A fusion protein comprising sorbitol dehydrogenase and thioredoxin

Abstract

The present invention relates to a novel form of sorbitol dehydrogenase, and its use in the treatment or prevention of disorders which result in excess sorbitol.

Description

[0001] Therapy I

[0002] Introduction

[0003] The present invention relates to a novel fusion protein comprising sorbitol dehydrogenase and thioredoxin. The present invention also relates to the use of sorbitol dehydrogenase, for example in the form of a novel fusion protein as described herein, in the treatment of a disorder associated with excess sorbitol. Also provided is a nucleic acid molecule encoding a fusion protein comprising sorbitol dehydrogenase and thioredoxin. The present invention also provides a composition comprising a fusion protein comprising sorbitol dehydrogenase and thioredoxin or a nucleic acid molecule encoding the same; and a cell or cell free system comprising a fusion protein comprising sorbitol dehydrogenase and thioredoxin or nucleic acid molecule encoding the same.

[0004] Background to the invention

[0005] Metabolites generated in the body are normally detoxified by the liver enzymes. When an enzyme deficiency is present, the body is unable to do so and the metabolites are released into circulation, where they may build up to toxic levels. Excess metabolite accumulation may be toxic to various organs, and may cause a range of symptoms, disabilities and early death.

[0006] Sorbitol dehydrogenase (“SDH”; EC 1.1.1.14) is a medium chain dehydrogenase enzyme, encoded by the SORD gene. It serves within the polyol pathway of glucose metabolism, where excess glucose are reduced to sorbitol by the enzyme aldose reductase. The sorbitol is then oxidised to fructose by sorbitol dehydrogenase.

[0007] Deficiencies in sorbitol dehydrogenase or other enzymes involved in sorbitol metabolism result in elevated levels of sorbitol, which are characterized by impairment to the retina, lens, kidney, nerve and heart. Sorbitol dehydrogenase deficiency may arise as a result of a recessive hereditary neuropathy in which a deficiency of SDH results in an inability to reduce sorbitol to fructose. Increased aldose reductase activity may also result in excess sorbitol. Sorbitol cannot pass across a cell membrane, and it therefore accumulates within the cell, leading to osmotic swelling, oxidative stress, and ultimately, tissue damage. Significantly high levels of sorbitol may cause axonal damage to peripheral nerves. Excess sorbitol may consequently lead to significant disability, loss of sensory function, neuromuscular dysfunction, neuropathic pain, and decreased mobility.

[0008] Sorbitol dehydrogenase deficiency affects approximately 1 :100,000 people worldwide.

[0009] In addition, it has been proposed that the polyol pathway is the one of the proposed mechanisms of diabetic retinopathy and associated diabetic tissue damage, directly due to sorbitol overload.

[0010] Traditional treatment of disorders associated with excess sorbitol have focused on monitoring and addressing symptoms, such as providing pain relief, orthotics or performing surgery to address deformities. However, long term outcomes are generally poor as the disease is progressive.

[0011] There are currently no drugs approved for use in the treatment of sorbitol dehydrogenase deficiency.

[0012] US2022249624 describes the detection of mutations in the SORD gene, and describes gene therapy for the treatment of inherited neuropathy. The authors also suggest administration of a SORD peptide for administration to subjects where SORD may be present at inadequate levels or may be absent.

[0013] US2023121312 describes the treatment of SORD deficiency by administration of aldose reductase inhibitors.

[0014] The present invention seeks to overcome or ameliorate problems in the art associated with disorders relating to excess sorbitol.

[0015] Summary of the invention

[0016] In a first aspect, there is provided a fusion protein comprising sorbitol dehydrogenase and thioredoxin. Suitably, the fusion protein is recombinant.

[0017] In a second aspect, there is provided a method for treatment of a disorder which results in excess sorbitol in a subject, wherein the method comprises administering a therapeutically effective amount of sorbitol dehydrogenase to a subject in need thereof. Suitably, the method comprises administering a fusion protein comprising sorbitol dehydrogenase and thioredoxin to a subject in need thereof. The present invention provides sorbitol dehydrogenase for use in the treatment of a disorder which results in excess sorbitol in a subject. Suitably, the sorbitol dehydrogenase is provided as a fusion protein comprising sorbitol dehydrogenase and thioredoxin, for example as described herein.

[0018] A disorder may be diabetic neuropathy or tissue damage due to sorbitol overload, or sorbitol dehydrogenase deficiency resulting from a genetic abnormality.

[0019] In a third aspect, the present invention provides a method for treatment of excess sorbitol in a subject, wherein the method comprises administering a therapeutically effective amount of sorbitol dehydrogenase to a subject in need thereof. Suitably, the sorbitol dehydrogenase is provided as a fusion protein comprising sorbitol dehydrogenase and thioredoxin, for example as described herein.

[0020] The present invention provides sorbitol dehydrogenase for use in the prevention or reduction of excess sorbitol in a subject. Suitably, the sorbitol dehydrogenase is provided as a fusion protein comprising sorbitol dehydrogenase and thioredoxin, for example as described herein.

[0021] In a fourth aspect, the present invention provides a nucleic acid molecule encoding a fusion protein comprising sorbitol dehydrogenase and thioredoxin. The nucleic acid molecule may be provided in an expression vector. Also provided are host cells comprising a nucleic acid molecule or expression vector of the invention. The cell may be a microbial cell, suitably a bacterial cell, suitably E. coli. The nucleic acid molecule, expression vector and / or host cell may be used in the recombinant manufacture of a fusion protein of the present invention.

[0022] In a fifth aspect of the invention, the present invention provides a composition comprising a fusion protein of the first aspect, or a host cell of the fourth aspect.

[0023] In a sixth aspect, there is provided a method of producing a fusion protein as described herein, wherein the method comprises i) providing a host cell or a cell free system comprising a nucleic acid sequence encoding the fusion protein; and ii) maintaining the host cell or cell free system under conditions suitable for expression of the fusion protein,; and optionally iii) isolating the fusion protein.

[0024] Detailed description of the invention

[0025] The present invention is based in part upon the development of a novel form of sorbitol dehydrogenase, which is suitable for recombinant manufacture and which has been shown to be effective in vivo at reducing sorbitol levels. Whilst enzyme replacement therapy has been suggested for treatment of sorbitol dehydrogenase deficiency, an enzyme suitable for therapeutic use has not been available.

[0026] The present invention is based in part upon the generation of a novel fusion protein which comprises the enzyme, sorbitol dehydrogenase, and thioredoxin. The fusion to thioredoxin has been shown to improve enzyme activity, increased stability and manufacturability. In particular a fusion protein as described herein has been shown to be capable of being concentrated.

[0027] A recombinant fusion protein comprising sorbitol dehydrogenase which is capable of being manufactured in vitro and reducing sorbitol levels in vivo, provides an effective treatment of disorders which cause or arise due to, excess sorbitol.

[0028] Definitions

[0029] A ’’nucleic acid molecule” as referred to herein refers to two or more nucleosides that are covalently linked together. The nucleosides may be ribonucleosides (such that the molecule is RNA), deoxyribonucleotides (such that the molecule is DNA) or a mixture of ribo- and deoxyribonucleosides. The nucleosides may be linked together by standard phosphodiester linkages or non-standard linkages, or a combination thereof. A nucleic acids molecule may be single-stranded or double-stranded, or may include both single-stranded regions and double-stranded regions. A nucleic acid molecule may comprise naturally occurring bases (i.e., adenine, guanine, uracil, thymine and cytosine), and may optionally include one or more modified and / or synthetic bases, such as, for example, inosine, xanthine, hypoxanthine, etc.

[0030] The term “protein” is used broadly used herein to include any proteinaceous molecule, including peptides and polypeptides, as well as protein or polypeptide fragments. A protein may comprise D- and L-amino acids, and mixtures of D- and L- amino acids.

[0031] An enzyme is a biological catalyst, capable of accelerating a reaction between one or more substrates to produce a product. The term “recombinant” (or “engineered” or “non-naturally occurring”) refers to a form of a molecule (such as a protein, or nucleic acid molecule or cell) which has been modified in a manner that would not otherwise exist in nature, or has been produced or derived from a form which has been modified. A recombinant protein may be identical to a wild type protein, but may have been produced using recombinant techniques.

[0032] “Percentage of sequence identity” refers to comparisons of nucleic acid or protein sequences, where the degree of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence for optimal alignment of the two sequences. Methods of calculating sequence identity are known and available in the art. The percentage may be calculated by determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0033] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wl), using default parameters provided.

[0034] A “reference sequence” refers to a defined sequence used as a basis for a sequence comparison. Two sequences to be compared may include a portion that is similar between the two sequences, and a sequence that is divergent. For this reason, sequence comparison may be performed over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” is therefore a conceptual segment of at least about 20 contiguous nucleotides or amino acid residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0035] A “variant” sequence may comprise one or more amino acid differences compared to a reference or wild type sequence. A “residue difference” or “mutation” refers to a change in the amino acid at a position of a sequence relative to the amino acid residue at a corresponding position in a reference sequence. Such positions may be referred to in a sequence as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based, and X refers to the residue of the reference sequence . If the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the variant polypeptide. In some embodiments, there more than one amino acid can appear in a specified residue position, the alternative amino acids can be listed in the form XnY / Z, where Y and Z represent alternate amino acid residues. In some instances, a variant sequence can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where changes are made relative to the reference sequence. A difference may be a conservative or non-conservative amino acid substitution.

