triphosphate, 2'-Fluoro-2'-deoxycytidine-5'-triphosphate, 5-Methylcytidine-5'-triphosphate, N4- Ethyl-cytidine-5'-triphosphate, N4-Methyl-cytidine-5'-triphosphate, N4-Acetyl- cytidine-5'-triphosphate, 2'-O-Methylcytidine-5'-triphosphate, y-[2'(2- Benzothiazoyl)-6'-hydroxybenzothiazole]-cytidine-5'-triphosphate, 6-Aza-CTP, 5- Bromo-CTP, and 5-azacytidine 5'-triphosphate. In some embodiments, the additional therapeutic agent is 6-Aza-CTP or 5-Bromo-CTP.

[0122] Also provided are uses of the products and compositions described herein for the manufacture of a medicament for preventing or treating a viral infection.

[0123] Accordingly, there is provided the use of:

[0124] (i) an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity;

[0125] (ii) one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity;

[0126] (iii) the fusion protein as described herein above and elsewhere;

[0127] (iv) the polynucleotide encoding the fusion protein as described herein above and elsewhere;

[0128] (v) the vector as described herein above and elsewhere;

[0129] (vi) the cell as described herein above and elsewhere;

[0130] (vii) the immunogenic composition as described herein above and elsewhere;

[0131] (viii) the antiviral vaccine as described herein above and elsewhere; or (ix) the ferritin nanocage as described herein above and elsewhere, for the manufacture of a medicament for preventing or treating a viral infection.

[0132] The description above is equally applicable to this aspect.

[0133] Further Definitions

[0134] The term "polypeptide", as used herein, refers to any peptide-bond-linked polymer of amino acids, regardless of size, length, secondary and tertiary structure, number of subunits or post-translational modification. Thus, the term "polypeptide" is to be understood as covering the terms "peptide", "protein", "amino acid chain", and "amino acid sequence". Polypeptides of the invention may include at least one chemical modification, such as lipidation, glycosylation and phosphorylation. Any of the polypeptides according to the invention may be isolated polypeptides. The term "isolated polypeptide", as used herein, refers to a polypeptide that is in a form or environment that does not occur in nature, such as (1) any polypeptide that does not naturally occur, (2) any polypeptide that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any polypeptide that is manually modified by man with respect to that polypeptide as found in nature in admixture with other components, such as other polypeptides, secondary metabolites, salts, et alia, or (4) any polypeptide modified by increasing the amount of the polypeptide in relation to other components with which it is naturally associated.

[0135] The term "amino acid", as used herein, refers to organic compounds containing the functional groups amine (-NH2) and carboxylic acid (-COOH) and its zwitterions, typically and preferably, along with a side chain specific to each amino acid. The term "amino acid" typically and preferably includes amino acids that occur naturally, such as proteinogenic amino acids (produced by RNA- translation), non-proteinogenic amino acids (produced by other metabolic mechanisms, e.g., posttranslational modification), standard or canonical amino acids (that are directly encoded by the codons of the genetic code) and nonstandard or non-canonical amino acids (not directly encoded by the genetic code). Naturally occurring amino acids include non-eukaryotic and eukaryotic amino acids. The term "amino acid", as used herein, also includes unnatural amino acids that are chemically synthesized. Moreover, the term covers alpha- (a-), beta- (b- ), gamma- (g-) and delta- (d-) etc. amino acids as well as mixtures thereof in any ratio, and, if applicable, any isomeric form of an amino acid, i.e., its D- and L- stereoisomers (alternatively addressed by the (R) and ( S) nomenclature) as well as mixtures thereof in any ratio, such as in a racemic ratio of 1: 1. Amino acids in this invention are typically in L-configuration. The term "D-stereoisomer", "L- stereoisomer", "D-amino acid" or "L-amino acid" refers to the chiral alpha carbon of the amino acids. Amino acids can include modifications and / or attached compounds and residues, for example residues used for peptide synthesis, such as Boc, Fmoc or both.

[0136] In the description above, the term "sequence identity" is used to refer to the similarity of two sequences. For the purpose of this invention, it is defined here that in order to determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment with a second amino or nucleic acid sequence). The nucleotide / amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e. overlapping positions) x 100). Generally, the two sequences are the same length. A sequence comparison is typically carried out over the entire length of the two sequences being compared.

[0137] The skilled person will be aware of the fact that several different computer programs are available to determine the identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two nucleic acid sequences is determined using the sequence alignment software Clone Manager 9 (Sci-Ed software - www.scied.com) using global DNA alignment; parameters: both strands; scoring matrix: linear (mismatch 2, OpenGap 4, ExtGap 1).

[0138] Alternatively, the percent identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. A further method to assess the percent identity between two amino acid or nucleic acid sequences can be to use the BLAST sequence comparison tool available on the National Center for Biotechnology Information (NCBI) website (www.blast.ncbi.nlm.nih.gov), for example using BLASTn for nucleotide sequences or BLASTp for amino acid sequences using the default parameters.

[0139] The term "polynucleotide" as used herein refers to any linear or sequential array of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleotides and derivatives thereof. This term may be used interchangeably with "nucleic acid" and "nucleic acid molecule". Polynucleotides may include derivatised or modified nucleotides and nucleosides such as, without limitation, halogenated nucleotides such as, but not limited to, 5-bromouracil and derivatized nucleotides such as nucleotides identified by biotin.

[0140] Any polynucleotide of the invention may be an isolated polynucleotide. The term "isolated polynucleotide", as used herein, covers, for example, (a) a DNA that has the sequence of part of a naturally occurring genomic molecule, but not flanked by at least one of the sequences flanking that part of the molecule in the genome of the species in which it occurs naturally and; (b) an isolated polynucleotide that has been incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote such that the resulting genomic DNA or vector is not identical to the naturally occurring DNA from which the nucleic acid isolate was obtained; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein; and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally occurring. Isolated nucleic acid molecules of the present invention can include, for example, naturally occurring allelic variants as well as isolated nucleic acid molecules modified by nucleotide deletions, insertions, inversions or substitutions. Isolated nucleic acid molecules of the invention can be DNA, RNA, or nucleic acid analogues. Nucleic acid analogues can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2'- deoxycytidine or 5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. Polynucleotides of the invention can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. Polynucleotides of the invention may be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3' to 5' direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Polynucleotides of the invention may be obtained by mutagenesis. For example, a reference sequence (e.g., the nucleotide sequence set out in SEQ ID NO: 10 or 13) can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology, Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992.

[0141] A skilled person will appreciate that all aspects of the invention, whether they relate to, for example, the medical uses, the polypeptides, the polynucleotides, or the fusion proteins, are equally applicable to all other aspects of the invention. In particular, aspects of the hSAND protein and CYB5R3 protein, for example, may have been described in greater detail than in other aspects of the invention, for example, the antiviral vaccine. However, the skilled person will appreciate where more detailed information has been given for a particular aspect of the invention, this information is generally equally applicable to other aspects of the invention.

[0142] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0143] Brief Descriotion of the Drawinas

[0144] Embodiments of the invention will now be further described by way of example only and with reference to the accompanying drawings.

[0145] Figure 1. CYB5R3 activates hSAND. (a) The structure of hSAND showing the catalytic [4Fe-4S](Cys)s cluster coordinated to SAM analogue S-adenosyl homocysteine (SAH) (PDB Code: 6Q2Q). (b) The catalytic conversion of CTP to ddhCTP by hSAM requires two electrons, (c) The AlphaFold predicted CYB5R3 and hSAND structures show an N-terminus hydrophobic membrane anchoring domain, (d) Purified CYB5R3 is catalytic active and catalysed reduction of NADH, providing electrons for the oxidation of the artificial electron acceptor DCIP. (e) CYB5R3 activated hSAND in vitro. (+Ctrl), positive control, in which the CYB5R3 / NADH system was replaced by the chemical-reducing agent sodium dithionite. (- Ctrl-1) Negative control-1 contains includes CBY5R3 / NADH. (- Ctrl-2) Negative control-2 contains NADH, SAM, and CTP. (RXN) Reaction contains all components, hSAND, CTP, SAM, CYB5R3, and NADH. (f) The amount of ddhCTP formed is dependent on the amount of NADH present. The number next to each chromatogram shows the NADH / CTP ratio, (e, f) Each trace shows the ddhCTP [M-H]'1(m / z) extracted ion chromatogram =464.0 ± 0.1. All measurements were repeated three times with a biologically independent sample to confirm reproducibility.

[0146] Figure 2. CYB5R3 is specific to hSAND. (a) CYB5R3 does not activate TtSAND in vitro. (+Ctrl), positive control, in which the CYB5R3 / NADH system was replaced by the chemical-reducing agent sodium dithionite. (- Ctrl-1) Negative control-1 contains CBY5R3 / NADH only. (- Ctrl-2) Negative control-2 contains NADH, SAM, and CTP. (RXN) Reaction contains all components, TtSAND, CTP, SAM, CYB5R3, and NADH. (b) In vitro, hSAND / CYB5R3 show a 30-40% higher activity than hSAND / CYB5R2. (c) A schematic presentation of the pull-down assay to study the interaction of hSAND-CYB5R3. (d) Western blot analysis of hSAND was collected after the pull-down assay to determine the interaction with CYB5R3 or CYB5R2. +C, is purified hSAND; W1 and W10 are wash 1 and wash 10; and E is elusion.

[0147] Figure 3. CYB5R3-hSAND shows specific activity inside cells. (a-g) Targeted metabolomics studies of (a, and c-g) ddhCTP formation and (b) ddhUTP formation in E. coli cells expressing a SAND and a CYB5R member as compared to cells expressing hSAND, hSAND-Vl or TtSAND alone, (g) Targeted metabolomic studies of ddhCTP in HEK293 cells. Cells were transfected with CYB5R3 / hSAND plasmids or with hSAND plasmid alone. Metabolites were extracted, and the formation of ddhCTP was measured using HR LC-MS. Data are an average of five independent biological samples ± standard deviation.

[0148] Figure 4. Comparison of the structure of 4 closely related members of the CYB5R family. All structures are predicted by AlfaFold. Both CYB5R1 and CYB5R3 have an N-terminus hydrophobic membrane anchoring domain. CYB4R4 has a heme domain.

[0149] Figure 5. CYB5R3 provides electrons to hSAND. (a) The UV-visible absorbance spectrum of CYB5R3 shows the absorbance spectrum of the FAD cofactor, (b) Progress curve of DCIP reduction after addition of NADH and CYB5R3. The progress curve shows the conversion of oxidized DCIP with a blue colour (a maximum at 600 nm) to reduced DCIP (colourless), (c) Formation of 5'-dA byproduct of the conversion of CTP to ddhCTP by hSAND in the presence of CYB5R3. Each trace shows the extracted ion chromatogram of 5'-dA [M+H]+ with a m / z=251.0. (d) Endpoint measurement of ddhCTP formation by hSAND in the presence of CYB5R3 at various concentrations of NADH. Data are averages of three independent measurements ± standard deviation, (e) Stacked UV-visible absorbance spectrum of CYB5R3 FAD cofactor in the presence of hSAND, CTP, SAM, and four different NADH concentrations.

[0150] Figure 6. Characterisation of CYB5R2. (a) UV-visible absorbance spectrum of CYB5R2 (1.4 pM) shows the peaks characteristic of the presence of FAD. (b) Michaelis-Menten kinetics of CYB5R2 in the presence of different concentrations of NADH. The concentration of CYB5R2 and DCIP was 0.0014 pM and 10 pM, respectively. The concentration of NADH varied from 0.25-10 pM. (c) ddhCTP formation by CYB5R2 / hSAND couple as compared to CYB5R3 / hSAND couple. Each trace shows the ddhCTP extracted ion chromatogram [M-H]-1(m / z) =464.0 ± 0.1.

[0151] Figure 7. Detection of CYB5R3 and hSAND interaction using a pull-down assay, (a) His-Ent-hSAND was incubated with Enterokinase (Ent) overnight and then, the mixture of untagged-hSAND and Ent was incubated with Ni-resin loaded with His-CBY5R3 in the presence or absence of NADH. As a control the untagged hSAND and Ent mixture was added to Ni-resin only, (b) His-CYB5R3 was mixed with Ent and the mixture was incubated with the Ni-resin loaded with hSAND. (a- b) The mixture with resin was incubated for 1-2 hours and subsequently washed 5-10 times. The proteins were eluted using elusion buffer. A sample from wash 1 (Wl) and wash 5 (W5) or wash (W10) were run together with elusion (E) and a positive control (+C), purified hSAND (a) or CYB5R3 (b). Ladder was MagicMark™ XP Western Protein Standard. Figure 8. hSAND-Vl is stable and contains a catalytically active cluster, (a) Invisible absorbance spectrum of the hSAND-Vl oxidised and reduced cluster (the hSAND-Vl and sodium dithionite concentrations were 11 pM). Total volume is 700 pl. (b) LC-MS measurement of ddhCTP by hSAND-Vl. Each trace shows the ddhCTP extracted ion chromatogram [M-H]'1(m / z) =464.0 ± 0.1. (Final concentrations: WT-hSAND = 53pM, MT-hSAND = 53pM, NADH = 10.6 mM, SAM = 21.2mM, CTP = 5.3 mM, sodium dithionite = 5.3mM), reaction volume is 660 Ml.

[0152] Figure 9. Expression of hSAND-CYB5R3 fusion protein in E. coli results in formation of soluble protein. The codon-optimized gene was purchased from Thermo fisher and cloned into pCYC-Duet-1 vector using EcoRI and Hindlll. This results in addition of 6x his tag to the N-terminus of the protein. Ni-resin was used for protein purification. Protein purification was carried out as explained for CYB5R3.

[0153] Figure 10. (a) The innate immune response two-enzyme system generating antiviral nucleoside analogue ddhCTP after a viral infection. The ANTA ddhCTP has broad-spectrum antiviral activity and boost the immune response, (b) The chemical synthesis of nucleoside analogue prodrugs (NAPs) and their conversion to ANTAs. This process has cellular toxicity. E.g. it can inhibit activity of cellular Ribonucleotide Reductase (RNR).