[0036] A “conservative amino acid substitution” is a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids.

[0037] A “non-conservative substitution” is a substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. An amino acid / residue difference may be a “deletion”, which is the removal of one or more amino acids from the reference sequence. A deletion can be removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered imine reductase enzyme. Deletions can be directed to the internal portions and / or terminal portions of the enzyme. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0038] An “insertion” refers to the addition of one or more amino acids in a variant enzyme compared to the wild type or reference enzyme. An insertion may be in the internal portion of the enzyme, or to the carboxy or amino terminus. Therefore, an insertion as used herein includes the generation of a fusion protein, comprising an enzyme as described herein. An insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the wild type enzyme.

[0039] A “fragment” refers to a protein that has an amino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Suitably, a fragment has the desired enzyme activity, for example as described herein. A fragment may be at least 150, 170, 190, 210, 230, 250, 260, 270, or 280 amino acids long, or up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length sequence.

[0040] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is non-random and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. Suitably, the polynucleotides encoding the enzymes described herein may be codon optimized for optimal production from the host organism selected for expression. A “control sequence” as referred to herein includes any components which may be required for or useful in the expression of a polynucleotide and / or polypeptide. A control sequence may be native or foreign to the nucleic acid sequence encoding the enzyme. Examples of control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, enhancer, promoter, signal peptide sequence, and transcription terminator. Typically, a promoter, and transcriptional and translational stop signals may be operably linked to a nucleic acid molecule, for expression thereof.

[0041] A “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0042] By “operably linked” is meant that a sequence is placed in a functional relationship with a nucleic acid molecule to be expressed, to enable the sequence to directs or regulate the expression of the nucleic acid molecule.

[0043] As used herein the terms “preventing”, "treating" or “treatment” refer to any and all uses which prevent, remedy or ameliorate a condition or a symptom thereof, or otherwise hinder, retard, reduce or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever. Thus the terms “preventing” and "treating" are to be considered in their broadest context. For example, treatment does not necessarily imply that a subject is treated until total recovery, but includes any improvement or amelioration in the condition of a patient or subject, or in a symptom of the disease or condition. In conditions which display or are characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, ameliorate, prevent, hinder, retard, reduce or reverse all of said symptoms, but may remedy, ameliorate, prevent, hinder, retard, reduce or reverse one or more of said symptoms. As used herein, "amelioration" refers to the lessening of severity of at least one indicator or symptom of a condition or disease. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

[0044] An “effective amount” or “therapeutically effective amount” as used herein refers to an amount of the fusion protein sufficient to effect beneficial or desirable biological and / or clinical results. Such response may be a beneficial result, including, without limitation, amelioration, reduction, prevention, or elimination of symptoms of a disease or disorder, including glutaric aciduria type 1.

[0045] The term “subject” and “patient” are used interchangeably herein and refer to both human and non-human animals, for example as described herein.

[0046] The term “enzyme replacement therapy (ERT)” refers to medical treatment which replaces an enzyme that is deficient or absent in the body.

[0047] A “host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence, or which harbors such a nucleic acid molecule, for example because the cell is the progeny of a transfected cell. Therefore the term includes the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A host cell may include a homogenous or heterogenous population of host cells.

[0048] The N-terminus (also known as the amino-terminus, NH^-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, and derives its name due to the free amine group (-NH2) located at the end of a polypeptide. The C- terminus (also known as the carboxyl-term in us, carboxy-terminus, C-terminal tail, C- terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). An N terminal extension sequence as described herein is a sequence which is provided at the N terminal end of a sequence, which typically is not native to the sequence. A linker sequence is a sequence which joins two individual or separate sequences. A linker may ne heterologous to one or both of the sequences it is joining. A linker may join two protein sequences, or a protein and a non-protein sequence, such as an N or C extension sequence or a other functional sequence such as a signal peptide.

[0049] The term “sorbitol dehydrogenase” refers to an enzyme which has the ability to reduce sorbitol to fructose.

[0050] Herein, a “fusion protein” refers to a protein consisting of at least two domains that are encoded by separate genes that have been joined so that they are transcribed and translated as a single unit, producing a single polypeptide.

[0051] Sorbitol dehydrogenase

[0052] Sorbitol dehydrogenase is a non-glycosylated protein, formed of four identical monomer subunits in a dimer-dimer configuration to form a tetramer. X ray crystallography has shown each monomer to be of 354-356 amino acids, having a molecular weight of 38-40kDa. A deep cleft exists between two beta-barrel domains in each subunit, with the active site located at the bottom of the cleft. The enzyme uses NAD+as a co-factor, which binds in the grooves of all four subunits. Binding of the NAD+leads to binding of the substrate, followed by removal of the product and then removal of the co-factor.

[0053] The term “sorbitol dehydrogenase” as used herein includes any protein having sorbitol dehydrogenase activity. Suitably, the protein may be “a protein having Sorbitol Dehydrogenase (SORD) activity”. Sorbitol dehydrogenase may also be referred to as (R, R)-butanediol dehydrogenase, L-iditol 2-dehydogenase, epidymis secretory sperm binding protein, polyol dehydrogenase, ribitol dehydrogenase, xylitol dehydrogenase. The enzyme may fall under the classification numbers EC 1.1.1.14, EC 1.1.1.4, EC 1.1.1.56, and EC 1.1.1.9. Sorbitol dehydrogenase is responsible for the reduction of reduce sorbitol to fructose. Together with aldose reductase, it provides a way for the body to produce fructose from glucose without using ATP. Suitably, such an enzyme may fall within enzyme classification EC1.1.1.14. Sorbitol dehydrogenase activity may be determined or measured using any suitable assay available to a person skilled in the art, for example Sorbitol Dehydrogenase Activity Assay Kit (Colorimetric, Abeam) which is based upon the utilization of a provided substrate while reducing NAD+to form NADH. NADH reacts with the developer, leading to the formation of a chromophore with strong absorbance at OD 450 nm. Other assays are available in the art, for example based upon the reduction of tetrazolium salt MTT (3-[4, 5-dimethylthiazol-2-y / ]-2, 5 diphenyl tetrazolium bromide) in a NADH-coupled enzymatic reaction. The reduced form of MTT exhibits an absorption maximum at 565 nm and the increase in absorbance at 565 nm is directly proportional to the enzyme activity.

[0054] Sorbitol dehydrogenase is expressed and found in the cellular cytoplasm. Its main expression is in somatic tissues, such as liver, kidney, glandular tissue, and eyes.

[0055] Herein, reference to the sorbitol dehydrogenase of the fusion protein of the invention may include any sorbitol dehydrogenase enzyme from any organism, including plant, microbial, mammal or non-mammal. The sorbitol dehydrogenase may be derived from a non-human animal (such as mouse, cow, rabbit, rat, monkey, chimpanzee, and dog etc), or from other sources including fungi, plants or bacteria. Suitably, the sorbitol dehydrogenase is a mammalian enzyme, most suitably a human sorbitol dehydrogenase. The degree of structural and functional homology between sorbitol dehydrogenase from different sources is high, for example the human enzyme shares 75% or higher protein homology with other mammalian sorbitol dehydrogenase enzymes (95% protein homology with old world monkey; 92% with new world monkey, 89% with cows, hippos, pigs and ruminants; 83% with rodents). Some fish, such as a red bellied prihana shares 75% with the human sequence. Therefore, the enzymes may in certain circumstances be used interchangeably with a human or mammalian sorbitol dehydrogenase. Nucleic acid and amino acid sequences of sorbitol dehydrogenase from non-human organisms are well known in the art and provided in freely-available databases, such as, for example, the National Center for Biotechnology Information (NCBI) Nucleotide (ncbi.nlm.nih.gov / nuccore) and Protein (ncbi.nlm.nih.gov / protein) databases. The human sorbitol dehydrogenase may be any enzyme falling within classification EC1.1.1.14. The sorbitol dehydrogenase may be the human sequence, as set forth in SEQ ID NO. 1. The sorbitol dehydrogenase may be encoded by the human sorbitol dehydrogenase gene (SORD) of SEQ ID NO. 2 . Non-human examples of sorbitol dehydrogenase include, as described above, include non-human animals such as monkey, pig, bat, cow, sheep; alternatively plant, fungi, bacterial or microbial.

[0056] Also included are variants of a native sorbitol dehydrogenase enzyme. Therefore, the sorbitol dehydrogenase enzyme of the fusion protein of the invention may include sequence variants and fragments of the native sorbitol dehydrogenase sequence. A variant sorbitol dehydrogenase protein may comprise one or more amino acid substitutions compared to the native sorbitol dehydrogenase protein sequence. Therefore, the sorbitol, dehydrogenase of the fusion protein of the invention may be a protein which shares 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a native sorbitol dehydrogenase protein. Suitably, the sorbitol dehydrogenase of the fusion protein is a protein which shares 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the human sorbitol dehydrogenase of SEQ ID NO. 1. Suitably, a sequence variant of sorbitol dehydrogenase retains at least at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the enzyme activity of the native protein, when measured using an a suitable enzyme activity test, for example as described herein.

[0057] Sequence identity may be determined across the full length of the native protein, or may be determined over a continuous window of 300, 310, 320, 330, 340, or 350 amino acids. Suitably, sequence identity is measured across a continuous window of at least 330 amino acids. The sequence identity over the defined window is at least 90% or 95%. The sorbitol dehydrogenase of the fusion protein of the present invention may be a fragment of a full length sorbitol dehydrogenase protein, for example the human sorbitol dehydrogenase of SEQ ID NO. 1 . Such a fragment may comprise one or more amino acid deletions compared to the native sorbitol dehydrogenase enzyme. A fragment may be 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of the native protein. A fragment is suitably enzymatically active and retains the ability to reduce sorbitol to fructose. Suitably, a fragment of sorbitol dehydrogenase retains at least at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the enzyme activity of the native protein, when measured using an a suitable enzyme activity test, for example for example as described herein. A fragment of a sorbitol dehydrogenase enzyme may be a mature form a of native sorbitol dehydrogenase enzyme, whereby the N terminal methionine residue has been cleaved (removed) by prost-translational processing. A native enzyme may be in the “precursor” form where the enzyme comprises the N terminal methionine residue. Therefore, by way of example, a human sorbitol dehydrogenase sequence may comprise a sequence which is the protein of SEQ ID NO. 1 , or which is the sorbitol dehydrogenase of SEQ ID NO. 1 which lacks the N terminal methionine residue.

[0058] It is well within the capabilities of a skilled person to modify a sorbitol dehydrogenase enzyme, such as the polypeptide set forth in SEQ ID NO:1 , to generate enzymatically-active variants for use in the methods provided herein. For example, a person skilled in the art would understand that modifications at positions involved in substrate binding, or in the active site, are less likely to be tolerated than modifications at positions outside these critical regions. Any sorbitol dehydrogenase enzyme can be tested using methods well known in the art, such as those described in the Examples below, to assess the ability of the sorbitol dehydrogenase enzyme to catalyze the conversion of sorbitol to fructose.

[0059] As described above, sorbitol dehydrogenase in its active form is a tetramer comprising four identical monomer units. The sorbitol dehydrogenase of SEQ ID NO.

[0060] 1 represents a monomer subunit.

[0061] Reference to a sorbitol dehydrogenase enzyme includes any form of enzymatically- active sorbitol dehydrogenase, including human and non-human forms. The sorbitol dehydrogenase is suitably recombinant. In some embodiments, the sorbitol dehydrogenase may be synthetic or may be isolated.

[0062] Thioredoxin

[0063] Thioredoxin (also referred to as TXN, TRDX, TRX, TRX1 , and Trx80) is a 12-kD (105 amino acid) oxidoreductase protein which reduces oxidized cysteine residues and mediates the cleavage of disulfide bonds. A thioredoxin acts as electron donors to peroxidases and ribonucleotide reductase. Thioredoxin has a tertiary protein structure comprising a active site which contains dithiols in a CXXC motif. These two cysteines are the key to the ability of thioredoxin to reduce other proteins. Herein, reference to thioredoxin may include any thioredoxin protein from any organism, including plant, microbial or mammal, or non-mammal. The thioredoxin may be derived from a non-human animal (such as mouse, cow, rabbit, rat, monkey, chimpanzee, and dog etc), or from other sources including fungi, plants or bacteria. Suitably, the thioredoxin is a mammalian protein, most suitably a human thioredoxin. for example the human enzyme shares 75% or higher protein homology with other mammalian sorbitol dehydrogenase enzymes (95% protein homology with monkey; 93-94% for cow or pig, 50-99% for bacteria and 50-100% for fungi. Therefore, Nucleic acid and amino acid sequences of thioredoxin from non-human organisms are well known in the art and provided in freely-available databases, such as, for example, the National Center for Biotechnology Information (NCBI) Nucleotide (ncbi.nlm.nih.gov / nuccore) and Protein (ncbi.nlm.nih.gov / protein) databases. The human thioredoxin is provided under UniProt No. P10599 (NP_001231867.1) as set forth in SEQ ID NO. 3. A thioredoxin as defined herein may be encoded by a nucleic acid sequence, for example the nucleic acid sequence of SEQ ID NO. 4, or a variant or fragment thereof. Non-human examples of thioredoxin include mouse, pig, monkey, cow, plant, bacterial, microbial or fungal.

[0064] Also included are variants of a native thioredoxin protein. Therefore, the thioredoxin protein of the fusion protein of the invention may include sequence variants and fragments of the native thioredoxin sequence. Such a variant may comprise one or more amino acid substitutions compared to the native thioredoxin protein sequence. Therefore, the thioredoxin of the fusion protein of the invention may be a protein which shares 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a native thioredoxin protein. Suitably, the thioredoxin of the fusion protein is a protein which shares 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the human thioredoxin protein of SEQ ID NO. 3. Suitably, a sequence variant of thioredoxin retains at least at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the function of the native thioredoxin, when measured using an a suitable functionality test, for example, with an assay that measures the reduction of oxidized cysteine residues or the cleavage of disulfide bonds. Sequence identity may be determined across the full length of the native protein, or may be determined over a continuous window of 80, 85, 90, 95, 100 amino acid residues. Suitably, sequence identity is measured across a continuous window of 95 to 100 amino acid residues.

[0065] A thioredoxin protein of a fusion protein of the present invention may include a fragment of the native thioredoxin protein. Therefore, a fragment of a thioredoxin protein may comprise one or more amino acid deletions compared to a native thioredoxin protein, such as the human thioredoxin protein of SEQ ID NO. 3. A fragments may be 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of the native protein. A fragment is suitably enzymatically active and retains the ability to reduce oxidised cysteine residues or cleave disulphide bonds. Suitably, a fragment of thioredoxin protein retains at least at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the activity of the native protein, when measured using an a suitable functionality test, for example, with an assay that measures the reduction of oxidized cysteine residues or the cleavage of disulfide bonds.

[0066] Fusion protein

[0067] The provision of thioredoxin as a fusion partner with sorbitol dehydrogenase has been shown by the present inventors to be advantageous in improving the stability and / or solubility of the sorbitol dehydrogenase enzyme when manufactured recombinantly. The fusion protein of the invention comprising sorbitol dehydrogenase and thioredoxin has also been shown by the present inventors to have in vitro and in vivo activity at reducing sorbitol levels.

[0068] The sorbitol dehydrogenase and thioredoxin fusion protein of the present invention may be the product of a nucleic acid sequence encoding the sorbitol dehydrogenase gene and a nucleic acid sequence encoding the thioredoxin protein, which are transcribed and translated as a single unit to provide sorbitol dehydrogenase and thioredoxin as single protein sequence.

[0069] The fusion protein of the present invention comprises a sorbitol dehydrogenase enzyme and a thioredoxin protein. Suitably, the sorbitol dehydrogenase enzyme is as described above, and most suitably is the human sorbitol dehydrogenase enzyme of SEQ ID NO. 1. Suitably, the thioredoxin is as described above, and most suitably is the human thioredoxin sequence of SEQ ID NO. 3. Suitably, the sorbitol dehydrogenase enzyme may be a fragment or a variant of the enzyme of SEQ ID NO. 1 , as described above and the thioredoxin protein may be the sequence of SEQ ID NO. 3. Suitably, the sorbitol dehydrogenase enzyme may be the enzyme of SEQ ID NO. 1 , as described above and the thioredoxin protein may be a fragment or a variant of the sequence of SEQ ID NO. 3. Suitably, the sorbitol dehydrogenase enzyme may be a fragment or a variant of the enzyme of SEQ ID NO. 1 and the thioredoxin protein may be a fragment or a variant of the sequence of SEQ ID NO. 3.

[0070] The thioredoxin sequence may be provided at the N terminal or at the C terminal of the sorbitol dehydrogenase enzyme. Most suitably, the thioredoxin is provided at the N terminal end of the sorbitol dehydrogenase enzyme.

[0071] The fusion protein may be a monomer, or may be a multimer. Where the fusion protein is a multimer, it may be a dimer, trimer, tetramer or may comprise 2 or more monomer subunits, for example 2 to 20, 2 to 16, 2 to 15, 2 to 14 or 2 to 12 subunits. Suitably, the fusion protein is a tetramer. A fusion protein of the invention may comprise a mixture of monomers and multimer, where the multimers may be substantially all of the same multimeric form or may comprise different multimeric forms, for example a mixture of dimers, tetramers etc. The multimers may range in size from 2 to 20 subunits.

[0072] A fusion protein of the present invention may consist of sorbitol dehydrogenase and thioredoxin. Alternatively, a fusion protein of the present invention may comprise sorbitol dehydrogenase and thioredoxin, and may additionally comprise one or more additional sequences, for example a linker sequence, a His tag, an uptake tag, a signal peptide, an N terminal methionine, an affinity tag for purification (e.g. a his-tag e.g. his6, MYC, FLAG, HA or GST tag), a leader sequence (such as the pelB leader sequence), a sequence for directing protein secretion, or a protein for stabilising and / or solubilising the fusion protein (e.g. maltose-binding protein (MBP)), or a protein for increasing in vivo half-life (e.g. albumin or Fc domains, or fragments thereof). Any additional sequences may be provided either at the N and / or C terminal end of the fusion protein of the present invention, and / or may be provided between the sorbitol dehydrogenase enzyme and the thioredoxin protein. Suitably, an “inter protein” linker sequence may be provided between the sorbitol dehydrogenase enzyme and the thioredoxin protein. Such an “inter protein” linker sequence may be any suitable length, for example 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22,23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or 35 amino acids in length. Suitably, am “inter protein” linker sequence between the sorbitol dehydrogenase enzyme and the thioredoxin protein may be 10 to 32 amino acids in length, suitably 10 to 30 amino acids in length. Exemplary “inter protein” linkers between the sorbitol dehydrogenase enzyme and the thioredoxin protein as shown herein may be 11 , 22 or 27 amino acids in length.