[0154] Figure 11. Creation of self-sufficient enzymes (SSEs). (a) The predicted structure of the hSAND-CYB5R3 fusion protein (fusion protein-00; created by linking the C-terminus of hSAND with the C-terminus of CYB5R3 using a linker peptide) producing broad-spectrum antiviral nucleoside triphosphate (ANTA), (b) The ability of the fusion protein to produce the antiviral nucleotide analogue ddhCTP as measured by LC-MS. The data show the extracted ion chromatogram of ddhCTP [M-H]-l with a m / z of 464. (Reaction) contains SSEs and NADH as a source of electrons, and (- Control) lacks the enzyme, (c) The chain-termination activity of the fusion protein can terminate the activity of viral polymerase and rescue cell growth by approximately 55% compared to the live control, which does not exhibit toxic viral RNA polymerase activity by producing a toxin protein (- viral RNA pol). (d) The chain-termination activity of the fusion protein-00 is similar to that of individual CYB5R3 and hSAND expressed together, (e) The chain-termination activity of the fusion protein-04 (created by linking the c- terminus of CYB5R3 with the N-terminus of hSAND) is close to that of individual CYB5R3 and hSAND expressed together. The + viral RNA polymerase control: cells exhibit toxic viral RNA polymerase activity by expressing the toxin LdrD protein, thereby inducing toxicity and abolishing growth.

[0155] Detailed Description of the Invention

[0156] The inventors have developed new antiviral therapeutics which can be used to generate the antiviral ANTA ddhCTP in cells through the catalytic mechanism of hSAND and its newly identified electron transfer partner CYB5R3. The therapeutic agents can be used to prevent viral infection by producing ddhCTP inside a cell before viral infection, thereby blocking early entry and replication of the virus. In addition, ddhCTP can be generated in cells to boost the immune response to existing viral infections.

[0157] Furthermore, the inventors have developed a method of producing ddhCTP in vitro to allow larger scale production of this antiviral compound.

[0158] Various modifications whether by way of addition, deletion, or substitution of features may be made to above-described embodiments to provide further embodiments, any and all of which are intended to be encompassed by the appended claims.

[0159] Example 1

[0160] The electron transfer (ET) from a partner protein to the active site of many metalloenzymes is required for their biological functions and applications in the chemical industry. The widely studied examples of enzymes requiring this intermolecular ET process include hydrogenases, nitrogenases, Cytochrome P450, and photosynthesis and respiratory complexes.1-6This intermolecular ET process is fundamental for activating several human iron-sulfur ([FeS]) enzymes and members of the radical S-adenosylmethionine (SAM) superfamily. These radical- SAM enzymes' functions are associated with immune response, and multiple human diseases such as cancer, viral infections, heart disease, and diabetes.7-9They have a highly conserved [4Fe-4S] cluster that acquires electrons from electron transfer partner proteins (ETPs) to perform critical steps in the biosynthesis of cofactors such as molybdopterin, modification of tRNA, or biosynthesis of antiviral metabolites, among others.7-9However, the ETP of these enzymes has remained elusive. Using a combination of bioinformatic, biochemical assays, cell biological methods, metabolomics, and spectroscopic techniques, a specific ETP of the human interferon-stimulated and antiviral enzyme radical-SAM dependent nucleotide dehydratase (hSAND) (also known as viperin) was identified. This finding shed light on a novel ET process associated with viral infections.

[0161] The antiviral enzyme hSAND, whose expression is induced by interferons,16uses two electrons20(Fig. lb) to catalyse the transformation of nucleoside triphosphate CTP to its antiviral and immunoregulatory analogue 3 '-deoxy-3 ',4'-didehydro- CTP (ddhCTP).21Using a multidisciplinary approach, the inventors identified CYB5R3, a member of the Cytochrome-bs reductases (CYB5R) family comprising 4 closely related proteins (CYB5R1-4), as the hSAND's ETP. Members of CYB5R are known to reduce Cytochrome b5 (Cyt-b5), a known electron transfer partner of Cyt-P450 or Cyt-c.22Members of CYB5R play a role in cancer, immune cell function, neurodegenerative disorders, diabetes, and cardiovascular disease.23-28

[0162] CYB5R3 activates hSAND in vitro

[0163] To find a potential ETP of hSAND, the inventors used the ProteinPrompt server29and predicted the possible interacting partners of all eight known human Radical- SAM enzymes. The inventors noted that four closely related members of CYB5R enzymes, each of which can localise to different cellular compartments, were predicted to interact with all eight human radical-SAM enzymes. These enzymes can generate electrons using NADH as a substrate, and all have the same structural fold for flavin adenine dinucleotide (FAD) binding (Fig. 4). Among these enzymes, CYB5R3 showed the highest score for binding to all the radical-SAM enzymes including hSAND. This enzyme has an N-terminus hydrophobic membrane anchoring domain like hSAND (Fig. 1c) and is known to localize to the ER membrane,30the primary hSAND localization site.31Therefore, the inventors hypothesized that CYB5R3 is a potential ETP of hSAND. Previous biochemical studies showed that CYB5R3 binds the FAD cofactor and generates electrons through the catalytic conversion of NADH to NAD+.32Consistently, the UV-visible absorbance spectrum of the soluble CYB5R3 lacking the N-terminus hydrophobic domain showed absorbance peaks very similar to those of fully oxidized FAD cofactor in other proteins (Fig. 5a).33-35To confirm that the purified enzyme is active, the inventors used DCIP as an artificial electron acceptor and recorded the progress curve of 2-electron reduction of DCIP (Fig. 5b) at different NADH concentrations and derived enzyme kinetics parameters (Fig. Id). The apparent Kmvalue for NADH was 0.9 ± 0.2 pM. This value is within the experimental error, similar to what was reported previously.32Therefore, the purified CYB5R3 is catalytic active. Next, the inventors tested if CYB5R3 can act as the ETP of hSAND. They used liquid chromatography-mass spectrometry (LC-MS) and recorded the formation of ddhCTP by hSAND (Fig. le). Only in the presence of all reaction components, hSAND converted CTP to ddhCTP like the positive control, in which instead of CYB5R3 and NADH an anaerobic solution of sodium dithionite was added to reduce the [4Fe-4S]2+cluster and provide electrons for catalysis. The formation of ddhCTP was concomitant with the production of 5 '- deoxyadenosine (5 '-dAH) byproduct (Fig. 5c). End-point measurements of the ddhCTP formation in the presence of ETP showed an increase in the amount of ddhCTP formed as a function of NADH concentration (Fig. If) with an apparent Kmvalue of 5.0 ± 3.2 pM (Fig. 5d). When the UV-visible absorbance spectrum was recorded at different NADH / SAM ratios in the presence of hSAND, CTP and SAM (Fig. 5e), the absorbance peak of fully oxidized FAD at 391, 460 and 485 nm was only observed at an NADH / SAM ratio of one or less. This observation suggested that the two electrons per molecule of SAM required for the catalytic conversion of CTP to ddhCTP by hSAND are provided by FAD, and when excess NADH is present (NADH / SAM > 1), the electrons generated from NADH oxidation cannot be used for catalysis and, the FAD remains in the reduced FADH2 state.

[0164] Specificity of CYB5R3 and hSAND activity

[0165] After establishing that CYB5R3 transfers two electrons to hSAND, the inventors investigated the specificity of this ET process. They first tested if CYB5R3 can provide electrons to activate the homologue of hSAND from the fungus Thielavia terrestris (TtSAND). This fungal SAND has about 40% identity with hSAND and is a radical-SAM enzyme37with the ability to perform catalytic transformation of UTP to ddhUTP.20The fungal TtSAND was not able to generate ddhUTP in the presence of CYB5R3 (Fig. 2a), thus suggesting that CYB5R3 interacts with hSAND specifically to provide electrons for the catalytic conversion of CTP to ddhCTP. Next, the inventors studied the activity of hSAND with the CYB5R2, another member of the CYB5R family. The purified R2 showed the absorbance peak characteristic of the FAD cofactor (Fig. 6a) like CYB5R3. The steady-state initial rate measurements of two-electron reduction of DCIP at different concentrations of NADH revealed Michaelis Menten kinetics behaviour for R2 (Fig. 6b) with an apparent Kmvalue of 1.1 ± 0.2. Subsequently, the inventors studied the activity of CYB5R2 with hSAND in vitro. The amount of ddhCTP formed by CYB5R2 was compared to that produced by CYB5R3 (Fig. 6c). The activity of CYB5R2 was circa 60% of that of CYB5R3 (Fig. 2b). This finding suggests that the hSAND and CYB5R3 interaction is stronger than hSAND and CYB5R3, as predicted by ProteinPrompt. To test this possibility, the inventors developed a pulldown assay (Fig. 2c), taking advantage of the fact that the N-terminus His-tag of hSAND has an enterokinase cleavage site (His-Ent-hSAND), but that of CYB5R3 or 2 does not have (His-CYB5R). The inventors first tested if their pulldown assay could detect the interaction of CYB5R3-hSAND. To this end, His-Ent-hSAND was subject to enterokinase cleavage. The untagged-hSAND was then incubated with Ni-resin previously loaded with His-CYB5R3 (Fig. 7a). Alternatively, His-CYB5R3 was mixed with enterokinase and then incubated with Ni-resin previously loaded with His-Ent-hSAND (Fig. 7b). In both cases, western blot analysis of the elution confirmed that hSAND and CYB5R3 interact physically and are co-purified. Next, the inventors repeated the experiment by adding the untagged-hSAND and enterokinase solution to the Ni-resin column previously loaded with CYB5R2 or R3 (Fig. 2d). Western blot analysis of wash and elution samples using a primary antibody specific to hSAND (Fig. 2d) confirmed that hSAND and CYB5R3 interaction was strong enough to be detected by the pull-down assay, but that of hSAND with CYB5R2 was not.

[0166] In vivo specificity of CYB5R3-hSAND activity

[0167] The inventors' biochemical data showed a specific interaction between CYB5R3 and hSAND, which was detected using the pull-down assay. To obtain insight into this specific interaction, the inventors used the AlfaFold structure of CYB5R4, in which the cytochrome b5 domain (Cyt-b5) directly interacts with the FAD-binding domain to receive electrons. Using this surface as a starting point, the inventors used HADDOCK server and predicted the complex between hSAND and CYB5R3 or TtSAND and CYB5R3, which unlike CYB5R3-hSAND does not have any activity in vitro (Fig. 2a). Comparing the structure of CYB5R4, those of hSAND in complex with CYB5R3 and TtSAND with CYB5R3 revealed a possible involvement of an hSAND surface loop (Q195GKK), absent in TtSAND, in the specific interaction between CYB5R3-hSAND. In the Cytochrome b5 domain of CYB5R4, a lysine residue (Lys25) is directly interacting with the FAD. Similarly, only in the complex between CYB5R3-hSAND is a lysine residue (Lysl97) of the Q195GKK loop in hSAND is predicted to directly interact with FAD in CYB5R3. Therefore, the inventors created a double variant (hSAND-Vl) in which two lysine residues of the loop were replaced with aspartic acid and valine, respectively. The purified hSAND-Vl had [4Fe-4S] cluster (Fig. 8a), which was reduced by sodium dithionite, and was catalytically active (Fig. 8b), generating ddhCTP. To test how the mutation affects activity and confirm hSAND-CYB5R3 specific interaction, the inventors used metabolomic studies. They first performed metabolomic studies using hSAND, TtSAND, and hSAND-Vl in E. coli cells. The inventors cotransformed cells with hSAND / CYB5R3, TtSAND / CYB5R3, hSAND / CYB5R2, hSAND-Vl / CYB5R3, hSAND / CYB5Rl, and hSAND / CYB5R4 plasmids. As a control, cells were transformed with hSAND, TtSAND, CYB5R1, CYB5R2, CYB5R3, or CYB5R4 alone. Targeted metabolomic studies of ddhCTP formation by hSAND or ddhUTP formation by TtSAND were then performed using LC-MS. The coexpression of CYB5R3-hSAND resulted in a three- to four-fold increase in the amount of ddhCTP generated as compared to cells expressing hSAND alone (Fig. 3a). However, the co-expression of CYB5R3-hSAND-Vl (Fig. 3f), CYB5R3 / TtSAND (Fig. 3b), CYB5Rl / hSAND (Fig. 3d), CYB5R2 / hSAND (Fig. 3c), or CYB5R4 / hSAND (Fig. 3e) did not show a statistically significant change in ddhUTP or ddhCTP formation, as compared to cells expressing TtSAND, hSAND, or hSAND-Vl alone. Therefore, CYB5R3 / hSAND interaction is specific and it requires the lysine residues of the hSAND surface loop (Q195GKK loop). The activity of soluble forms of hSAND and CYB5R3 was further confirmed in human HEK293 cells using targeted metabolomic studies. Cells were transfected with hSAND, or CYB5R3, or both hSAND and CYB5R3. Analysis of ddhCTP formation using high resolution (HR) LC-MS confirmed that consistent with the results obtained using E. coli cells, expression of hSAND and CYB5R3 together led to an approximately two-fold increase in the formation of ddhCTP as compared to hSAND alone (Fig. 3g), which did not show significant formation of ddhCTP relative to the background signal observed when cells were transfected with CYB5R3 alone. HEK293T cells have endogenous expression of CYB5R3. The observation that the expression of hSAND alone does not lead to an increase in ddhCTP beyond the background analytical noise suggest that co-localisation of hSAND and CYB5R3 is essential for their interaction and activity. The results together establish that CYB5R3 is the specific ETP of hSAND capable of activating the enzyme inside cells.

[0168] Methods

[0169] Chemical and reagents. All chemicals were reagent grade and were purchased from Sigma or ThermoFisher. Enterokinase from the porcine intestine (40 UN) was from Merck (E0885-40UN) and prepared as explained previously.39 Cloning and overexpression of proteins in E. coli. The cloning of human SAND (hSAND) and the fungal TtSAND was described before.20'40The soluble human SAND (expression in E. coli or in human) lacked 68 amino acid residues of the N-terminus hydrophobic domain (SEQ ID NO: 3). The soluble CYB5R3 (expression in E. coli or in human) lacked 27 amino acid residues of the N- terminus hydrophobic domain (SEQ ID NO: 7F). The E. coli codon-optimized synthetic gene for all CYB5R members was purchased from GeneArt (Life Science Tech.). The synthesis gene was cloned into the pCYC-Duet-1 expression vector using EcoRI and Hindlll restriction enzyme. The presence of correct insert was confirmed using double digestion and agarose gel electrophoresis. Overexpression of hSAND was achieved as explained previously for TtSAND. Briefly, TOPIO competed cells were transformed with hSAND-pBAD His / C plasmid. Cells were spread on LB-Agar plate containing 100 pg / ml ampicillin and incubated overnight at 37°C. Next day, a single colony was picked and inoculated in 50 ml LB media supplemented with 100 pg / ml ampicillin. Cells were grown overnight in a shaker (ThermoFisher) (37°C and 185 rpm) for approximately 18 hours and subsequently inoculated in 2 L flask containing 500 ml TB medium. After the OD at 600 nm reached a value between 0.5-0.8 expression of protein was induced by addition of 0.08% arabinose (final concentration) and the growth was continued (37 C and 185 rpm) for another 7 hours. Finally, cells were collected by centrifugation at 3750 rpm for 20 minutes and stored in -80°C freezer for purification of proteins.

[0170] For expression of CYB5R enzymes, BL21 E. coli cells were transformed with the pCYC-Duet-1 plasmid encoding of the enzymes. Cells were spread on LB-agar plates supplemented with 25 pg / ml chloramphenicol and the plates were incubated at 37°C overnight. The next day, a single colony was picked and inoculated into a small flask containing 50 mL LB medium and 25 pg / ml chloramphenicol. The culture was incubated in a shaker / incubator (at 37°C, 185 rpm) overnight for approximately 18 hours. The next day, the overnight culture was inoculated into 2 L flask containing 500 ml TB medium. Protein production was induced by the addition of 1 mM isopropyl p-d-l-thiogalactopyranoside (IPTG) (final concentration) when the ODeoo reached 0.5 - 0.8 (after around 1.5 hours). Subsequently, the cells were grown for another 7 hours and then collected using centrifugation at 3750 rpm for 20 minutes. Collected cells were stored at - 80°C. Purification of proteins. Purification of hSAND and TtSAND was carried out as explained previously.20BL21 cells overexpressing CYB5R were removed from the freezer and thawed on ice at room temperature. The lysis buffer was 50 mM TrisBase, 300 mM NaCI, 2% (v / v) Triton X-100 and 20.0 mg lysozyme (Thermo Fisher Scientific) at pH 8.0 with the addition of 0.05 mg Phenylmethylsulphonyl Fluoride (PMSF) (Thermo Fisher Scientific) to block protease activity and 0.05 mg DNase (Sigma Aldrich). The latter was added after pH adjustment. Collected cells were divided into several centrifuge tubes, filled up with lysis buffer up to 5 mL and then vortexed briefly to ensure all cells were dissolved. Cells were lysed using sonication (Brand), which was performed in 10 cycles at 30% amplitude. Each cycle comprises of 30 seconds sonication at 30% amplitude followed with 15 seconds of break. During sonication, cells were kept on ice. Next, lysed cells were subject to centrifugation at 3750 rpm, 20 minutes, 4 °C to remove cell debris and the supernatant containing soluble proteins was collected. Purification of the CYB5R proteins was performed using His-tag affinity chromatography. A gravity column (ThermoFisher) was packed with His-Pur™ Ni-NTA resin (Thermo Fisher Scientific), 500 pL resin per 500 mL of bacterial culture. First, the resin was washed with equilibration buffer (50 mM Tris-Base, 300 mM NaCI, 10 mM Imidazole, 0.2% v / v Triton X-100 at pH 7.5) (5 steps each 1 ml of equilibration buffer). Then, the resin was mixed with supernatant containing a CYB5R protein and the mixture was incubated using a rotating mixer at 4 °C for 2 hours. This solution was then poured to the gravity column, washed with wash buffer (50 mM Tris-Base, 300 mM NaCI, 50 mM Imidazole, 0.2% v / v Triton X-100 at pH 7.5) for 5 times of the resin volume. At the end, protein was separated using elution buffer (50 mM Tris-Base, 300 mM NaCI, 500 mM Imidazole, 0.2% v / v Triton X- 100 at pH 7.5). Finally, the elution buffer was exchanged with 50 mM Tris-300 mM NaCI at pH 7.5 using a PD10 desalting column (Cytiva). All purification steps were performed under aerobic conditions.

[0171] The purity of proteins were confirmed using SDS-Page assay (SureCast™ Handcast System, Thermo Fisher Scientific) and the proteins concentrations were determined using BCA Protein Assay Kit (Millipore).

[0172] Expression of hSAND and CYB5R3 in HEK293 cells. Human embryonic kidney (HEK293) cells were cultured in a T75 flask using Dulbecco's Modified Eagle Medium (IX) (DMEM; Thermo Fisher Scientific) supplemented with 1 mM L- glutamine (Gibco), 10% fetal bovine serum (FBS; Gibco), and 1% penicillin- streptomycin (Pen / Strep; Thermo Fisher Scientific). Cells were incubated at 37°C in a humidified incubator with 5% CO2 until they reached approximately 80-90% confluency. The cells were then split and transferred into four T25 flasks, each receiving fresh media to a total volume of 5 mL. The seeding density was adjusted to 0.7 x 106cells per T25 flask. The cells were incubated at 37°C for 24 hours to allow them to adhere and spread. The following day, cell confluency was assessed, ensuring that the cells covered approximately 50-60% of each T25 flask. Two hours before transfection, the old media was replaced with fresh media DMEM (Thermo Fisher Scientific) supplemented with 2 mM L-glutamine, 10% FBS, and 1% Pen / Strep. Transfection was performed using Lipofectamine 3000 Transfection Kit (Thermo Fisher Scientific) as described below:

[0173] Reagent A: 16.5 pL of Lipofectamine™ 3000 (Thermo Fisher Scientific) was diluted in 250 pL of Opti-MEM™ (Thermo Fisher Scientific). The solution was mixed thoroughly by pipetting up and down.

[0174] Reagent B: A master mix of 14 pg of plasmid DNA was diluted in 250 pL of Opti-MEM™. To this, 22 pL of P3000™ reagent (Thermo Fisher Scientific) was added, and the solution was mixed thoroughly by pipetting. For doubled transfections, each plasmid DNA (14 pg) was added to the solution.

[0175] Reagent A (250 pL) was combined with Reagent B (250 pL), and the mixture was incubated at room temperature for 15 minutes. Following the incubation, 500 pL of the reagent mixture was added to each T25 flask. Four conditions were prepared, each for 3 repeats except GFP control.

[0176] GFP control: Transfected with a plasmid encoding GFP. hSAND-only expression: Transfected with a plasmid encoding hSAND.

[0177] NB5R3-only expression: Transfected with a plasmid encoding NB5R3.

[0178] Double expression: Co-transfected with plasmids encoding hSAND and NB5R3.

[0179] After the addition of transfection reagents, cells were incubated for 24 hours at 37°C in an incubator with 5% CO2. After 24 hours, the cells were then split and transferred all into four T75 flasks, each receiving fresh media to a total volume of 15 mL. The cells were then incubated for an additional 24 hours at 37°C in a humidified incubator with 5% CO2.

[0180] Preparation of metabolomic samples using HEK293 cells. HEK293T cells were incubated for 48 hours post transfection and metabolites were extracted. Firstly, all media was carefully removed from each flask. 25 mL of phosphate- buffered saline pH 7.5 (PBS; Gibco) was added to each flask. The flasks were gently shaken by hand to wash and remove any residual media or non-adherent cells. The PBS was then removed. Immediately after washing, 25 mL of liquid nitrogen was added to each flask to rapidly freeze the cells. Cells were left to freeze completely for one minute, ensuring uniform freezing across the flask surface. After freezing, 350 pL of dry ice-cold 100% methanol (Sigma-Aldrich) was added to each flask. An L-shaped cell spreader was used to carefully scrape the frozen cells from the flask surface. The cell suspension was then transferred into pre-chilled vials using a pipette to minimize any cell degradation. These vials were immediately placed on ice. The collected cell suspensions were centrifuged at 13,000 rpm for 10 minutes at 4°C. Following centrifugation, the supernatant was carefully transferred into new pre-labeled vials for further analysis. The DNA concentration in each vial was quantified using a NanoDrop spectrophotometer (IMPLEN). DNA concentrations were adjusted to match the sample with the lowest concentration by diluting it with 100% methanol. This ensured consistency in DNA concentrations across all samples for subsequent analyses. Then 350 pL of the sample was added to a 0.5 mL Amicon Ultra Centrifugal Filters 3K (Merck) and centrifuged at 13000rpm for 30 minutes. The resulting flowthrough was then carefully transferred to an HPLC vial (Fisher Scientific) and sealed tightly with a Polypropylene cap (Fisher Scientific). The filtered and concentrated samples were stored at -80°C and later were subject to analysis using HR LC-MS.

[0181] UV-visible spectrophotometry and enzyme kinetics measurements of CYB5R. The concentration of the purified CYB5R protein was measured using BCA assay. The purified protein was diluted to a final concentration of 1.14 pM using 50 mM MOPS buffer pH 7.0, containing 300 mM NaCI pH 7.0). DCIP (Dichlorophenolindophenol) was used as an artificial electron acceptor. The molar extinction coefficient of DCIPox (e600) used was 22,000 M'1cm-1to calculate the concentration of reduced DCIP in various NADH concentration based on Lambert- Beer law. Samples for kinetic studies consisted of a stock solution of DCIP (5 mM), a stock solution of CYB5R (1.14 pM) and freshly prepared various NADH concentrations (0.1 - 30 mM). All materials were kept on ice during measurement. The 1-mL reaction were prepared from 992 pL buffer (50 mM MOPS-300 mM NaCI pH 7.0), 2 pL DCIP, 5 pL NADH and 1 pL CYB5R all from stock solutions prepared. The measurement with UV-visible spectrophotometer was set on TimeDrive mode (continuous measurement) for 5 minutes at 600 nm. Initially, DCIP and NADH were added to the reaction in the absence of CYB5R and absorbanc was recorded for approximately 100 s to ensure baseline stability. Subsequently, CYB5R was added and decrease in absorbance due to two electron reduction of DCIP was recorded. The initial slope of the curve was used to derive the rate of DCIP reduction and NADH oxidation. Measurement for variations in NADH concentration were repeated for three times. All abs / s slope data were converted to enzyme unit from three replicates with standard deviations based on the following equation:

[0182] Enzyme Unit=(Abs / s slope22,000 ) x (1,000) x (60)0.00005 (pmol / min) / mg

[0183] The 0.00005 factor was based on the diluted 1 pL RedX enzyme (initially diluted 20 times for stock solution) to a final 1000 pL solution inside the cuvette. Eventually, the enzyme unit data were plotted against NADH concentration by using OriginLab data analysis and graphing software to obtain MM constants (KM) and maximum rate Vmax.

[0184] Pull-down assay and Western blot analysis. For the pull-down assay, three reaction groups were set up: (1) interaction between NB5R3 and hSAND, (2) interaction between NB5R2 and hSAND, and (3) a control group. hSAND was first thawed at room temperature inside a glovebox and subsequently, subjected to a 2-minute mini-centrifugation to remove any precipitate. The supernatant was then diluted to a concentration of 20 pM using an anaerobic buffer containing 50 mM MOPS, 100 mM NaCI, pH 7.0. Next, 10 pl of stock solution of enterokinase was added 1 ml of hSAND solution, and the mixture was incubated overnight at room temperature in the glovebox. The next day, at first 100 pl of Ni resin for each group was washed five times, each time with 200 pl of anaerobic buffer (50 mM MOPS 100 mM NaCI pH 7.0). The stock solution of CYB5R2 or CYB5R3 was thawed in the glove box and diluted to a concentration of 20 pM using anaerobic MOPS buffer. Next, 500 pl of CYB5R3 (sample 1), CYB5R2 (sample 2), or buffer (50 mM MOPS 100 mM NaCI pH 7.0) (Control) was added to the resin and the solution was incubated for 1 hour. After incubation, the resin was washed five times, each time with 200 pl of buffer (50 mM MOPS 100 mM NaCI pH 7.0). Subsequently, 300 pl of the overnight incubated hSAND was added to each group, followed by another 1-hour incubation. The resin was then washed 5-10 times, each time with 500 pl of buffer (50 mM MOPS 100 mM NaCI pH 7.0). The first and the last wash were collected for further analysis. Finally, the proteins were eluted using an elution buffer (50 mM Tris 300 mM NaCI 500 mM Imidazole 0.2% Triton pH 7.5). The samples collected after the first and last wash and elusion were subject to western blot analysis.

[0185] 12% SDS-PAGE gels were prepared using the SureCast Gel Handcast System (Invitrogen), according to the manufacturer's protocol. Samples and 1-3 pl of MagicMark™ XP Western Protein Standard (ThermoFisher) and 3 pl of PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (ThermoFisher) were used. Following electrophoresis, the proteins were transferred to a PVDF Membrane (ThermoFisher) using Power Blotter system (ThermoFisher). Antibody incubation was performed using iBind Flex system (ThermoFisher). The primary antibody was: RSAD2 Rabbit polyclonal Antibody (proteintech) (1: 1000) as confirmed previously using SAND (RSAD2) knockout cells,41the secondary antibody was Anti-rabbit IgG HRP-linked Antibody (Cell Signalling) (1 :2000). The membrane was imaged using the iBright imaging system (ThermoFisher).

[0186] Preparation of samples for LC-MS measurements. Anaerobic buffer, 50 mM MOPS, pH 7.0, containing 100 mM NaCI, was used to prepare stock solutions of SAM, CTP and sodium dithionite. An aliquot of chemicals was transported into the glovebox and mixed with the buffer under anaerobic conditions. The buffer was prepared a day prior to the experiments and stored in the glovebox overnight to ensure complete removal of dioxygen. Four samples were prepared. The complete reaction had 500 pL of purified hSAND solution (concentration of approximately 70 pM), 50 pL of cone NB5R3 20 pL of 700 mM SAM chloride (S-(5'-Adenosyl)-L- methionine-(S-methyl-13C) chloride), 20 pL of 175 mM CTP (Cytidine 5'- triphosphate disodium salt), and 20 pL of 175 mM Na2S2O4 (sodium dithionite). The anaerobic MOPS buffer pH 7.0 was added to reach a final volume of 660 pL. A negative control sample was prepared. In the negative control, hSAND was not added, and it was replaced with buffer and NADH. The reaction mixture was incubated anaerobically overnight. The following day, the sample was centrifuged at 13000 rpm for 3 minutes. Then 450 pL of the reaction was added to a 0.5 mL Amicon Ultra Centrifugal Filters 3K (Merck) and centrifuged at 13000rpm for 30 minutes. The resulting flowthrough was then carefully transferred to an HPLC vial (Fisher Scientific) and sealed tightly with a Polypropylene cap (Fisher Scientific). Each experiment was repeated at least two times with different batches of protein to test reproducibility.