[0073] An “inter protein” (or first) linker sequence between the sorbitol dehydrogenase enzyme and the thioredoxin protein may have any suitable sequence. An interprotein linker sequence may be any suitable linker which provides suitable structural, spatial and / or spherical properties to the fusion protein which enables the enzyme to be expressed, fold, and to function effectively. Such a linker sequence may comprise one or more G and or H residues. Such a linker sequence may comprise a GG, GGG, GGGG (SEQ ID NO. 17) or GGGGG (SEQ ID NO. 18) sequence. Suitably, such a linker may comprise a GGGG (SEQ ID NO. 17) sequence. Suitably, such a linker may comprise two or more, suitably three GGGG (SEQ ID NO. 17) sequences. Any two or more multi-G sequences may be contiguous, or may be separated by one or more non-G residues. A suitable G and non-G sequence may be GGGGS (SEQ ID NO. 19). A linker sequence between the sorbitol dehydrogenase enzyme and the thioredoxin protein may comprise a multi-H sequence, for example a HH, HHH, HHHH (SEQ ID NO. 20), HHHHH (SEQ ID NO. 21) or HHHHHH (SEQ ID NO. 22) sequence. Suitably, such a linker may comprise a HHHHH (SEQ ID NO. 21) sequence. Suitably, such a linker may comprise two or more HHHHH (SEQ ID NO. 21) sequences. Any two or more multi-H sequences may be contiguous, or may be separated by one or more non-H residues. In a suitable embodiment, a linker between the sorbitol dehydrogenase enzyme and the thioredoxin protein may comprise a multi H sequence (e.g. HHHHH, (SEQ ID NO. 21)) between multi-G sequences (e.g. GGGG (SEQ ID NO. 17)), thereby providing a linker comprising the sequence GGGG - HHHHH - GGGG (SEQ ID NO. 23), wherein the GGGG (SEQ ID NO. 17) and HHHHH (SEQ ID NO. 21) sequence may be contiguous or may be separated by other non-H or non-G amino acids. In a suitable embodiment, a linker between the sorbitol dehydrogenase enzyme and the thioredoxin protein may comprise a multi H sequence (e.g. HHHHH, (SEQ ID NO. 21)) adjacent, or contiguous with, a multi-G sequences (e.g. GGGG, (SEQ ID NO. 17)), thereby providing a linker comprising or consisting of the sequence GGGG - HHHHH (SEQ ID NO. 24).

[0074] Suitably, a known linker may be used, for example EAAAK (SEQ ID NO. 25), PAPAP (SEQ ID NO. 26), or a variation thereof, or a dipeptide sequence.

[0075] Suitable “inter protein” linker sequences may include GGGGXGGGGXHHHHHXGGGGX (SEQ ID NO. 11) , or

[0076] GGGGXGGGGXHHHHHXHHHHHXGGGGX (SEQ ID NO. 12) or XHHHHHGGGGX (SEQ ID NO. 13) where each X is independently 0, 1 , 2 or 3 amino acids. Suitably, each instance of X may be independently selected from S, or M, or A or MA, or a combination thereof. Suitable “inter protein” linker sequences may include GGGGSGGGGSXHHHHHXGGGGS (SEQ ID NO. 14), or GGGGSGGGGSHHHHHXHHHHHXGGGGS (SEQ ID NO. 15) or XHHHHHGGGGS (SEQ ID NO. 16) where X is 0, 1 , 2 or 3 amino acids. Suitably, each instance of X may be 0 amino acid, or may be 1 amino acid, or may be 2 amino acids, such as MA.

[0077] Where the inter-protein linker is GGGGX1GGGGX2HHHHHX3GGGGX4, X1may be 1 amino acid, and suitably may be S. X2may be 1 , 2 or 3 amino acids, and suitably may be S, or MA, or SMA X3may be 0, X4may be 1 amino acid, and suitably may be S. An exemplary “inter protein” linker sequence may be GGGGSGGGGSMAHHHHHGGGGS (Sord 15; SEQ ID NO. 8).

[0078] Where the inter-protein linker is GGGGX1GGGGX2HHHHHX3HHHHHX4GGGGX5, X1m and X2may each independently be 1 amino acid, and suitably may be S. X3may be 2 amino acids, and suitably may be MA. X4may be 0. X5may be 1 amino acid, and suitably may be S. An exemplary “inter protein” linker sequence may be GGGGSGGGGSHHHHHMAHHHHHGGGGS (Sord 17) SEQ ID NO. 9).

[0079] Where the inter-protein linker is XHHHHHGGGGX, X1may be 1 amino acid, and suitably may be A. X2may be 1 amino acid and suitably may be S. An exemplary “inter protein” linker sequence may be AHHHHHGGGGS (Sord 8) SEQ ID NO. 10). Therefore, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the sorbitol dehydrogenase may each independently be the native sequence as described herein or a variant or fragment thereof as described herein:

[0080] Thioredoxin (e.g. SEQ ID NO. 3)- GGGGXGGGGXHHHHHXGGGGX (SEQ ID NO. 11) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0081] Thioredoxin (e.g. SEQ ID NO. 3)- GGGGXGGGGXHHHHHXHHHHHXGGGGX (SEQ ID NO. 12) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0082] Thioredoxin (e.g. SEQ ID NO. 3)- XHHHHHGGGGX (SEQ ID NO. 13) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0083] Suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows:

[0084] Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSXHHHHHXGGGGS (SEQ ID NO. 14) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0085] Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSHHHHHXHHHHHXGGGGS (SEQ ID NO. 15) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0086] Thioredoxin (e.g. SEQ ID NO. 3)- XHHHHHGGGGS (SEQ ID NO. 16) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0087] Suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the sorbitol dehydrogenase may each independently be the native sequence as described herein or a variant or fragment thereof as described herein:

[0088] Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGS (SEQ ID NO.

[0089] 8) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1) Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSHHHHHMAHHHHHGGGGS - (SEQ ID NO. 9) sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0090] Thioredoxin (e.g. SEQ ID NO. 3)- AHHHHHGGGGS (SEQ ID NO. 10) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0091] A fusion protein of the present invention may comprise an N terminal extension sequence. An N terminal extension sequence may be a His tag, an uptake tag, a signal peptide, an N terminal methionine, an affinity tag for purification, a leader sequence, a sequence for directing protein secretion or subcellular targeting or organ / tissue targeting, or a protein or sequence (such as an mRNA or oligonucleotide sequence) for stabilising and / or solubilising the fusion protein, or a protein for increasing in vivo half-life. Suitably, the N terminal extension sequence may be an uptake tag. An cysteine or arginine residue may be provided on the N terminal end of the fusion protein to enable conjugation of an N terminal extension sequence such as a tag, for example where an N terminal extension sequence is added to the fusion protein by conjugation rather than recombinantly. By way of example, a malemeide- labelled peptide may be conjugated to a cysteine or arginine amino acid).

[0092] Any suitable uptake tag may be used, for example, pentapeptides to direct uptake in to cells, tricyclic charged peptides, or charged peptides to enhance cellular uptake, such as arginine-rich peptides, tat, and penetratin. A most suitable uptake tag may be the sequence MPTLK (SEQ ID NO. 27). Such an uptake tag may improve cellular uptake in a subject. Suitably, an N terminal extension may be an N terminal methionine residue, optionally with a cysteine residue. For example, an N terminal extension sequence may be MXC, where X is 0, 1 , 2, 3, 4, or 5 amino acids, of any sequence. Suitably, X is 0, 1 or 2. Suitably, X is 1 . Suitably, X may be any amino acid. Suitably, X is alanine. Suitably, an N terminal extension sequence is MAC. An N terminal extension sequence may comprise an uptake tag and an N terminal methionine, or may comprise an uptake tag or an N terminal methionine residue. The fusion protein may comprise a linker sequence between the thioredoxin and the sorbitol dehydrogenase, as described above.