[0187] Preparation of metabolomic samples from E. coli cells. Metabolites were extracted from three separate conditions. E. coli BL21-AI were transformed with plasmids expressing hSAND only, NB5R only, or both hSAND and NB5R3 following the manufacturer's protocol (Thermo Fischer Scientific). After transformation, cells were spread on the LB-agar plates containing either ampicillin (100 pg / ml) (hSAND or NB5R transformed cells) or ampicillin (100 pg / ml) and chloramphenicol (hSAND / NB5R double-transformed cells). A single colony from each condition was picked and inoculated into 5 mL LB medium containing 100 pg / ml ampicillin (hSAND), chloramphenicol (NB5R), or 100 pg / ml ampicillin and chloramphenicol (hSAND and NB5R). The vials were incubated in a shaker at 37 °C and 185 rpm for 24 hours. Subsequently, the mini culture was inoculated in 50 mL LB medium, and immediately 0.04% (final concentration) of L-arabinose (hSAND), 1 mM (final concentration) IPTG (NB5R), or 1 mM (final concentration) IPTG and 0.04% (final concentration) of L-arabinose (NB5R and hSAND) was added. This culture was incubated at 37 °C in a shaker 185 rpm overnight. The following day, cells were collected by centrifugation at 5000 rpm, 4 °C for 10 minutes. The medium was discarded, and cells were washed two times, each time with 50 mL ice-cold Tris buffer (50 mM Tris, 150 mM NaCI at pH 7.4) and centrifuged at 5000 rpm, 4 °C for 10 minutes to remove the buffer. After discarding the buffer, cells were stored in a -80 °C freezer. To extract metabolites, cells were thawed on ice, and 500 pL of dry-ice cold 100% methanol was added to the cell pellet in 50 ml falcon tube and mixed well. Sonication was then performed on ice for 2 minutes with 40% amplitude, each cycle lasted for 10 seconds and was followed by a 10-second break. After sonication, three cycles of 10 minutes of freeze and 10 minutes of thaw were performed using dry ice. Lysed cells were added to 1.5 mL Eppendorf tubes and centrifuge for 10 minutes at 13000 rpm to remove cell derbies and denatured proteins. After centrifugation, the supernatants were added into new 1.5 mL tubes, and the protein concentration was measured using the BOA assay. The concentrations of hSAND and hSAND / NB5R samples were adjusted to be the same. All the samples were filtered with the Amicon Ultra-15 Centrifugal Filter 3 kDa MWCO Millipore (Sigma Aldrich) and collected in recovery vials for LC-MS analysis detecting ddhCTP. All the steps were done on ice. Liquid Chromatography-Mass spectrometry (LC-MS). Measurements were performed using an Agilent InfinityLab G6160A LC / MSD iQ mass spectrometer equipped with [Agilent 1260 Infinity II] liquid chromatography system. Instrument control and data processing were performed using OpenLab CDS. The system was calibrated on the day of the analysis. Electrospray source conditions were adjusted to maximise sensitivity, and the detection mode was set to detect negative (-) ions. For each run, 50 pL of solution was injected with a flow rate of 0.2 mL / min and an oven temperature of 45 °C:

[0188] • Buffer A: 90 vol.% MeCN (Fisher, 99.9%): 10 vol.% LC-MS grade water (Fisher), 20 mM ammonium acetate, pH 7.4-7.5.

[0189] • Buffer B: 10 vol.% LC-MS grade water (Fisher), 20 mM ammonium acetate, pH 7.4-7.5.

[0190] • Column:

[0191] • Flow rate: 0.2 mL / min

[0192] • Gradient: [0-1 min]: 100:0 (A:B); [1-10 min] 100:0 (A:B) linearly changed to 10:90 (A:B); [10-15 min] 10:90 (A:B); [15: 17] linearly changed to 100:0 (A:B) and [17-35 min] 100-0 (A:B).

[0193] Analysis of the LC-MS data was performed using OpenLab CDS software.

[0194] Additional results are shown in Figure 11b, demonstrating the ability of the fusion protein to produce the antiviral nucleotide analogue ddhCTP. The data show the extracted ion chromatogram of ddhCTP [M-H]-l with a m / z of 464.

[0195] High resolution LC-MS. An Agilent 6546 LC / Q-TOF was coupled to a 1290 Infinity II Bio LC (Agilent Technologies, Santa Clara, CA) with an Agilent Jet Stream electrospray ionisation source (AJS). The instrument was run in negative ion mode with an m / z range of 100-1100 and a scan rate of 1 spectra per second in MSI mode. Source parameters were as follows; gas temperature, 225°C; gas flow, 9 L / min; Nebulizer, 30 psig; sheath gas temperature, 375 °C, sheath gas flow 12 L / min; capillary voltage, 3 kV; nozzle voltage 500 V and fragmentor voltage 175 V. For each run, 5 pL of sample was injected with a flow rate of 0.2 mL / min and an oven temperature of 45 °C:

[0196] • Buffer A: 90 vol.% MeCN (Fisher, 99.9%): 10 vol.% LC-MS grade water (Fisher), 20 mM ammonium acetate, pH 7.4-7.5.

[0197] • Buffer B: 10 vol.% LC-MS grade water (Fisher), 20 mM ammonium acetate, pH 7.4-7.5. • Column:

[0198] • Flow rate: 0.2 mL / min

[0199] • Gradient: [0-1 min]: 100:0 (A:B); [1-10 min] 100:0 (A:B) linearly changed to 10:90 (A:B); [10-15 min] 10:90 (A:B); [15: 17] linearly changed to 100:0 (A:B) and [17-35 min] 100-0 (A:B).

[0200] Example 2

[0201] Testing antiviral activity by measuring viral polymerase chain-termination reaction The inventors previously developed and described the VITAS ((Viral polymerase- Inhibition Toxin-Associated Selection) assay to measure viral RIMA polymerase chain-termination (Alharbi AF et al. Chem. Commun., 2023, 59, 5419-5422). The assay relies on activity of viral T7 RNA polymerase expressing toxin protein LdrD. If the fusion enzyme produces an antiviral nucleotide analogue, acting as a chainterminator of viral RNA polymerase, then the toxin LdrD will not be expressed, and cells will survive. This assay was modified by introducing the plasmid for the expression of CYB5R3-hSAND fusion enzyme. Briefly, E. coli BL21-AI competent cells were used and transformed with plasmid expressing fusion hSAND-CYB5R3 alone, toxin LdrD (dead control) alone, fusion hSAND-CYB5R3 and toxin LdrD together, or three plasmids, expressing CYB5R3 and hSAND separately together with toxin LdrD. To transform E. coli cells, 3 pl of 100 ng / pl stock mini-prep prepared plasmid was used. After transformation, 1% arabinose (final concentration) was added to the cells, and the cells were spread on an agar plate containing suitable antibiotics. The fusion hSAND-CYB5R3 plasmid (pBAD / His C), LdrD plasmid (pET18b), and CYB5R3 plasmid (pCYC-Duet 1) are ampicillin, kanamycin, and chloramphenicol resistant, respectively. After overnight incubation at 37 °C, a single colony was picked the next day and inoculated into 2 ml of LB media containing 0.01% arabinose. Cells were grown for an additional 5- 6 hours in a shaker (200 rpm) at 37 °C for six hours, and subsequently, the bacterial growth was recorded by measuring OD at 600 nm using 1.5 ml plastic cuvettes. The growth was plotted relative to its live control cells, i.e., cells not transformed with LdrD plasmid and therefore do not exhibiting toxic viral RNA polymerase activity.

[0202] The results are shown in Figure 11c and d. The chain-termination activity of the fusion protein can terminate the activity of viral polymerase and rescue cell growth by approximately 55% compared to the live control, which does not exhibit toxic viral RNA polymerase activity by producing a toxin protein (- viral RNA pol). The chain-termination activity of the fusion protein-00 (created by linking the C-terminus of hSAND with the C-terminus of CYB5R3 using a linker peptide) is similar to that of individual CYB5R3 and hSAND expressed together. The chain-termination activity of the fusion protein-04 (created by linking the c- terminus of CYB5R3 with the N-terminus of hSAND) is close to that of individual CYB5R3 and hSAND expressed together. The + viral RNA polymerase control: cells exhibit toxic viral RNA polymerase activity by expressing the toxin LdrD protein, thereby inducing toxicity and abolishing growth.

[0203] Testing antiviral activity in vitro by plague assay

[0204] Plaque assays are carried out according to the detailed protocols in Mendoza et al. ("Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS- CoV-2." Curr Protoc Microbiol. 2020 Jun;57(l):ecpmcl05). Briefly, HPAEpiC and Huh7 cells are respectively seeded in 48-well plates (200 pL / well) at 8xl05cells / well and grown overnight. Cells are transfected with 1-1000 ng mRNA for hSAND, CYB5R3, or an hSAND-CYB5R3 fusion protein for 24 or 48 h, and then infected with SARSCoV-2 at an MOI of 0.01. After 1 h of incubation at 37°C, the virus is removed and replaced with fresh medium. In 24, 48, and 72 h, the cell supernatants are collected, and plaque formation will be measured using two assays described by Mendoza et al.

[0205] Experiments are repeated at least 5 times. The experiments are repeated for other viruses to demonstrate broad spectrum antiviral activity.

[0206] Testing antiviral activity in vitro by RT-gPCR

[0207] RT-qPCR assays are carried out according to published protocols (Jingxin Qiao et al., SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model. Science 371, 1374-1378 (2021)). Briefly, HPAEpiC and Huh7 cells are respectively seeded in 48-well plates (200 pL / well) at 8xl05cells / well and grown overnight. Cells are transfected with 1-1000 ng mRNA for hSAND, CYB5R3, or an hSAND-CYB5R3 fusion protein for 24 or 48 h, and then infected with SARSCoV-2 at an MOI of 0.01. After 1 h of incubation at 37°C, the virus is removed and replaced with fresh medium. In 24, 48, 72 h, the cell supernatants are collected to extract viral RNA, which is subjected to RT-qPCR analysis.

[0208] TaqMan primers for SARS-CoV-2 are 5'- GGGGAACTTCTCCTGCTAGAAT-3' and 5'- CAGACATTTTGCTCTCAAGCTG-3' with SARS-CoV-2 probe FAM- TTGCTGCTGCTTGACAGATT-TAMRA-3'. The EC50 values are calculated by using a dose-response model in GraphPad Prism 8.0 software. Experiments are repeated at least 5 times.

[0209] The experiments are repeated for other viruses using known TaqMan primers and probes appropriate to those viruses to demonstrate broad spectrum antiviral activity.

[0210] Example 3

[0211] In vivo assays are carried out to test the antiviral activity of the therapeutic agent. A detailed protocol of an in vivo assay using a mouse model for SARS- CoV-2 is provided in Jingxin Qiao et al., SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model. Science371, 1374-1378(2021).

[0212] The in vivo assay is adjusted to test different viruses and different formats of the therapeutic agent (mRNA, mRNA-lipid nanoparticle, mRNA-ferritin nanocage).

[0213] In vivo mouse model of vaccination and infection

[0214] The whole angiotensin-converting enzyme 2 (ACE2) humanized mice (hACE2 mice) are purchased from Gempharmatech. Co., Ltd ( T037659). The mice are treated with mRNA or mRNA-lipid nanoparticle (mRNA-LNP) 24 h prior to infection. Then the mice are anesthetized by inhalation of isofluorane (RWD Life Science, Shenzhen, China) and infected with 2x l06TCID50 (low dose) or 5x l06TCID50 (high dose) of SARS-CoV-2 by intranasal instillation. The conditions of mice, including body weight, is monitored daily until sacrifice. Lung tissues (n = 3, per dpi group) are collected on days 1, 3 and 5 post infection. RNA is extracted from lung tissues using the TRIzol™ Reagent (Invitrogen) according to the manufacturer's instructions. Viral RNA is quantified by THUNDERBIRD® Probe One-step qRT-PCR Kit (Toyobo) according to the manufacturer's instructions using the TaqMan primers for SARS-CoV-2: 5’- GGGGAACTTCTCCTGCTAGAAT-3’ and 5’- CAGACATTTTGCTCTCAAGCTG-3’, with SARS-CoV-2 probe FAM- TTGCTGCTGCTTGACAGATT-TAMRA-3’ .

[0215] Relevant control groups are included (vehicle; control mRNA-LNP; LNP).

[0216] Sequences

[0217] SEQ ID NO: 1 - hSAND DNA sequence (expression in human) >ENA|AC017076|AC017076.14 Homo sapiens BAC clone RP11-439M11 from 2, complete sequence.

[0218] SEQ ID NO: 2 - hSAND amino acid sequence

[0219] MWVLTPAAFAGKLLSVFRQPLSSLWRSLVPLFCWLRATFWLLATKRRKQQLVLRGPDETK EEEEDPPLPTTPTSVNYHFTRQCNYKCGFCFHTAKTSFVLPLEEAKRGLLLLKEAGMEKI NFSGGEPFLQDRGEYLGKLVRFCKVELRLPSVSIVSNGSLIRERWFQNYGEYLDILAISC DSFDEEVNVLIGRGQGKKNHVENLQKLRRWCRDYRVAFKINSVINRFNVEEDMTEQIKAL NPVRWKVFQCLLIEGENCGEDALREAERFVIGDEEFERFLERHKEVSCLVPESNQKMKDS YLILDEYMRFLNCRKGRKDPSKSILDVGVEEAIKFSGFDEKMFLKRGGKYIWSKADLKLD W

[0220] SEQ ID NO: 3 - hSAND hydrophobic domain amino acid sequence

[0221] MWVLTPAAFAGKLLSVFRQPLSSLWRSLVPLFCWLRATFWLLATKRRKQQLVLRGPDETK EEEEDPPL

[0222] SEQ ID NO: 4 - Modified hSAND amino acid sequence

[0223] MPTTPTSVNYHFTRQCNYKCGFCFHTAKTSFVLPLEEAKRGLLLLKEAGMEKI

[0224] NFSGGEPFLQDRGEYLGKLVRFCKVELRLPSVSIVSNGSLIRERWFQNYGEYLDILAISC DSFDEEVNVLIGRGQGKKNHVENLQKLRRWCRDYRVAFKINSVINRFNVEEDMTEQIKAL NPVRWKVFQCLLIEGENCGEDALREAERFVIGDEEFERFLERHKEVSCLVPESNQKMKDS YLILDEYMRFLNCRKGRKDPSKSILDVGVEEAIKFSGFDEKMFLKRGGKYIWSKADLKLD W

[0225] SEQ ID NO: 5 - CYB5R3 DNA sequence

[0226] >ENA|AY341030|AY341030.1 Homo sapiens diaphorase (NADH) (cytochrome b-5 reductase) (DIA1) gene, complete cds.

[0227] SEQ ID NO: 6 - CYB5R3 amino acid sequence

[0228] MGAQLSTLGHMVLFPVWFLYSLLMKLFQRSTPAITLESPDIKYPLRLIDREIISHDTRRF RFALPSPQHILGLPVGQHIYLSARIDGNLVVRPYTPISSDDDKGFVDLVIKVYFKDTHPK FPAGGKMSQYLESMQIGDTIEFRGPSGLLVYQGKGKFAIRPDKKSNPIIRTVKSVGMIAG GTGITPMLQVIRAIMKDPDDHTVCHLLFANQTEKDILLRPELEELRNKHSARFKLWYTLD RAPEAWDYGQGFVNEEMIRDHLPPPEEEPLVLMCGPPPMIQYACLPNLDHVGHPTERCFVF

[0229] SEQ ID NO: 7 - CYB5R3 hydrophobic domain amino acid sequence

[0230] MGAQLSTLGHMVLFPVWFLYSLLMKLF

[0231] SEQ ID NO: 8 - Modified CYB5R3 amino acid sequence

[0232] MQRSTPAITLESPDIKYPLRLIDREIISHDTRRF

[0233] RFALPSPQHILGLPVGQHIYLSARIDGNLVVRPYTPISSDDDKGFVDLVIKVYFKDTHPK FPAGGKMSQYLESMQIGDTIEFRGPSGLLVYQGKGKFAIRPDKKSNPIIRTVKSVGMIAG GTGITPMLQVIRAIMKDPDDHTVCHLLFANQTEKDILLRPELEELRNKHSARFKLWYTLD RAPEAWDYGQGFVNEEMIRDHLPPPEEEPLVLMCGPPPMIQYACLPNLDHVGHPTERCFV F

[0234] SEQ ID NO: 9 - Modified hSAND DNA sequence (codon-optimised for expression in E. coli)

[0235] CAGCTAGCATGCCGACCACACCGACCAGCGTTAATTATCATTTTACCCGTCAGTGCAACT ACAAATGCGGTTTTTGTTTTCATACCGCCAAAACCAGCTTTGTTCTGCCGCTGGAAGAAG CAAAACGCGGTCTGCTGCTGCTGAAAGAAGCCGGTATGGAAAAGATTAACTTTAGCGGT GGTGAACCGTTTCTGCAGGATCGTGGTGAATATCTGGGTAAACTGGTTCGTTTTTGCAAA GTTGAACTGCGTCTGCCGAGCGTTAGCATTGTTAGCAATGGTAGCCTGATTCGTGAACGT TGGTTTCAGAATTATGGCGAGTATCTGGATATTCTGGCAATTAGCTGTGATAGCTTTGAT GAAGAAGTGAACGTTCTGATTGGTCGTGGTCAGGGTAAAAAGAATCATGTTGAAAACCT GCAAAAACTGCGTCGTTGGTGTCGTGATTATCGTGTTGCCTTTAAAATCAACAGCGTGAT CAATCGCTTTAACGTGGAAGAAGATATGACCGAGCAGATTAAAGCACTGAATCCGGTTC GTTGGAAAGTTTTTCAGTGTCTGCTGATTGAAGGTGAAAATTGTGGTGAAGATGCACTGC GTGAAGCAGAACGTTTTGTTATTGGTGATGAAGAGTTTGAACGTTTTCTGGAACGTCATA

[0236] AAGAAGTTAGCTGTCTGGTTCCGGAAAGCAACCAGAAAATGAAAGATTCCTATCTGATCC TGGATGAATACATGCGTTTTCTGAATTGCCGTAAAGGTCGTAAAGATCCGAGCAAAAGCA TTCTGGATGTTGGTGTTGAAGAAGCCATCAAATTTAGCGGTTTCGACGAGAAAATGTTTC TGAAACGTGGTGGCAAATACATTTGGAGCAAAGCAGATCTGAAACTGGATTGGTAA SEQ ID NO: 10 - Modified hSAND DNA sequence (codon-optimised for expression in humans)

[0237] CAGCTAGCATGCCCACCACACCTACCAGCGTGAACTACCACTTCACCCGGCAGTGCAAC

[0238] TACAAGTGCGGCTTCTGCTTCCACACCGCCAAGACCAGCTTTGTGCTGCCTCTGGAAGA

[0239] GGCCAAGAGAGGCCTGCTGCTGCTGAAAGAGGCCGGCATGGAAAAGATCAACTTCTCTG

[0240] GCGGCGAGCCCTTTCTGCAGGACAGAGGCGAGTATCTGGGAAAGCTCGTGCGGTTCTG

[0241] CAAGGTGGAACTGAGACTGCCCAGCGTGTCCATCGTGTCCAATGGCAGCCTGATCAGAG

[0242] AGCGGTGGTTCCAGAACTACGGCGAGTACCTGGACATCCTGGCCATCAGCTGCGACAGC

[0243] TTCGACGAGGAAGTGAACGTCCTGATCGGCAGAGGCCAGGGCAAGAAAAACCACGTGG

[0244] AAAATCTGCAGAAACTGCGGCGGTGGTGCCGGGATTACAGAGTGGCCTTCAAGATCAAC

[0245] AGCGTGATCAACCGGTTCAACGTGGAAGAGGACATGACCGAGCAGATCAAGGCCCTGAA

[0246] TCCTGTGCGGTGGAAGGTGTTCCAGTGCCTGCTGATCGAGGGCGAGAATTGTGGCGAG

[0247] GATGCCCTGAGAGAAGCCGAGAGATTTGTGATCGGCGACGAAGAGTTCGAGCGGTTCCT

[0248] GGAACGGCACAAAGAGGTGTCCTGTCTGGTCCCCGAGAGCAACCAGAAGATGAAGGAC

[0249] AGCTACCTGATCCTGGACGAGTACATGCGGTTTCTGAACTGCCGGAAGGGCAGAAAGGA

[0250] CCCCAGCAAGAGCATCCTGGATGTGGGCGTTGAGGAAGCCATCAAGTTCAGCGGCTTCG

[0251] ATGAGAAGATGTTCCTGAAGAGAGGCGGCAAGTACATCTGGTCCAAGGCCGACCTGAAG CTGGATTGGTAG

[0252] SEQ ID NO: 11 - Modified hSAND mRNA sequence

[0253] AUGCCCACCACACCUACCAGCGUGAACUACCACUUCACCCGGCAGUGCAACUACAAGU

[0254] GCGGCUUCUGCUUCCACACCGCCAAGACCAGCUUUGUGCUGCCUCUGGAAGAGGCCA

[0255] AGAGAGGCCUGCUGCUGCUGAAAGAGGCCGGCAUGGAAAAGAUCAACUUCUCUGGCG

[0256] GCGAGCCCUUUCUGCAGGACAGAGGCGAGUAUCUGGGAAAGCUCGUGCGGUUCUGCA

[0257] AGGUGGAACUGAGACUGCCCAGCGUGUCCAUCGUGUCCAAUGGCAGCCUGAUCAGAG

[0258] AGCGGUGGUUCCAGAACUACGGCGAGUACCUGGACAUCCUGGCCAUCAGCUGCGACA

[0259] GCUUCGACGAGGAAGUGAACGUCCUGAUCGGCAGAGGCCAGGGCAAGAAAAACCACG

[0260] UGGAAAAUCUGCAGAAACUGCGGCGGUGGUGCCGGGAUUACAGAGUGGCCUUCAAGA

[0261] UCAACAGCGUGAUCAACCGGUUCAACGUGGAAGAGGACAUGACCGAGCAGAUCAAGG

[0262] CCCUGAAUCCUGUGCGGUGGAAGGUGUUCCAGUGCCUGCUGAUCGAGGGCGAGAAUU

[0263] GUGGCGAGGAUGCCCUGAGAGAAGCCGAGAGAUUUGUGAUCGGCGACGAAGAGUUC

[0264] GAGCGGUUCCUGGAACGGCACAAAGAGGUGUCCUGUCUGGUCCCCGAGAGCAACCAG

[0265] AAGAUGAAGGACAGCUACCUGAUCCUGGACGAGUACAUGCGGUUUCUGAACUGCCGG

[0266] AAGGGCAGAAAGGACCCCAGCAAGAGCAUCCUGGAUGUGGGCGUUGAGGAAGCCAUC

[0267] AAGUUCAGCGGCUUCGAUGAGAAGAUGUUCCUGAAGAGAGGCGGCAAGUACAUCUGG

[0268] UCCAAGGCCGACCUGAAGCUGGAUUGGUAG

[0269] SEQ ID NO: 12 - Modified CYB5R3 DNA sequence (codon-optimised for expression in E. coli)

[0270] CGAATTCGATGCAGCGTAGCACACCGGCAATTACCCTGGAAAGTCCGGATATCAAATATC

[0271] CGCTGCGTCTGATTGATCGTGAAATTATCAGCCATGATACCCGTCGTTTTCGTTTTGCACT

[0272] GCCGAGTCCGCAGCATATTCTGGGTCTGCCGGTTGGTCAGCATATTTATCTGAGCGCAC

[0273] GTATTGATGGTAATCTGGTTGTTCGTCCGTATACACCGATTAGCAGTGATGATGATAAAG

[0274] GCTTTGTTGATCTGGTGATCAAGGTGTATTTCAAAGATACGCATCCGAAATTTCCGGCAG

[0275] GCGGTAAAATGAGCCAGTATCTGGAAAGCATGCAGATTGGTGATACCATTGAATTTCGTG

[0276] GTCCGAGCGGTCTGCTGGTTTATCAAGGTAAAGGTAAATTTGCAATCCGTCCGGATAAAA

[0277] AGAGCAATCCGATTATTCGTACCGTTAAAAGCGTTGGTATGATTGCTGGCGGTACAGGTA

[0278] TTACCCCGATGCTGCAGGTTATTCGTGCAATTATGAAAGATCCTGATGATCATACCGTTT

[0279] GCCATCTGCTGTTTGCAAATCAGACCGAAAAAGATATTCTGCTGCGTCCGGAACTGGAAG

[0280] AACTGCGTAATAAACATAGCGCACGTTTCAAACTGTGGTATACCCTGGATCGTGCACCGG

[0281] AAGCATGGGATTATGGTCAGGGTTTTGTTAACGAAGAAATGATCCGTGATCATCTGCCTC

[0282] CGCCTGAAGAAGAACCGCTGGTTCTGATGTGTGGTCCGCCTCCGATGATTCAGTATGCA

[0283] TGTCTGCCGAATCTGGATCATGTTGGTCATCCGACCGAACGTTGTTTTGTGTTTTAA

[0284] SEQ ID NO: 13 - Modified CYB5R3 DNA sequence (codon-optimised for expression in humans)

[0285] CGAATTCGATGCAGCGTAGCACACCGGCAATTACCCTGGAAAGTCCGGATATCAAATATC

[0286] CGCTGCGTCTGATTGATCGTGAAATTATCAGCCATGATACCCGTCGTTTTCGTTTTGCACT GCCGAGTCCGCAGCATATTCTGGGTCTGCCGGTTGGTCAGCATATTTATCTGAGCGCAC

[0287] GTATTGATGGTAATCTGGTTGTTCGTCCGTATACACCGATTAGCAGTGATGATGATAAAG

[0288] GCTTTGTTGATCTGGTGATCAAGGTGTATTTCAAAGATACGCATCCGAAATTTCCGGCAG

[0289] GCGGTAAAATGAGCCAGTATCTGGAAAGCATGCAGATTGGTGATACCATTGAATTTCGTG

[0290] GTCCGAGCGGTCTGCTGGTTTATCAAGGTAAAGGTAAATTTGCAATCCGTCCGGATAAAA

[0291] AGAGCAATCCGATTATTCGTACCGTTAAAAGCGTTGGTATGATTGCTGGCGGTACAGGTA

[0292] TTACCCCGATGCTGCAGGTTATTCGTGCAATTATGAAAGATCCTGATGATCATACCGTTT

[0293] GCCATCTGCTGTTTGCAAATCAGACCGAAAAAGATATTCTGCTGCGTCCGGAACTGGAAG

[0294] AACTGCGTAATAAACATAGCGCACGTTTCAAACTGTGGTATACCCTGGATCGTGCACCGG

[0295] AAGCATGGGATTATGGTCAGGGTTTTGTTAACGAAGAAATGATCCGTGATCATCTGCCTC

[0296] CGCCTGAAGAAGAACCGCTGGTTCTGATGTGTGGTCCGCCTCCGATGATTCAGTATGCA

[0297] TGTCTGCCGAATCTGGATCATGTTGGTCATCCGACCGAACGTTGTTTTGTGTTTTAA

[0298] SEQ ID NO: 14 - Modified CYB5R3 mRNA sequence

[0299] AUGCAGCGUAGCACACCGGCAAUUACCCUGGAAAGUCCGGAUAUCAAAUAUCCGCUGC

[0300] GUCUGAUUGAUCGUGAAAUUAUCAGCCAUGAUACCCGUCGUUUUCGUUUUGCACUGC

[0301] CGAGUCCGCAGCAUAUUCUGGGUCUGCCGGUUGGUCAGCAUAUUUAUCUGAGCGCAC

[0302] GUAUUGAUGGUAAUCUGGUUGUUCGUCCGUAUACACCGAUUAGCAGUGAUGAUGAUA

[0303] AAGGCUUUGUUGAUCUGGUGAUCAAGGUGUAUUUCAAAGAUACGCAUCCGAAAUUUC

[0304] CGGCAGGCGGUAAAAUGAGCCAGUAUCUGGAAAGCAUGCAGAUUGGUGAUACCAUUG

[0305] AAUUUCGUGGUCCGAGCGGUCUGCUGGUUUAUCAAGGUAAAGGUAAAUUUGCAAUCC

[0306] GUCCGGAUAAAAAGAGCAAUCCGAUUAUUCGUACCGUUAAAAGCGUUGGUAUGAUUG

[0307] CUGGCGGUACAGGUAUUACCCCGAUGCUGCAGGUUAUUCGUGCAAUUAUGAAAGAUC

[0308] CUGAUGAUCAUACCGUUUGCCAUCUGCUGUUUGCAAAUCAGACCGAAAAAGAUAUUCU

[0309] GCUGCGUCCGGAACUGGAAGAACUGCGUAAUAAACAUAGCGCACGUUUCAAACUGUG

[0310] GUAUACCCUGGAUCGUGCACCGGAAGCAUGGGAUUAUGGUCAGGGUUUUGUUAACGA

[0311] AGAAAUGAUCCGUGAUCAUCUGCCUCCGCCUGAAGAAGAACCGCUGGUUCUGAUGUG

[0312] UGGUCCGCCUCCGAUGAUUCAGUAUGCAUGUCUGCCGAAUCUGGAUCAUGUUGGUCA UCCGACCGAACGUUGUUUUGUGUUUUAA

[0313] SEQ ID NO: 15 - hSAND-CYB5R3 fusion DNA sequence (codon-optimised for expression in E. coli; Fusion protein-OO)