[0093] Therefore, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the sorbitol dehydrogenase may each independently be the native sequence as described herein or a variant or fragment thereof as described herein:

[0094] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGXGGGGXHHHHHXGGGGX (SEQ ID NO. 11) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0095] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGXGGGGXHHHHHXHHHHHXGGGGX (SEQ ID NO. 12) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0096] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- AHHHHHGGGGX (SEQ ID NO. 13) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0097] Suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the sorbitol dehydrogenase may each independently be the native sequence as described herein or a variant or fragment thereof as described herein:

[0098] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSXHHHHHXGGGGS (SEQ ID NO. 14) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0099] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSHHHHHXHHHHHXGGGGS (SEQ ID NO. 15) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0100] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- XHHHHHHGGGGS (SEQ ID NO. 16) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0101] Suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the sorbitol dehydrogenase may each independently be the native sequence as described herein or a variant or fragment thereof as described herein:

[0102] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGS (SEQ ID NO. 8) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0103] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSHHHHHMAHHHHHGGGGS (SEQ ID NO. 9) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0104] N-terminal extension sequence (e.g. MAC or MPTLK) - Thioredoxin (e.g. SEQ ID NO. 3)- AHHHHHGGGGS (SEQ ID NO. 10) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0105] Suitably, an exemplary sequence may comprise or consist of

[0106] MAC - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGS (SEQ ID NO. 8) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0107] MPTLK - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSHHHHHMAHHHHHGGGGS (SEQ ID NO. 9) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0108] MPTLK - Thioredoxin (e.g. SEQ ID NO. 3)- AHHHHHGGGGS (SEQ ID NO. 10) - sorbitol dehydrogenase (e.g. SEQ ID NO. 1)

[0109] Suitably, an N terminal (or second) linker sequence comprising GGGG (SEQ ID NO. 17) may be provided between the thioredoxin protein and an N terminal extension sequence. Any suitable linker sequence, such as one comprising

[0110] G or GG, GGG, GGGG (SEQ ID NO. 17), HHHH (SEQ ID NO. 20), EAAAK (SEQ ID NO. 25), PAPAP (SEQ ID NO> 26) or a dipeptide may be used. Such a linker sequence may be any suitable length, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14 or 15 or more amino acids. Suitably, the N terminal (or second) linker may be 2- 30 amino acids in length, suitably 3 to 10, more suitably 4 to 10 amino acids in length. Exemplary N terminal (or second) linkers as shown herein may be 5 or 9 amino acids in length. A suitable linker may comprise one or more G and or H residues. Such a linker sequence may comprise a GG, GGG, GGGG (SEQ ID NO. 17) or GGGGG (SEQ ID NO. 18) sequence. Suitably, such a linker may comprise a GGGG (SEQ ID NO. 17) sequence. Suitably, such a linker may comprise a GGGGS (SEQ ID NO. 19) sequence. A N terminal (or second) linker sequence may comp rise a multi-H sequence, for example a HH, HHH, HHHH (SEQ ID NO. 20), HHHHH (SEQ ID NO. 21) or HHHHHH (SEQ ID NO. 22) sequence. Suitably, such a linker may comprise a HHHH (SEQ ID NO. 20) sequence. In a suitable embodiment, an N terminal (or second) linker between the N terminal extension sequence and the thioredoxin protein may comprise a multi H sequence (e.g. HHHHH, SEQ ID NO. 21) and a multi-G sequences (e.g. GGGG, SEQ ID NO. 17), thereby providing a linker comprising the sequence G(X) - H(X), where X is independently 0, 1 , 2, 3 or 4. Suitably, an N terminal (or second) linker may comprise or consist of the sequence GGGGHHHH (SEQ ID NO. 28) or GGGGSHHHH (SEQ ID NO. 29).

[0111] Suitably, the N terminal sequence upstream of the thioredoxin protein may comprise or consist of the sequence N-terminal extension sequence (e.g. MAC or MPTLK, SEQ ID NO. 27) - N terminal (or second) linker. Suitably, the N terminal sequence upstream of the thioredoxin protein may comprise or consist of the sequence N- terminal extension sequence (e.g. MAC or MPTLK SEQ ID NO. 27) - GGGGS (SEQ ID NO. 19). Suitably, the N terminal sequence upstream of the thioredoxin protein may comprise or consist of the sequence N-terminal extension sequence (e.g. MAC or MPTLK, SEQ ID NO. 27) - GGGGSHHHH (SEQ ID NO. 29). Suitably, the N terminal sequence upstream of the thioredoxin protein may comprise or consist of the sequence N-terminal extension sequence MAC - GGGGS (SEQ ID NO. 30). Suitably, the N terminal sequence upstream of the thioredoxin protein may comprise or consist of the sequence MPTLK - GGGGSHHHH (SEQ ID NO. 31).

[0112] Amino acid linkers enable preparation of the fusion protein by recombinant means but non-amino acid-based linkers might also be used, again according to principles and techniques well known in the art and described in the literature. A linker may be cleavable (e.g. enzymatically) or non-cleavable.

[0113] Suitably, a fusion protein of the present invention consists of or comprises the sequence of SEQ ID NO. 5 (rhSORD8).

[0114] Suitably, a fusion protein of the present invention consists of or comprises the sequence of SEQ ID NO. 6 (rhSORD17).

[0115] Suitably, a fusion protein of the present invention consists of or comprises the sequence of SEQ ID NO. 7 (rhSORD15).

[0116] A fusion protein of the invention may be prepared as a naked protein or may undergo post-translational modification, for example coupling or conjugating to a further moiety or chemical group or substance. Exemplary modifications include, but are not limited to, pegylation, albumination or glycosylation, lipidation, myristilation, ubiquitination, or other known modifications. For example, in some instances, a fusion protein for use in the described methods is pegylated using standard methods well known in the art. This may serve, for example, to improve the half-life of the fusion protein in use. Thus, suitably the fusion protein may be provided as a conjugate with a polymer such as polyethylene glycol (PEG) or a poly- or oligosaccharide or polynucleotide. Conjugates with PEG are particularly suitable. As indicated above the preparation of such conjugates is well known in the art and described in the literature. Thus, PEGs of various sizes may be used to prepare the conjugates e.g. ranging from 100 Daltons to 100 kD, but more often from 5kD to 100 kD, for example 12 or 15 kD to 60 or 80 kD, such as 15 to 50, 15 to 40, or 15 to 30 kD. Further, the PEG may be attached or linked to the fusion protein in various ways, and more than one PEG may be attached to each single protein. It may be linked directly or indirectly, e.g. via a linker as described, for fusion proteins above or by any molecular or chemical group which may provide a linker function. Thus, the PEG may be linked at one or both of the N- or C- termini, or internally in the fusion protein, for example at the amino group of one or more lysine residues in the fusion protein or at any other chemical moiety or residue in the protein molecule. Methods for coupling or conjugating polymers such as PEG to proteins are well known in the art and described in the literature (see for example Roberts et al. 2012, Advanced Drug Delivery Reviews, 64 (supplement) 116-127 and Veronese 2001 , Biomaterials 22, 405-417). A PEG conjugate comprising a PEG linked to the N terminus of a fusion protein represents one preferred embodiment of the present invention. The fusion protein may be pegylated in monomeric and / or multimeric form. Thus, for convenience a preparation comprising both monomeric and various multimeric forms of the fusion protein may be subjected to pegylation.

[0117] Nucleic acid sequence The present invention provides a nucleic acid sequence which encodes a protein which consists of or comprises a fusion protein as described herein.

[0118] The nucleic acid sequence may be a DNA, RNA, or cDNA sequence. A nucleic acid sequence may be codon optimized for expression in a particular system, for example a bacterial cell, for example E. coli.

[0119] Nucleic acid sequence for thioredoxin and sorbitol dehydrogenase are known in the art and provided in freely-available databases, such as, for example, the National Center for Biotechnology Information (NCBI) Nucleotide (ncbi.nlm.nih.gov / nuccore). The nucleic acid sequences are provided as SEQ ID NO. 2 (sorbitol dehydrogenase) and 4 (thioredoxin).

[0120] Nucleic acid encoding a protein or linker sequence for use in generating a fusion protein of the present invention can be obtained by any suitable method, including, but not limited to, RT-PCR of RNA and synthetic nucleotide synthesis. Primers for amplification can be designed based on known sequences.

[0121] A nucleic acid sequence of the present invention may be cloned into an expression vector, suitable for an expression system of choice. The present invention therefore provides an expression vector, comprising a nucleic acid sequence encoding a fusion protein of the present invention as described herein. A nucleic acid sequence encoding a fusion protein of the present invention may be operably linked to one or more regulatory sequences that facilitate expression of the fusion protein. A regulatory sequence may be a transcriptional promoter, enhancer, translational signal, or transcriptional and translational termination signal.

[0122] The choice of expression vector is influenced by the choice of host expression system. Many expression vectors suitable for the expression of a fusion protein as described herein are available and known to those of skill in the art. Such selection is well within the level of skill of the skilled artisan. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vectors in the cells. Suitably, an expression vector viral or non-viral. By way of example, a suitable viral expression vector may be derived from a virus selected from the group consisting of paramyxovirus, retrovirus, adenovirus, lentivirus, pox virus, alphavirus, and herpes virus. Other suitable viral vectors will be known to those skilled in the art.

[0123] Suitable non-viral expression vectors may be selected from the group consisting of inorganic particle expression vectors (such as calcium phosphate, silica, and gold), lipid based particle expression vectors (for example cationic lipids, lipid nano emulsions, and solid lipid nanoparticles) and polymer based particle expression vectors (for example peptides, polyethylenimine, chitosan, and dendimers). Other suitable non-viral expression vectors will be known to those skilled in the art.

[0124] Production

[0125] A fusion protein as provided herein may be produced recombinantly, by the expression of nucleic acid sequences encoding different polypeptides, operably linked in a manner to produce a single protein sequence comprising the different polypeptides. The fusion protein of the present invention may be a recombinant protein.

[0126] Therefore the present invention provides a host cell or a cell free system comprising a nucleic acid encoding a fusion protein of the invention. A host cell may be prokaryotic, eg. E. coli, or eukaryotic, for example mammalian or yeast. A host cell may be recombinant. A population of host cells may be provided, for recombinant manufacture of a fusion protein of the invention, or as a vehicle for administration of a fusion protein to a subject.