[0314] CGAATTCGATGCCGACCACACCGACCAGCGTTAATTATCATTTTACCCGTCAGTGCAACT

[0315] ACAAATGCGGTTTTTGTTTTCATACCGCCAAAACCAGCTTTGTTCTGCCGCTGGAAGAAG

[0316] CAAAACGCGGTCTGCTGCTGCTGAAAGAAGCCGGTATGGAAAAGATTAACTTTAGCGGT

[0317] GGTGAACCGTTTCTGCAGGATCGTGGTGAATATCTGGGTAAACTGGTTCGTTTTTGCAAA

[0318] GTTGAACTGCGTCTGCCGAGCGTTAGCATTGTTAGCAATGGTAGCCTGATTCGTGAACGT

[0319] TGGTTTCAGAATTATGGCGAGTATCTGGATATTCTGGCAATTAGCTGTGATAGCTTTGAT

[0320] GAAGAAGTGAACGTTCTGATTGGTCGTGGTCAGGGTAAAAAGAATCATGTTGAAAACCT

[0321] GCAAAAACTGCGTCGTTGGTGTCGTGATTATCGTGTTGCCTTTAAAATCAACAGCGTGAT

[0322] CAATCGCTTTAACGTGGAAGAAGATATGACCGAGCAGATTAAAGCACTGAATCCGGTTC

[0323] GTTGGAAAGTTTTTCAGTGTCTGCTGATTGAAGGTGAAAATTGTGGTGAAGATGCACTGC

[0324] GTGAAGCAGAACGTTTTGTTATTGGTGATGAAGAGTTTGAACGTTTTCTGGAACGTCATA

[0325] AAGAAGTTAGCTGTCTGGTTCCGGAAAGCAACCAGAAAATGAAAGATTCCTATCTGATCC

[0326] TGGATGAATACATGCGTTTTCTGAATTGCCGTAAAGGTCGTAAAGATCCGAGCAAAAGCA

[0327] TTCTGGATGTTGGTGTTGAAGAAGCCATCAAATTTAGCGGTTTCGACGAGAAAATGTTTC

[0328] TGAAACGTGGTGGCAAATACATTTGGAGCAAAGCAGATCTGAAACTGGATTGGGGTGGT

[0329] GGTGGTAGCGAAGCAGCAGCCAAAGGTGGTGGCGGTTCTGAAGCAGCCGCAAAAGGCG

[0330] GAGGTGGTAGTCCGGCAGTTCTGAAAGATTATCGCGAAGAAGAAAAGAAAGTTCTGAAT

[0331] GGTATGCTGCCGAAAAGCCAGGTTACCGATACACTGGCAAAAGAAGGTCCGCAGCGTAG

[0332] CACACCGGCAATTACCCTGGAAAGTCCGGATATCAAATATCCGCTGCGTCTGATTGATCG

[0333] CGAAATTATCAGCCATGATACCCGTCGTTTTCGTTTTGCACTGCCGAGTCCGCAGCATAT

[0334] TCTGGGTCTGCCGGTTGGTCAGCATATTTATCTGAGCGCACGTATTGATGGTAATCTGGT

[0335] TGTTCGTCCGTATACACCGATTAGCAGTGATGATGATAAAGGCTTTGTTGATCTGGTGAT

[0336] CAAGGTGTATTTCAAAGATACGCATCCGAAATTTCCGGCAGGCGGTAAAATGTCACAGTA

[0337] CCTGGAAAGCATGCAGATTGGTGATACCATTGAATTTCGTGGTCCGAGTGGTCTGCTGG

[0338] TTTATCAAGGTAAAGGTAAATTTGCAATCCGTCCGGATAAAAAGAGCAATCCGATTATTC GTACCGTTAAAAGCGTTGGTATGATTGCTGGCGGTACAGGTATTACCCCGATGCTGCAG GTTATTCGTGCAATTATGAAAGATCCTGATGATCATACCGTTTGCCATCTGCTGTTTGCAA ATCAGACCGAAAAAGATATTCTGCTGCGTCCGGAACTGGAAGAACTGCGTAATAAACATA GCGCACGTTTCAAACTGTGGTATACCCTGGATCGTGCACCGGAAGCATGGGATTATGGT CAGGGCTTTGTTAACGAAGAAATGATCCGTGATCATCTGCCTCCGCCTGAAGAAGAACC GCTGGTTCTGATGTGTGGTCCGCCTCCGATGATTCAGTATGCATGTCTGCCGAATCTGGA TCATGTTGGTCATCCGACCGAACGTTGTTTTGTGTTTTAA

[0339] SEQ ID NO: 16 - hSAND-CYB5R3 fusion mRNA sequence

[0340] AUGCCGACCACACCGACCAGCGU UAAU UAUCAUU U UACCCGUCAGUGCAACUACAAAU GCGGUUUUUGUUUUCAUACCGCCAAAACCAGCUUUGUUCUGCCGCUGGAAGAAGCAA AACGCGGUCUGCUGCUGCUGAAAGAAGCCGGUAUGGAAAAGAUUAACUUUAGCGGUG GUGAACCGUUUCUGCAGGAUCGUGGUGAAUAUCUGGGUAAACUGGUUCGUUUUUGCA AAGUUGAACUGCGUCUGCCGAGCGUUAGCAUUGUUAGCAAUGGUAGCCUGAUUCGUG AACGUUGGUUUCAGAAUUAUGGCGAGUAUCUGGAUAUUCUGGCAAUUAGCUGUGAUA GCUUUGAUGAAGAAGUGAACGUUCUGAUUGGUCGUGGUCAGGGUAAAAAGAAUCAUG UUGAAAACCUGCAAAAACUGCGUCGUUGGUGUCGUGAUUAUCGUGUUGCCUUUAAAA UCAACAGCGUGAUCAAUCGCUUUAACGUGGAAGAAGAUAUGACCGAGCAGAUUAAAG CACUGAAUCCGGUUCGUUGGAAAGUUUUUCAGUGUCUGCUGAUUGAAGGUGAAAAUU GUGGUGAAGAUGCACUGCGUGAAGCAGAACGUUUUGUUAUUGGUGAUGAAGAGUUU GAACGUUUUCUGGAACGUCAUAAAGAAGUUAGCUGUCUGGUUCCGGAAAGCAACCAG AAAAUGAAAGAUUCCUAUCUGAUCCUGGAUGAAUACAUGCGUUUUCUGAAUUGCCGU AAAGGUCGUAAAGAUCCGAGCAAAAGCAUUCUGGAUGUUGGUGUUGAAGAAGCCAUC AAAUUUAGCGGUUUCGACGAGAAAAUGUUUCUGAAACGUGGUGGCAAAUACAUUUGG AGCAAAGCAGAUCUGAAACUGGAUUGGGGUGGUGGUGGUAGCGAAGCAGCAGCCAAA GGUGGUGGCGGUUCUGAAGCAGCCGCAAAAGGCGGAGGUGGUAGUCCGGCAGUUCU GAAAGAUUAUCGCGAAGAAGAAAAGAAAGUUCUGAAUGGUAUGCUGCCGAAAAGCCA GGUUACCGAUACACUGGCAAAAGAAGGUCCGCAGCGUAGCACACCGGCAAUUACCCU GGAAAGUCCGGAUAUCAAAUAUCCGCUGCGUCUGAUUGAUCGCGAAAUUAUCAGCCA UGAUACCCGUCGUUUUCGUUUUGCACUGCCGAGUCCGCAGCAUAUUCUGGGUCUGCC GGUUGGUCAGCAUAUUUAUCUGAGCGCACGUAUUGAUGGUAAUCUGGUUGUUCGUCC GUAUACACCGAUUAGCAGUGAUGAUGAUAAAGGCUUUGUUGAUCUGGUGAUCAAGGU GUAUUUCAAAGAUACGCAUCCGAAAUUUCCGGCAGGCGGUAAAAUGUCACAGUACCU GGAAAGCAUGCAGAUUGGUGAUACCAUUGAAUUUCGUGGUCCGAGUGGUCUGCUGGU UUAUCAAGGUAAAGGUAAAUUUGCAAUCCGUCCGGAUAAAAAGAGCAAUCCGAUUAUU CGUACCGUUAAAAGCGUUGGUAUGAUUGCUGGCGGUACAGGUAUUACCCCGAUGCUG CAGGUUAUUCGUGCAAUUAUGAAAGAUCCUGAUGAUCAUACCGUUUGCCAUCUGCUG UUUGCAAAUCAGACCGAAAAAGAUAUUCUGCUGCGUCCGGAACUGGAAGAACUGCGU AAUAAACAUAGCGCACGUUUCAAACUGUGGUAUACCCUGGAUCGUGCACCGGAAGCA UGGGAUUAUGGUCAGGGCUUUGUUAACGAAGAAAUGAUCCGUGAUCAUCUGCCUCCG CCUGAAGAAGAACCGCUGGUUCUGAUGUGUGGUCCGCCUCCGAUGAUUCAGUAUGCA UGUCUGCCGAAUCUGGAUCAUGUUGGUCAUCCGACCGAACGUUGUUUUGUGUUUUAA SEQ ID NO: 17 - hSAND-CYB5R3 fusion amino acid sequence (Fusion protein-OO) MPTTPTSVNYHFTRQCNYKCGFCFHTAKTSFVLPLEEAKRGLLLLKEAGMEKI NFSGGEPFLQDRGEYLGKLVRFCKVELRLPSVSIVSNGSLIRERWFQNYGEYLDILAISC DSFDEEVNVLIGRGQGKKNHVENLQKLRRWCRDYRVAFKINSVINRFNVEEDMTEQIKAL NPVRWKVFQCLLIEGENCGEDALREAERFVIGDEEFERFLERHKEVSCLVPESNQKMKDS YLILDEYMRFLNCRKGRKDPSKSILDVGVEEAIKFSGFDEKMFLKRGGKYIWSKADLKLD WGGGGSEAAAKGGGGSEAAAKGGGGSPAVLKDYREEEKKVLNGMLPKSQVTDTLAKEGP QRSTPAITLESPDIKYPLRLIDREIISHDTRRFRFALPSPQHILGLPVGQHIYLSARIDGNLVVR PYTPISSDDDKGFVDLVIKVYFKDTHPKFPAGGKMSQYLESMQIGDTIEFRGPSGLLVYQGK GKFAIRPDKKSNPIIRTVKSVGMIAGGTGITPMLQVIRAIMKDPDDHTVCHLLFANQTEKDIL LRPELEELRNKHSARFKLWYTLDRAPEAWDYGQGFVNEEMIRDHLPPPEEEPLVLMCGPPPM IQYACLPNLDHVGHPTERCFVF

[0341] SEQ ID NO: 18 - Fusion peptide linker amino acid sequence GGGGSEAAAKGGGGSEAAAKGGGGSPAVLKDYREEEKKVLNGMLPKSQVTDTLAKEGP SEQ ID NO: 19 - hSAND ferritin nanocage monomer

[0342] MPTTPTSVNYHFTRQCNYKCGFCFHTAKTSFVLPLEEAKRGLLLLKEAGMEKINFSGGEPFLQ DRGEYLGKLVRFCKVELRLPSVSIVSNGSLIRERWFQNYGEYLDILAISCDSFDEEVNVLIGR GQGKKNHVENLQKLRRWCRDYRVAFKINSVINRFNVEEDMTEQIKALNPVRWKVFQCLLIE GENCGEDALREAERFVIGDEEFERFLERHKEVSCLVPESNQKMKDSYLILDEYMRFLNCRKG RKDPSKSILDVGVEEAIKFSGFDEKMFLKRGGKYIWSKADLKLDWAEAAAKEAAAKEAAAK EAAAKALEAEAAAKEAAAKEAAAKEAAAKAGGGGSMLSERMLKALNDQLNRELYSAYLYFA MAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFE AAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILF MLDKELSARAPKLPGLLMQGGESMTGGQQMGRDLYDDDDKDRWGSEMGGSHHHHHHG MASMTGGQQMMLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEI GHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYS TRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE

[0343] SEQ ID NO: 20 - CYB5R3 ferritin nanocage monomer

[0344] MQRSTPAITLESPDIKYPLRLIDREIISHDTRRFRFALPSPQHILGLPVGQHIYLSARIDGNLVV RPYTPISSDDDKGFVDLVIKVYFKDTHPKFPAGGKMSQYLESMQIGDTIEFRGPSGLLVYQG KGKFAIRPDKKSNPIIRTVKSVGMIAGGTGITPMLQVIRAIMKDPDDHTVCHLLFANQTEKDI LLRPELEELRNKHSARFKLWYTLDRAPEAWDYGQGFVNEEMIRDHLPPPEEEPLVLMCGPPP MIQYACLPNLDHVGHPTERCFVFAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAA KEAAAKAGGGGSMLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEE EIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKD YSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGES MTGGQQMGRDLYDDDDKDRWGSEMGGSHHHHHHGMASMTGGQQMMLSERMLKALND QLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPK PPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILD KLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE

[0345] SEQ ID NO: 21 - hSAND plasmid (expression in human)

[0346] GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATG CCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGC GCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCT GCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGAC ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAG TGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGT TTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGC ACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGG GCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAA CCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGC TAGCGTTTAAACTTAAGCTTGGTACCAGCTAGCATGCCCACCACACCTACCAGCGTGAAC TACCACTTCACCCGGCAGTGCAACTACAAGTGCGGCTTCTGCTTCCACACCGCCAAGACC AGCTTTGTGCTGCCTCTGGAAGAGGCCAAGAGAGGCCTGCTGCTGCTGAAAGAGGCCG GCATGGAAAAGATCAACTTCTCTGGCGGCGAGCCCTTTCTGCAGGACAGAGGCGAGTAT CTGGGAAAGCTCGTGCGGTTCTGCAAGGTGGAACTGAGACTGCCCAGCGTGTCCATCGT GTCCAATGGCAGCCTGATCAGAGAGCGGTGGTTCCAGAACTACGGCGAGTACCTGGACA TCCTGGCCATCAGCTGCGACAGCTTCGACGAGGAAGTGAACGTCCTGATCGGCAGAGGC CAGGGCAAGAAAAACCACGTGGAAAATCTGCAGAAACTGCGGCGGTGGTGCCGGGATT ACAGAGTGGCCTTCAAGATCAACAGCGTGATCAACCGGTTCAACGTGGAAGAGGACATG ACCGAGCAGATCAAGGCCCTGAATCCTGTGCGGTGGAAGGTGTTCCAGTGCCTGCTGAT CGAGGGCGAGAATTGTGGCGAGGATGCCCTGAGAGAAGCCGAGAGATTTGTGATCGGC GACGAAGAGTTCGAGCGGTTCCTGGAACGGCACAAAGAGGTGTCCTGTCTGGTCCCCGA GAGCAACCAGAAGATGAAGGACAGCTACCTGATCCTGGACGAGTACATGCGGTTTCTGA

[0347] ACTGCCGGAAGGGCAGAAAGGACCCCAGCAAGAGCATCCTGGATGTGGGCGTTGAGGA

[0348] AGCCATCAAGTTCAGCGGCTTCGATGAGAAGATGTTCCTGAAGAGAGGCGGCAAGTACA

[0349] TCTGGTCCAAGGCCGACCTGAAGCTGGATTGGTAGGAATTCGGATCCACTAGTCCAGTG

[0350] TGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTT

[0351] TAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCC

[0352] TCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT

[0353] GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG

[0354] GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGT

[0355] GGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCAC

[0356] GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG

[0357] CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCAC

[0358] GTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAG

[0359] TGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGC

[0360] CATCGCCCTGATAGACGGTmTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG

[0361] GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATA

[0362] AGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAAC

[0363] GCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCA

[0364] GCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTC

[0365] CCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCA

[0366] TAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTC

[0367] CGCCCCATGGCTGACTAATmTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTG

[0368] AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCC

[0369] CGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCA

[0370] TGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTC

[0371] GGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTC

[0372] AGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAAC

[0373] TGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGC

[0374] TGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCG

[0375] GGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGAT

[0376] GCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAA

[0377] ACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATC

[0378] TGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCG

[0379] CATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCA

[0380] TGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGAC

[0381] CGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATG

[0382] GGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTT

[0383] CTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAA

[0384] GCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTG

[0385] GGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCAT

[0386] GCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC

[0387] AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC

[0388] CAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCG

[0389] TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACA

[0390] TACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACA

[0391] TTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT

[0392] TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTC

[0393] CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACT

[0394] CAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA

[0395] GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA

[0396] TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA

[0397] ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT

[0398] CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG

[0399] GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG

[0400] CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA

[0401] ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCT

[0402] AACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACC

[0403] TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTT

[0404] TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC

[0405] TTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATG

[0406] AGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT

[0407] CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC

[0408] TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATA

[0409] ACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCC

[0410] ACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA

[0411] GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA

[0412] GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCG

[0413] TGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGC

[0414] GAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG

[0415] TTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATT

[0416] CTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT

[0417] CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGAT

[0418] AATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG

[0419] CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA

[0420] CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGA

[0421] AGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACT

[0422] CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATA

[0423] TTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTG

[0424] CCACCTGACGTC

[0425] SEQ ID NO: 22 - CYB5R3 plasmid (expression in human)

[0426] GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATG

[0427] CCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGC

[0428] GCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCT

[0429] GCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGAC

[0430] ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA

[0431] TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG

[0432] ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT

[0433] TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAG

[0434] TGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC

[0435] ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT

[0436] CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGT

[0437] TTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGC

[0438] ACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGG

[0439] GCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAA

[0440] CCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGC

[0441] TAGCGTTTAAACTTAAGCTTGGTACCAGCTAGCATGCAGAGAAGCACCCCTGCCATCACA

[0442] CTGGAAAGCCCCGACATCAAGTACCCTCTGCGGCTGATCGACAGAGAGATCATCAGCCA

[0443] CGACACCCGGCGGTTCAGATTCGCTCTGCCTTCTCCACAGCACATCCTGGGACTGCCTG

[0444] TGGGCCAGCACATCTACCTGAGCGCCAGAATCGACGGCAATCTGGTCGTGCGGCCCTAC

[0445] ACACCTATCAGCAGCGACGACGATAAGGGCTTCGTGGACCTGGTCATCAAGGTGTACTT

[0446] CAAGGACACACACCCTAAGTTCCCCGCTGGCGGCAAGATGAGCCAGTACCTGGAATCCA

[0447] TGCAGATCGGCGACACCATCGAGTTCAGAGGCCCTTCTGGCCTGCTGGTGTATCAAGGC

[0448] AAGGGCAAGTTCGCCATCAGACCCGACAAGAAGTCTAACCCCATCATCCGGACCGTGAA

[0449] GTCCGTGGGAATGATTGCTGGCGGAACCGGCATCACCCCTATGCTGCAAGTGATCCGGG

[0450] CCATCATGAAGGACCCCGACGATCACACCGTGTGCCATCTGCTGTTTGCCAACCAGACC

[0451] GAGAAGGACATCCTGCTGAGGCCCGAGCTGGAAGAACTGCGGAACAAGCACAGCGCCC

[0452] GGTTCAAGCTGTGGTACACCCTGGATAGAGCCCCTGAGGCCTGGGATTATGGCCAGGG

[0453] CTTTGTGAACGAGGAAATGATCAGGGACCATCTGCCTCCACCTGAAGAGGAACCCCTGG

[0454] TGCTGATGTGTGGCCCTCCACCTATGATCCAGTACGCCTGCCTGCCTAACCTGGACCAC GTGGGACACCCTACCGAGAGATGCTTCGTGTTCTGAGAATTCGGATCCACTAGTCCAGT

[0455] GTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCG

[0456] TTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC

[0457] CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA

[0458] ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTG

[0459] GGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCG

[0460] GTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCC

[0461] ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGAC

[0462] CGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCC

[0463] ACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTT

[0464] AGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGG

[0465] GCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAG

[0466] TGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTA

[0467] TAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA

[0468] ACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCC

[0469] CAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAG

[0470] TCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAC

[0471] CATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTC

[0472] TCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTC

[0473] TGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCT

[0474] CCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCG

[0475] CATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTAT

[0476] TCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTG

[0477] TCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGA

[0478] ACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCA

[0479] GCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGC

[0480] CGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTG

[0481] ATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCG

[0482] AAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGA

[0483] TCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCG

[0484] CGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATAT

[0485] CATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGG

[0486] ACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAA

[0487] TGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGC

[0488] CTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGAC

[0489] CAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAG

[0490] GTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATC

[0491] TCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATA

[0492] AAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGT

[0493] TTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCT

[0494] TGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACA

[0495] CAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAAC

[0496] TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC

[0497] TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

[0498] GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC

[0499] TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA

[0500] TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTT

[0501] TTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG

[0502] GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC

[0503] GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAA

[0504] GCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCT

[0505] CCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT

[0506] AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT

[0507] GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG

[0508] GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG

[0509] GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTT

[0510] TGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGG

[0511] TCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAA

[0512] ATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG

[0513] GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT

[0514] AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGA

[0515] GACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGA

[0516] GCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGA

[0517] AGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGG

[0518] CATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATC

[0519] AAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCC

[0520] GATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCA

[0521] TAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC

[0522] AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACG

[0523] GGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC

[0524] GGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTC

[0525] GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAA

[0526] CAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC

[0527] ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGAT

[0528] ACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAA

[0529] AAGTGCCACCTGACGTC

[0530] SEQ ID NO:23 - hSAND-CYB5R3 fusion protein in PCYC-Deut 1

[0531] GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGA

[0532] GATATACCATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGAATTCGATGCC

[0533] GACCACACCGACCAGCGTTAATTATCATTTTACCCGTCAGTGCAACTACAAATGCGGTmT

[0534] GTnTCATACCGCCAAAACCAGCTTTGTTCTGCCGCTGGAAGAAGCAAAACGCGGTCTGC

[0535] TGCTGCTGAAAGAAGCCGGTATGGAAAAGATTAACTTTAGCGGTGGTGAACCGTTTCTGC

[0536] AGGATCGTGGTGAATATCTGGGTAAACTGGTTCGTTTTTGCAAAGTTGAACTGCGTCTGCC

[0537] GAGCGTTAGCATTGTTAGCAATGGTAGCCTGATTCGTGAACGTTGGTTTCAGAATTATGGC

[0538] GAGTATCTGGATATTCTGGCAATTAGCTGTGATAGCTTTGATGAAGAAGTGAACGTTCTGAT

[0539] TGGTCGTGGTCAGGGTAAAAAGAATCATGTTGAAAACCTGCAAAAACTGCGTCGTTGGTG

[0540] TCGTGATTATCGTGTTGCCTTTAAAATCAACAGCGTGATCAATCGCTTTAACGTGGAAGAA

[0541] GATATGACCGAGCAGATTAAAGCACTGAATCCGGTTCGTTGGAAAGTTnTCAGTGTCTGC

[0542] TGATTGAAGGTGAAAATTGTGGTGAAGATGCACTGCGTGAAGCAGAACGTTTTGTTATTGG

[0543] TGATGAAGAGTTTGAACGTTTTCTGGAACGTCATAAAGAAGTTAGCTGTCTGGTTCCGGAA

[0544] AGCAACCAGAAAATGAAAGATTCCTATCTGATCCTGGATGAATACATGCGTTTTCTGAATTG

[0545] CCGTAAAGGTCGTAAAGATCCGAGCAAAAGCATTCTGGATGTTGGTGTTGAAGAAGCCAT

[0546] CAAATTTAGCGGTTTCGACGAGAAAATGTTTCTGAAACGTGGTGGCAAATACATTTGGAGC

[0547] AAAGCAGATCTGAAACTGGATTGGGGTGGTGGTGGTAGCGAAGCAGCAGCCAAAGGTGG

[0548] TGGCGGTTCTGAAGCAGCCGCAAAAGGCGGAGGTGGTAGTCCGGCAGTTCTGAAAGATT

[0549] ATCGCGAAGAAGAAAAGAAAGTTCTGAATGGTATGCTGCCGAAAAGCCAGGTTACCGATA

[0550] CACTGGCAAAAGAAGGTCCGCAGCGTAGCACACCGGCAATTACCCTGGAAAGTCCGGAT

[0551] ATCAAATATCCGCTGCGTCTGATTGATCGCGAAATTATCAGCCATGATACCCGTCGTTTTCG

[0552] TnTGCACTGCCGAGTCCGCAGCATATTCTGGGTCTGCCGGTTGGTCAGCATATTTATCTG

[0553] AGCGCACGTATTGATGGTAATCTGGTTGTTCGTCCGTATACACCGATTAGCAGTGATGATG

[0554] ATAAAGGCTTTGTTGATCTGGTGATCAAGGTGTATTTCAAAGATACGCATCCGAAATTTCCG

[0555] GCAGGCGGTAAAATGTCACAGTACCTGGAAAGCATGCAGATTGGTGATACCATTGAATTTC

[0556] GTGGTCCGAGTGGTCTGCTGGTTTATCAAGGTAAAGGTAAATTTGCAATCCGTCCGGATAA

[0557] AAAGAGCAATCCGATTATTCGTACCGTTAAAAGCGTTGGTATGATTGCTGGCGGTACAGGT

[0558] ATTACCCCGATGCTGCAGGTTATTCGTGCAATTATGAAAGATCCTGATGATCATACCGTTTG

[0559] CCATCTGCTGTTTGCAAATCAGACCGAAAAAGATATTCTGCTGCGTCCGGAACTGGAAGAA

[0560] CTGCGTAATAAACATAGCGCACGTTTCAAACTGTGGTATACCCTGGATCGTGCACCGGAAG

[0561] CATGGGATTATGGTCAGGGCTTTGTTAACGAAGAAATGATCCGTGATCATCTGCCTCCGCC

[0562] TGAAGAAGAACCGCTGGTTCTGATGTGTGGTCCGCCTCCGATGATTCAGTATGCATGTCTG CCGAATCTGGATCATGTTGGTCATCCGACCGAACGTTGTTTTGTGTTTTAAAAGCTTGCGG

[0563] CCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCCGCATAATCGAAATT

[0564] AATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTT

[0565] AAGTATAAGAAGGAGATATACATATGGCAGATCTCAATTGGATATCGGCCGGCCACGCGAT

[0566] CGCTGACGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCA

[0567] GCACATGGACTCGTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCA

[0568] ATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTmTTGCTGAAACCT

[0569] CAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAG

[0570] CAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTG

[0571] CTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGGCGTTTA

[0572] AGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGT

[0573] TGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAA

[0574] TCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATAGTGAAAACGGG

[0575] GGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGG

[0576] ATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCA

[0577] CCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATT

[0578] CACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAA

[0579] CACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTC

[0580] ATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGG

[0581] TCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGA

[0582] CTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAG

[0583] TGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGC

[0584] CCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCT

[0585] CATnTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTT

[0586] ATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGC

[0587] TGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCT

[0588] ATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCC

[0589] GGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCA

[0590] GTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGT

[0591] GATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCG

[0592] GCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATAC

[0593] TTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTn CCATAGGCTCCGCCCCCCTG