[0127] The fusion protein may be produced using any suitable expression system. Prokaryotes, especially E. coli, provide a system for producing large amounts of recombinant protein. Transformation of E. coli is a simple and rapid technique well known to those of skill in the art. Expression vectors for E. coli can contain inducible promoters that are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated APL promoter.

[0128] In other examples, eukaryotic expression systems are used to produce the fusion protein, such as baculovirus expression systems. Typically, expression vectors use a promoter such as the polyhedrin promoter of baculovirus for high level expression. Commonly used baculovirus systems include baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus (DpNI). For high level expression, the nucleotide sequence encoding the fusion protein is fused immediately downstream of the polyhedrin initiation codon of the virus.

[0129] Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris can also be used expression hosts for expression of a nucleic acid encoding a fusion protein of the invention. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters, such as include GALI, GAL7, and GAL5, are used to regulate gene expression. Yeast expression vectors often include a selectable marker such as LELI2, TRPI, HIS3, and LIRA3 for selection and maintenance of the transformed DNA.

[0130] Mammalian expression systems also can be used to express a fusion protein of the present invention. An expression construct may be transferred to a mammalian cell by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. Such vectors often include transcriptional promoter-enhancers for high level expression, for example the SV40 promoterenhancer, the human cytomegalovirus (CMV) promoter, and the long terminal repeat of Rous sarcoma virus (RSV). Exemplary cell lines available for mammalian expression include, but are not limited to, mouse, rat, human, monkey, and chicken and hamster cells, such as BHK, 293-F, CHO, Balb / 3T3, HeLa, MT2, mouse NSO (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2 / 0, COS, NIH3T3, HEK293, 293S, 293T, 2B8, and HKB cells.

[0131] Following expression in a host cell, a fusion protein of the present invention may be purified, using any suitable method available to those of skill in the art including, but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography and affinity chromatography. Affinity purification techniques can be used to improve the efficiency and purity of the preparations. For example, antibodies and other molecules that bind sorbitol dehydrogenase or thioredoxin can be used in affinity purification. As discussed above, expression constructs can be engineered to add an affinity tag such as a his, myc, FLAG or HA tag or GST moiety to the fusion protein, which can then be affinity purified with Ni-resin, myc antibody, HA antibody, FLAG antibody or glutathione resin, respectively. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques, such as SDS page and Size Exclusion Chromatography (SEC).

[0132] An affinity tag and / or linker sequence may be removed prior to use of the fusion protein. In certain conditions, it may be preferable to leave the tag in place and use the fusion protein with a tag and / or linker attached.

[0133] Conditions and disorders, subjects

[0134] A “subject” as used herein includes any human or non-human animal and particularly refers to mammals, including for example humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (e.g. foxes, kangaroos, deer). The subject may be a juvenile (child) or an adult. Suitably, the subject is human or a laboratory test animal. Most suitably, the subject is a human, and may be either an adult human or a child. Suitably, a subject may have a genetic and / or metabolic disorder that alters sorbitol metabolism or causes over production of sorbitol. Suitably, a subject may have a deficiency or absence of sorbitol dehydrogenase, an excess of aldose reductase, or a deficiency or absence of fructokinase. Most suitably, the subject has a deficiency or absence of sorbitol dehydrogenase, for example due to a genetic abnormality.

[0135] The term “excess sorbitol” includes any condition in which intracellular sorbitol is above a normal or healthy level. The intracellular level may therefore be higher as compared to the level of sorbitol in a subject without a condition or symptom which is the result of excess sorbitol. Excess sorbitol in a subject may arise due to a condition resulting in sorbitol dehydrogenase deficiency, increased aldose reductase activity, fructokinase deficiency. A sorbitol dehydrogenase deficiency may be a genetic abnormality. Sorbitol may be used as a biomarker for diabetic complications, such as cell edema and diabetic neuropathy. A healthy or normal intracellular sorbitol level may be in the region of 10mg / l or below. A high or excess sorbitol level may therefore be above 10mg / l. A sorbitol level of 10mg / l or above may indicate a predisposition or the presence of a disorder associated with excess sorbitol. Sorbitol levels in a subject may be determined using any method as described herein.

[0136] Symptoms of excess sorbitol in a subject may include one or more of cataracts, neuropathy including diabetic neuropathy, retinopathy including diabetic retinopathy, cardiomyopathy, nephropathy including diabetic nephropathy, microvascular complications, physical deformities (for example of limbs, such as hand, foot, arm, leg), physical disability including foot tremors, limb weakness and difficulty walking, reduced or impaired mobility, sensory impairment, atherosclerosis and other cardiovascular complications, albuminuria, hyperglycaemia and diabetes, shortness of breath and lung issues; muscle effects including muscle weakness; digestive system disorders including diarrhoea; signs of allergic reactions such as rash, itching, wheezing, throat issues; brain effects such as mood alteration, confusion and / or brain disorders associated with these symptoms.

[0137] Sorbitol levels in a subject may be measured or determined using any suitable method. Appropriate samples for determination of levels of sorbitol include any appropriate or desired sample in which the excess sorbitol may occur. These may be any appropriate or desired tissue or body fluid sample. An example of a suitable tissue is kidney, liver, glandular, and / or muscle tissue. Conveniently, a sample may be any body fluid sample for example plasma, serum, cerebrospinal fluid, or a stool or tissue sample, biopsy sample, a lavage or washing fluid sample, urine, or saliva, or suitably may be a blood or any blood-derived sample e.g. plasma or serum etc. This may depend of course on the precise nature of the condition to be treated etc.

[0138] Sorbitol may be measured using any suitable method available to a skilled person. Such methods may include a microplate reader, high performance liquid chromatography (HPLC), and Gas Chromatography - Mass spectrometry (GC / MS). A simple measurement method of sorbitol is the F-kit, which is enzyme colorimetric assay and quantifies sorbitol by measuring the color change of the formazan dye. Additionally, electrochemical sensors, optical sensors, and quartz crystal microbalance sensors among others have been devised for sorbitol measurement.

[0139] Methods of treatment

[0140] The present invention provides a method for the prophylaxis or treatment of a disorder which results in excess sorbitol in a subject, wherein the method comprises administering a therapeutically effective amount of a fusion protein comprising a sorbitol dehydrogenase enzyme and a thioredoxin protein to a subject in need thereof. Suitably, the fusion protein is as described herein.

[0141] The present invention also provides a fusion protein comprising a sorbitol dehydrogenase enzyme and a thioredoxin protein for use in the prophylaxis or treatment of a disorder which results in excess sorbitol in a subject.

[0142] A method of the first aspect may result in a decrease in sorbitol accumulation in a tissue of a subject.

[0143] Therefore, the present invention also provides a method for the prevention or reduction of excess sorbitol in a subject, wherein the method comprises administering a therapeutically effective amount of a fusion protein comprising a sorbitol dehydrogenase enzyme and a thioredoxin protein to a subject in need thereof. Suitably, the fusion protein is as described herein. The present invention also provides a fusion protein comprising a sorbitol dehydrogenase enzyme and a thioredoxin protein for use in the prevention or reduction of excess sorbitol in a subject. Suitably, the fusion protein is as described herein.

[0144] The fusion protein may be provided in the form of a composition, as described herein.

[0145] Thus for example in the case of a sorbitol dehydrogenase deficiency, treatment according to the present invention may not treat the underlying genetic disorder, but rather the resulting clinical condition of excess sorbitol.

[0146] In a suitable embodiment, an effective treatment according to the present invention is one which provides an effective relief from one or more symptoms selected from one or more of cataracts, neuropathy including diabetic neuropathy, retinopathy including diabetic retinopathy, cardiomyopathy, nephropathy including diabetic nephropathy, microvascular complications, physical deformities (for example of limbs, such as hand, foot, arm, leg), physical disability including foot tremors, limb weakness and difficulty walking, reduced or impaired mobility, sensory impairment, atherosclerosis and other cardiovascular complications, albuminuria, hyperglycaemia and diabetes, shortness of breath and lung issues; muscle effects including muscle weakness; digestive system disorders including diarrhoea; signs of allergic reactions such as rash, itching, wheezing, throat issues; brain effects such as mood alteration, confusion and / or brain disorders associated with these symptoms. An effective treatment may reduce intracellular sorbitol levels in a selected tissue of a subject to at or below 10mg / l. The sorbitol level of a subject may be measured as described herein, for example by assaying a biological sample of the subject.

[0147] The present methods and uses as described herein may be useful in maintenance of sorbitol levels at any normal or healthy range, for example at or below 10mg / l.

[0148] Treatment, according to the present invention, may provide an increase in the amount of active sorbitol dehydrogenase in a tissue of the subject to be treated. Such an increase may be in a suitable tissue, for example a somatic tissue such as liver, kidney, eye or glandular tissue. The increased level of sorbitol dehydrogenase may be sufficient to reduce sorbitol in a tissue of the subject to a normal or healthy range. It will also be appreciated that normal or healthy levels of sorbitol may be subject specific, and depend on factors such as the subject’s weight, diet, sex and age. Normal or healthy levels of sorbitol may be at or below 10mg / l.