[0594] ACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAA

[0595] GATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTT

[0596] ACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCC

[0597] GGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCG

[0598] CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACC

[0599] ACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTA

[0600] AGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTT

[0601] CAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTmTCGTT

[0602] TTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAG

[0603] ATAAAATATTTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCC

[0604] ATACGATATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGAC

[0605] TGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACT

[0606] TACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG

[0607] CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGG

[0608] TmTCTnTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGA

[0609] GAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGT

[0610] GGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATG

[0611] TCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTG

[0612] ATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGT

[0613] TGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGC

[0614] GAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGG

[0615] CCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGC

[0616] GTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAA ATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCG GATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTAC AGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGG CGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTG GCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATG TAATTCAGCTCCGCCATCGCCGCTTCCACTmTCCCGCGTTTTCGCAGAAACGTGGCTGG CCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCC ATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGC GACTCCTGCATTAGGAAATTAATACGACTCACTATA

[0617] SEQ ID NO:24 - CYB5R3-hSAND fusion amino acid sequence (Fusion protein-04)

[0618] QRSTPAITLESPDIKYPLRLIDREIISHDTRRFRFALPSPQHILGLPVGQHIYLSARIDGNLVVR PYTPISSDDDKGFVDLVIKVYFKDTHPKFPAGGKMSQYLESMQIGDTIEFRGPSGLLVYQGK GKFAIRPDKKSNPIIRTVKSVGMIAGGTGITPMLQVIRAIMKDPDDHTVCHLLFANQTEKDIL LRPELEELRNKHSARFKLWYTLDRAPEAWDYGQGFVNEEMIRDHLPPPEEEPLVLMCGPPPM IQYACLPNLDHVGHPTERCFGGGGSPAPAPPAVLKDYREEEKKVLNGMLPKSQVTDTLAKE GPMPTTPTSVNYHFTRQCNYKCGFCFHTAKTSFVLPLEEAKRGLLLLKEAGMEKINFSGGEPF LQDRGEYLGKLVRFCKVELRLPSVSIVSNGSLIRERWFQNYGEYLDILAISCDSFDEEVNVLI GRGQGKKNHVENLQKLRRWCRDYRVAFKINSVINRFNVEEDMTEQIKALNPVRWKVFQCL LIEGENCGEDALREAERFVIGDEEFERFLERHKEVSCLVPESNQKMKDSYLILDEYMRFLNCR KGRKDPSKSILDVGVEEAIKFSGFDEKMFLKRGGKYIWSKADLKLDW

[0619] SEQ ID NO:25 - CYB5R3-hSAND fusion DNA sequence (Fusion protein-04)

[0620] ATGCAGCGTAGCACACCGGCAATTACCCTGGAAAGTCCGGATATCAAATATCCGCTGCG TCTGATTGATCGTGAAATTATCAGCCATGATACCCGTCGTTTTCGTTTTGCACTGCCGAGT CCGCAGCATATTCTGGGTCTGCCGGTTGGTCAGCATATTTATCTGAGCGCACGTATTGAT GGTAATCTGGTTGTTCGTCCGTATACACCGATTAGCAGTGATGATGATAAAGGCTTTGTT GATCTGGTGATCAAGGTGTATTTCAAAGATACGCATCCGAAATTTCCGGCAGGCGGTAAA ATGAGCCAGTATCTGGAAAGCATGCAGATTGGTGATACCATTGAATTTCGTGGTCCGAGC GGTCTGCTGGTTTATCAAGGTAAAGGTAAATTTGCAATCCGTCCGGATAAAAAGAGCAAT CCGATTATTCGTACCGTTAAAAGCGTTGGTATGATTGCTGGCGGTACAGGTATTACCCCG ATGCTGCAGGTTATTCGTGCAATTATGAAAGATCCTGATGATCATACCGTTTGCCATCTG CTGTTTGCAAATCAGACCGAAAAAGATATTCTGCTGCGTCCGGAACTGGAAGAACTGCGT

[0621] AATAAACATAGCGCACGTTTCAAACTGTGGTATACCCTGGATCGTGCACCGGAAGCATG GGATTATGGTCAGGGTTTTGTTAACGAAGAAATGATCCGTGATCATCTGCCTCCGCCTGA AGAAGAACCGCTGGTTCTGATGTGTGGTCCGCCTCCGATGATTCAGTATGCATGTCTGCC GAATCTGGATCATGTTGGTCATCCGACCGAACGTTGTTTTGGTGGTGGTGGCAGTCCGG CACCGGCACCGCCTGCAGTTCTGAAAGATTATCGCGAAGAAGAAAAGAAAGTTCTGAAT GGTATGCTGCCGAAAAGCCAGGTTACCGATACACTGGCAAAAGAAGGTCCGATGCCGAC CACACCGACCAGCGTTAATTATCATTTTACACGTCAGTGCAACTACAAATGCGGCTTTTGT TTTCATACCGCCAAAACCAGCTTTGTTCTGCCGCTGGAAGAAGCAAAACGGGGTCTGCT GCTGCTGAAAGAAGCCGGTATGGAAAAGATTAACTTTAGCGGTGGTGAACCGTTTCTGC AGGATCGTGGTGAATATCTGGGTAAACTGGTTCGTTTTTGCAAAGTTGAACTGCGTCTGC

[0622] CGAGCGTTAGCATTGTTAGCAATGGTAGCCTGATTCGTGAACGTTGGTTTCAGAATTATG GCGAATACCTGGATATTCTGGCAATTAGCTGTGATAGCTTTGATGAAGAAGTGAACGTTC TGATTGGTCGCGGTCAGGGTAAAAAGAATCATGTTGAAAATCTGCAAAAACTGCGTCGTT GGTGTCGTGATTATCGTGTTGCCTTTAAAATCAACAGCGTGATCAATCGCTTTAACGTGG AAGAAGATATGACCGAGCAGATTAAAGCACTGAATCCGGTTCGTTGGAAAGTTTTTCAGT GTCTGCTGATTGAAGGTGAAAATTGTGGTGAAGATGCACTGCGTGAAGCAGAACGTTTT GTTATTGGTGATGAAGAGTTTGAACGTTTTCTGGAACGTCATAAAGAAGTTAGCTGTCTG GTTCCGGAAAGCAACCAGAAAATGAAAGATTCCTATCTGATCCTGGATGAATACATGCGT TTTCTGAATTGCCGTAAAGGTCGTAAAGATCCGAGCAAAAGCATTCTGGATGTTGGTGTT GAAGAAGCCATCAAATTTAGCGGTTTCGACGAGAAAATGTTTCTGAAACGTGGTGGCAA

[0623] ATACATTTGGAGCAAAGCAGATCTGAAACTGGATTGGTAA References

[0624] 1. Hudson, J. M. et al. Electron Transfer and Catalytic Control by the Iron-Sulfur Clusters in a Respiratory Enzyme, E. coli Fumarate Reductase. J. Am. Chem. Soc. 127, 6977-6989 (2005).

[0625] 2. Nelson, N. & Ben-Shem, A. The complex architecture of oxygenic photosynthesis. Nat. Rev. Mol. Cell Biol. 5, 971-982 (2004).

[0626] 3. Volbeda, A. et al. Crystal Structure of the O2-Tolerant Membrane-Bound Hydrogenase 1 from Escherichia coli in Complex with Its Cognate Cytochrome b. Structure 21, 184-190 (2013).

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Claims

Claims1. An hSAND protein and a CYB5R3 protein for use in the prevention or treatment of a viral infection in a subject, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity.

2. One or more polynucleotides encoding an hSAND protein and a CYB5R3 protein for use in the prevention or treatment of a viral infection in a subject, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity.

3. The hSAND protein and the CYB5R3 protein for use according to claim 1 or the one or more polynucleotides for use according to claim 2, wherein:(i) the viral infection is selected from: an infection caused by a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-sense ssRNA virus, a negative-sense ssRNA virus, an RNA virus that reverse transcribes, or a DNA virus that reverse transcribes;(ii) the viral infection is an infection caused by a virus from a family selected from: Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Bornaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picobirnaviridae, Picobirna, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Togaviridae, or Delta; optionally wherein the viral infection is an infection caused a virus from the Coronaviridae family; optionally wherein the viral infection is caused by a severe acute respiratory syndrome (SARS) coronavirus or a MERS coronavirus; optionally wherein the viral infection is caused by SARS coronavirus 2 (SARS-CoV-2);(iii) the viral infection is a respiratory viral infection; optionally wherein the respiratory viral infection is caused by a rhinovirus, a respiratory syncytial virus (RSV), an adenovirus, a bocavirus, a coronavirus, a metapneumovirus, or a parainfluenza virus; and / or(iv) the subject is a mammal, optionally wherein the subject is human.

4. The hSAND protein and the CYB5R3 protein for use according to claim 1 or 3 or the one or more polynucleotides for use according to claim 2 or 3, wherein:(i) the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity to SEQ ID NO: 4; and / or(ii) the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 or an amino acid sequence having at least 80% identity to SEQ ID NO: 8.

5. The hSAND protein and the CYB5R3 protein for use according to claim 4 or the one or more polynucleotides for use according to claim 4, wherein:(i) the hSAND protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 4, optionally at least 95% identity to SEQ ID NO: 4, optionally at least 98% identity to SEQ ID NO: 4, optionally at least 99% identity to SEQ ID NO: 4; and / or(ii) the CYB5R3 protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 8, optionally at least 95% identity to SEQ ID NO: 8, optionally at least 98% identity to SEQ ID NO: 8, optionally at least 99% identity to SEQ ID NO: 8.

6. The one or more polynucleotides for use according to any one of claims 2- 5, wherein the one or more polynucleotides comprises the nucleotide sequence set out in SEQ ID NO: 10 and the nucleotide sequence set out in SEQ ID NO: 13.

7. The one or more polynucleotides for use according to claim 6, wherein the one or more polynucleotides are codon-optimised to enhance expression in humans.

8. A fusion protein comprising an hSAND protein and a CYB5R3 protein fused together, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity.

9. The fusion protein according to claim 8, wherein :(i) the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and(ii) the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 or an amino acid sequence having at least 80% identity to SEQ ID NO: 8.

10. The fusion protein according to claim 9, wherein:(i) the hSAND protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 4, optionally at least 95% identity to SEQ ID NO: 4, optionally at least 98% identity to SEQ ID NO: 4, optionally at least 99% identity to SEQ ID NO: 4; and / or(ii) the CYB5R3 protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 8, optionally at least 95% identity to SEQ ID NO: 8, optionally at least 98% identity to SEQ ID NO: 8, optionally at least 99% identity to SEQ ID NO: 8.

11. The fusion protein according to any one of claims 8-10, wherein the hSAND protein and the CYB5R3 protein are fused together via a linker, optionally wherein:(i) the linker is an amino acid linker, optionally wherein the linker comprises or consists of the amino acid sequence set out in SEQ ID NO: 18; and / or(ii) the linker is 1-30 amino acids in length, optionally wherein the linker is 1- 10 amino acids in length.

12. The fusion protein according to any one of claims 8-11, wherein the fusion protein comprises or consists of the amino acid sequence set out in SEQ ID NO: 17 or 24 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17 or 24.

13. A polynucleotide encoding the fusion protein according to any one of claims 8-12.

14. The polynucleotide according to claim 13 having the nucleotide sequence set out in SEQ ID NO: 15.

15. A vector comprising the polynucleotide according to claim 13 or 14.

16. A vector comprising one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein thereof has electron transfer partner activity.

17. A host cell comprising:(i) the fusion protein according to any one of claims 8-12;(ii) the polynucleotide according to claim 13 or 14; and / or(iii) the vector according to claim 15 or 16.

18. The host cell of claim 18, wherein the host cell is an antigen presenting cell, optionally wherein the host cell is a dendritic cell or a macrophage.

19. An immunogenic composition comprising:(i) an hSAND protein having nucleotide dehydratase activity and a CYB5R3 protein having electron transfer partner activity, optionally wherein the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 or an amino acid sequence having at least 80% identity to SEQ ID NO: 8; or(ii) the fusion protein according to any one of claims 8-12.

20. An immunogenic composition comprising:(i) one or more polynucleotides encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity, optionally wherein the one or more polynucleotides comprises the nucleotide sequence set out in SEQ ID NO: 10 and the nucleotide sequence set out in SEQ ID NO: 13;(iii) the polynucleotide according to claim 13 or 14; or(iv) the vector according to claim 15 or 16.

21. An antiviral vaccine comprising at least one ribonucleic (RNA) polynucleotide having an open reading frame encoding an hSAND protein and a CYB5R3 protein, wherein the hSAND protein has nucleotide dehydratase activity and the CYB5R3 protein has electron transfer partner activity.

22. The antiviral vaccine according to claim 21, wherein the hSAND protein has the amino acid sequence set out in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and wherein the CYB5R3 protein has the amino acid sequence set out in SEQ ID NO: 8 or an amino acid sequence having at least 80% identity to SEQ ID NO: 8.

23. An antiviral vaccine comprising at least one RIMA polynucleotide having an open reading frame encoding the fusion protein according to any one of claims 8- 12.

24. The antiviral vaccine according to any one of claims 21-23, wherein:(i) the antiviral vaccine is formulated in a lipid nanoparticle, optionally wherein the lipid nanoparticle comprises a cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid; optionally wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid and the sterol is a cholesterol;(ii) the antiviral vaccine further comprises an adjuvant; and / or(iii) the at least one RNA polynucleotide is a messenger RNA (mRNA) comprising a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly(A) tail, optionally wherein the 5' cap is a 5' terminal cap 7mG(5')ppp(5')NlmpNp.

25. The fusion protein according to any one of claims 8-12, or the polynucleotide according to claim 13 or 14, or the vector according to claim 15 or 16, or the cell according to claim 17 or 18, or the immunogenic composition according to claim 19 or 20, or the antiviral vaccine according to any one of claims 21-24 for use in the prevention or treatment of a viral infection in a subject.

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