[0149] An "effective amount” of a fusion protein for the reduction of excess sorbitol is an amount which is non-toxic to the subject, but is sufficient to provide the desired effect. Exemplary therapeutically effective amounts may be determined by a skilled person. The exact amount or dose required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not appropriate to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. A effective amount can be an amount that decreases sorbitol levels at least by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or more, e.g., about 100% (e.g., compared to pre-treatment level). The therapeutically effective amount can be sufficient to normalize sorbitol levels in a subject with a genetic and / or metabolic disorder that alters sorbitol metabolism or causes over production of sorbitol, such as SDH deficiency.

[0150] Methods for measuring tissue sorbitol are described herein, and may be used in the diagnosis of a condition which includes excess sorbitol as a symptom; and / or may be used in the monitoring of a disorder associated with excess sorbitol; or in the prevention of a disorder associated with excess sorbitol. A method of measuring sorbitol in a biological sample from a subject may be performed one, or more than once, at regular or irregular intervals. Regular intervals can include, for example, approximately daily (for example every 12-24 hours, suitably every 24 hours), weekly, bi-weekly, monthly, or any other interval. Selecting a treatment protocol is well within the level of skill of the skilled artisan. For example, a protocol can be determined based upon studies in animal models. In other example, repeat doses of the composition(s) can be administered to a subject if the ammonia level in the blood, is above a predetermined level.

[0151] Measuring the amount of sorbitol in a biological sample of a subject may also be used to determine the suitability of the present invention as a treatment for the subject, and / or to determine the effectiveness of the treatment. Such measurements may also be used to determine a suitable effective dose in a subject.

[0152] In a suitable embodiment, administration of a fusion protein comprising fusion protein comprising a sorbitol dehydrogenase enzyme and a thioredoxin protein may prevent, ameliorate or treat diabetic neuropathy. The fusion protein may be a fusion protein as described herein. The term “diabetic neuropathy” as used herein refers to peripheral, sensory, autonomic and / or motor neuropathy. Suitably, in the context of the present invention, “treatment” may refer to a reduction in numbness, tingling, burning sensations, discomfort and shooting pains, loss of feeling or sensations, loss of coordination, loss of bladder control, irregular heart beat, impotence, heat intolerance, gastroparesis, and / or muscle weakness.

[0153] In a suitable embodiment, administration of a fusion protein comprising fusion protein comprising a sorbitol dehydrogenase enzyme and a thioredoxin protein may prevent, ameliorate or treat SORD deficiency (also referred to as SORD-CMT or dHMN). The fusion protein may be a fusion protein as described herein. Suitably, in the context of the present invention, the administration of a fusion protein according to the present invention may prevent or reduce symptoms of excess sorbitol, as described herein, including for example one or more of cataracts, neuropathy including diabetic neuropathy, retinopathy including diabetic retinopathy, cardiomyopathy, nephropathy including diabetic nephropathy, microvascular complications, physical deformities (for example of limbs, such as hand, foot, arm, leg), physical disability including foot tremors, limb weakness and difficulty walking, reduced or impaired mobility, sensory impairment, atherosclerosis and other cardiovascular complications, albuminuria, hyperglycaemia and diabetes, shortness of breath and lung issues; muscle effects including muscle weakness; digestive system disorders including diarrhoea; signs of allergic reactions such as rash, itching, wheezing, throat issues; brain effects such as mood alteration, confusion and / or brain disorders associated with these symptoms.

[0154] Composition

[0155] In a further aspect the present invention provides a composition comprising sorbitol dehydrogenase, for example in the form of a fusion protein of the present invention. Also provided is a composition comprising a nucleic acid encoding a fusion protein of the present invention, an expression vector comprising such a nucleic acid, or a host cell comprising a nucleic acid or expression vector of the present invention.

[0156] Suitably, the composition may be a pharmaceutical or nutritional composition, for example a medicament or supplement. Such a composition may comprise one or more physiologically or pharmaceutically acceptable carriers or excipients.

[0157] Pharmaceutical compositions are physiologically acceptable and typically include the active compound and a carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Non-limiting examples of such pharmaceutical carriers include liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers may also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Other examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (Alfonso Gennaro ed., Krieger Publishing Company (1997); Remington's: The Science and Practice of Pharmacy, 21st Ed. (Lippincot, Williams & Wilkins (2005); Modern Pharmaceutics, vol. 121 (Gilbert Banker and Christopher Rhodes, CRC Press (2002). Selection of a suitable carrier or excipient is within the skill of the administering profession and can depend upon a number of parameters, such as the mode of administration.

[0158] A carrier may include a buffer, for example an alkaline buffers, e.g., ammonium buffer, acidic buffers, e.g., ethanoates, citrates, lactates, acetates, etc., or zwitterionic buffers, such as, glycine, alanine, valine, leucine, isoleucine and phenylalanine, Kreb's-Ringer buffer, TRIS, MES, ADA, ACES, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, TAPSO, acetamidoglycine, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, TRIZMA, PBS, Ammonium phosphate, Glycinamide, Glycyl-glycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0159] The composition can be in any suitable form, for example a tablet, capsule, solution, emulsion, suspension, gel, sol, or colloid that is physiologically and / or pharmaceutically acceptable.

[0160] In some examples the composition is provided as a fluid. In such an embodiment, a carrier may be a solvent or dispersion medium such as water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. A suitable fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. If desired tonicity adjusting agents can be included, such as, for example, sugars, sodium chloride or combinations thereof. In other instances, the composition is provided in a dried form, such as desiccated or freeze-dried form. Such dried forms can be rehydrated prior to administration by the addition of a suitable solution, such as water, buffer, saline or other suitable solution. The composition provided herein can be formulated for direct administration or can be formulated for dilution or other modification. Accordingly, the composition can be formulated in single (or unit) dosage forms or multiple dosage forms. Examples of single dose forms include ampoules and syringes. Examples of multiple dose forms include vials and bottles that contain multiple unit doses.

[0161] The compositions may also include additional ingredients, such as acceptable surfactants, co-solvents, emollients, agents to adjust the pH and osmolarity and / or antioxidants to retard oxidation of one or more component.

[0162] The compositions can be prepared for administration by any suitable route such as ocular (including periocular and intravitreal administration), oral, parenteral, intranasal, anal, vaginal, topical, subcutaneous, intravenous, intra-arterial, intrathecal and intraperitoneal administration.

[0163] Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, edible carriers or combinations thereof. Examples of pharmaceutically acceptable carriers may include, for example, water or saline solution, polymers such as polyethylene glycol, carbohydrates and derivatives thereof, oils, fatty acids, or alcohols. Surfactants such as, for example, detergents, are also suitable for use in the formulations. Specific examples of surfactants include polyvinylpyrrolidone, polyvinyl alcohols, copolymers of vinyl acetate and of vinylpyrrolidone, polyethylene glycols, benzyl alcohol, mannitol, glycerol, sorbitol or polyoxyethylenated esters of sorbitan; lecithin or sodium carboxymethylcellulose; or acrylic derivatives, such as methacrylates and others, anionic surfactants, such as alkaline stearates, in particular sodium, potassium or ammonium stearate; calcium stearate or triethanolamine stearate; alkyl sulfates, in particular sodium lauryl sulfate and sodium cetyl sulfate; sodium dodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or fatty acids, in particular those derived from coconut oil, cationic surfactants, such as water-soluble quaternary ammonium salts of formula N R'R"RmR'"'Y", in which the R radicals are identical or different optionally hydroxylated hydrocarbon radicals and Y" is an anion of a strong acid, such as halide, sulfate and sulfonate anions; cetyltrimethylammonium bromide is one of the cationic surfactants which can be used, amine salts of formula NR'R'R", in which the R radicals are identical or different optionally hydroxylated hydrocarbon radicals; octadecylamine hydrochloride is one of the cationic surfactants which can be used, non-ionic surfactants, such as optionally polyoxyethylenated esters of sorbitan, in particular Polysorbate 80, or polyoxyethylenated alkyl ethers; polyethylene glycol stearate, polyoxyethylenated derivatives of castor oil, polyglycerol esters, polyoxyethylenated fatty alcohols, polyoxyethylenated fatty acids or copolymers of ethylene oxide and of propylene oxide, amphoteric surfactants, such as substituted lauryl compounds of betaine.

[0164] If desired, an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc., or combinations thereof containing two or more of the foregoing.

[0165] Sterile injectable solutions may be prepared using an appropriate solvent. Generally, dispersions are prepared by incorporating the various sterilized amino acid components into a sterile vehicle, which contains the basic dispersion medium and / or the other ingredients. Suitable formulation methods for any desired mode of administration are well known in the art (see, generally, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).

[0166] Suitably, a composition is sterile.

[0167] In some embodiments, the composition is in unit dose form such as a tablet, capsule or single-dose vial. Suitable unit doses, i.e., therapeutically effective amounts, may be determined during clinical trials designed appropriately for each of the conditions for which administration of a chosen compound is indicated and will, of course, vary depending on the desired clinical endpoint.

[0168] It may be included in such foods as a polypeptide (e.g. purified enzyme) or as part of an expressing host cell or organism. Thus for example microbial (e.g. yeast or bacterial or fungal) host cells or plants (including plants cells) may be engineered to express the fusion protein and may be administered as such, e.g. a whole cells or extracts or other processed products (in which enzymatic activity may be retained), or may be incorporated into nutritional compositions. Thus, for example, bacterial or yeast cells suitable for human or non-human animal consumption may be engineered to express the fusion protein. Alternatively, plants may be engineered in an analagous manner and appropriate plant parts etc (e.g. seeds, leaves, tubers etc) may be provided for administration. It is known in the art which microoganisms (yeasts, bacteria, algae or fungi for example) are suitable for human or other animal consumption and many such organisms are used today, for example in probiotic formulations. Any such probiotic organisms or formulations could be used e.g. based on lactic acid bacteria such as Bifidobacterium or Lactobacillus sp. (e.g. L. acidophilus) etc. Thus, according to the present invention such organisms or preparations may be formulated for and administered directly into the Gl tract, e.g. by injection or infusion, or enema or rectal administration etc.

[0169] The precise amount or dose of the fusion protein administered to the subject depends on the activity of the sorbitol dehydrogenase, the route of administration, the disease or condition being treated, the number of dosages administered, and other considerations, such as the weight, age and general state of the subject. Particular dosages and administration protocols can be empirically determined or extrapolated from, for example, studies in animal models. Exemplary therapeutically effective doses of the fusion protein include, but are not limited to, from or from about 0.1 pg / kg body weight per day to or to about 10000 pg / kg body weight per day, including from or from about 1 pg / kg to or to about 1000 pg / kg body weight per day, or from or from about 10 pg / kg to or to about 100 pg / kg body weight per day Thus, for example, a subject can be administered 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, 10000, or 20000 pg or more the GS per kg body weight per day. Suitably, the fusion protein is administered intravenously or subcutaneously, at weekly or biweekly intervals.

[0170] Administration

[0171] The fusion protein of the present invention may be administered using any suitable method, including for example any route which delivers the protein to the desired tissue. Suitable methods include parenteral, intravenous, intramuscular, intradermal, transdermal, sublingual, nasal, vaginal, buccal, subcutaneous, rectal, oral, or intraperitoneal administration, or any combination thereof. Subcutaneous administration is a suitable mode of administration. Administration may be via any suitable mechanism, for example by injection, infusion, inhalation, sublingual, ointments, tablets, capsules, oral liquid, patches, enema, and transdermal. Suitably, systemic administration may be used. Suitable systemic administration methods will be known to those skilled in the art. By way of example, systemic administration may be achieved by parenteral route of administration, such as intravenous or subcutaneous route. It will be appreciated that “systemic administration” allows the fusion protein (or nucleic acid, expression vector or host cells, or composition) to be provided to multiple sites in the subject.

[0172] The fusion protein may be formulated as a pro-drug, such that it is formulated as an agent which metabolises into or undergoes in vivo hydrolysis to form the active fusion protein.

[0173] Combinations

[0174] The fusion protein may also be administered in conjunction or combination with one or more other therapeutic or active agents, notably a second or further therapeutic agent which may treat (e.g. to improve) or prevent a condition associated with excess sorbitol. The additional therapeutic agent may be administered separately, sequentially or simultaneously with the fusion protein, including in the same formulation or composition, or in a separate composition or formulation.

[0175] Accordingly, the present invention provides a combination of a fusion protein as described herein and a further therapeutic agent, as a combined preparation for separate, simultaneous or sequential use in treating or preventing a condition associated with excess sorbitol. The second or further agent may be administered by the same administration route or by a different administration route.

[0176] Kits

[0177] Also provided is a kit comprising (a) a fusion protein as described herein and (b) a further therapeutic agent. Suitably, the further therapeutic may be effective against a disorder which causes or is due to excess sorbitol.

[0178] Such a kit may be provided for use in treating or preventing such a disorder, which is associated with excess sorbitol or for preventing or reducing excess sorbitol. The components of the kit may be provided as separate pharmaceutical compositions comprising the agent(s) in question together with one or more pharmaceutically- acceptable carriers or excipients. The composition(s) of the invention can be administered once or more than once. If the composition(s) are administered more than one time, they can be administered at regular intervals or as needed, for example as determined by a clinician.

[0179] It will be appreciated that (except where the context requires otherwise), embodiments described with reference to one aspect of the invention may be generally applicable to any of the other aspects of the invention.

[0180] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “compound of Formula (I)” includes a single compound as well as two or more of the same or different compounds; reference to an “excipient” includes a single excipient as well as two or more of the same or different excipients, and the like.

[0181] The word “about” means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as “about 49, about 50, about 55, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. The present invention will now be further described with reference to the following non-limiting Examples and Figures in which:

[0182] Figure 1 shows reducing SDS PAGE of the Sord.8, Sord.15 and Sord.17 proteins. The arrow indicates the presence of the proteins at the correct sizes. BSA (Bovine Serum albumin) is used as a control. MW = Molecular weight marker with sizes (kilodalton = kDa) shown on the left hand side.

[0183] Figure 2 shows an enzyme activity assay line graph with the wt (wild type) Sord Enzyme versus Sord.8, Sord.15 and Sord.17 fusion proteins at indicated concentrations. The fusion proteins are all far more active at various concentrations compared to the wild type Sord enzyme.

[0184] Figure 3 shows the enzyme activity assay at various concentrations in a bar graph for the wild type (wt) Sord enzyme protein versus the fusion Sord proteins Sord.8, Sord.15 and Sord.17. All 3 fusion proteins showed far superior activity at various concentrations versus the wild type Sord enzyme. Graphing performed by GraphPad Prism with ttest comparison between vehicle and dosed groups at each timepoint. Error bars as SEM. ttest with n / s = non-significant; **p < 0.001 , ***p <0.0001

[0185] Figure 4_shows rhSord.8 enzyme candidate Sorbitol urine levels (uM) over time (i.p. and s.c. groups) in BI / 6 male mice over 12 days. Dosing points indicated by blue arrows (on days 3, 5, 7,9,11). Graphing performed by GraphPad Prism with ttest comparison between vehicle and dosed groups at each timepoint. Error bars as SEM. ttest with n / s = non-significant; **p < 0.001 , ***p <0.0001

[0186] Figure 5 shows rhSord.8 and rhSord.17 enzyme candidate Sorbitol blood levels (micromolar - uM) at 24 hrs post-dose (i.p. and s.c. groups) in BI / 6 male mice. Graphing performed by GraphPad Prism with ttest comparison between vehicle and dosed groups at each timepoint. Error bars as SD. ttest with n / s = non-significant; **p < 0.001 , ***p <0.0001 , ****p < 0.00001

[0187] Figure 6 shows the role of the Sorbitol Dehydrogenase in the Glucose detoxification pathway. Sorbitol dehydrogenase catalyzes the oxidation of D-sorbitol to d-fructose with the cofactor NAD, producing NADH. Figure 7 shows the three dimensional structure of the sorbitol dehydogenase tetramer.

[0188] SEQ ID NO. 5. (rhSORD.8):

[0189] N-terminal extension with a cysteine (first 3 residues, bold) + linker (underline) - thioredoxin (italics) - linker His sequence (underline) - Sord protein

[0190] MACGGGGS MVKQIESKTA FQEALDAAGD KLVVVDFSATWCGPCKMIKP FFHSLSEKYS NVIFLEVDVDDCQDVASECE VKCMPTFQFF KKGQKVGEFS GANKEKLEA TIN EL VGGGGSGGGGSM AH H H H HGGGGSM AAAAKPN N LSLVVHG PGDLRLENYPIPEPGPNEVLLRMHSVGICGSDVHYWEYGRIGNFIVKKPMVLGHE ASGTVEKVGSSVKHLKPGDRVAIEPGAPRENDEFCKMGRYNLSPSIFFCATPPDD GNLCRFYKHNAAFCYKLPDNVTFEEGALIEPLSVGIHACRRGGVTLGHKVLVCGA GPIGMVTLLVAKAMGAAQVWTDLSATRLSKAKEIGADLVLQISKESPQEIARKVEG QLGCKPEVTIECTGAEASIQAGIYATRSGGNLVLVGLGSEMTTVPLLHAAIREVDIK GVFRYCNTWPVAISMLASKSVNVKPLVTHRFPLEKALEAFETFKKGLGLKIMLKCD PSDQNP**

[0191] SEQ ID NO. 6 ( rhSORD.17)

[0192] MPTLK uptake tag (first 5 residues, bold) - Linker / His (underline)-Thioredoxin fusion (itlaics)- linker and His runs (underline) - SORD protein

[0193] MPTLKGGGGSHHHHMVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKP FFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKE KLEA TINEL VGGGGSGGGGSHHHHHMAHHHHHGGGGSMAAAAKPN N LSLVVHG PGDLRLENYPIPEPGPNEVLLRMHSVGICGSDVHYWEYGRIGNFIVKKPMVLGHE ASGTVEKVGSSVKHLKPGDRVAIEPGAPRENDEFCKMGRYNLSPSIFFCATPPDD GNLCRFYKHNAAFCYKLPDNVTFEEGALIEPLSVGIHACRRGGVTLGHKVLVCGA GPIGMVTLLVAKAMGAAQVVVTDLSATRLSKAKEIGADLVLQISKESPQEIARKVEG QLGCKPEVTIECTGAEASIQAGIYATRSGGNLVLVGLGSEMTTVPLLHAAIREVDIK GVFRYCNTWPVAISMLASKSVNVKPLVTHRFPLEKALEAFETFKKGLGLKIMLKCD PSDQNP**

[0194] SEQ ID NO. 7 (rhSORD.15) MPTLK uptake tag (first 5 residues, bold) - Linker (underline) - Thioredeoxin fusion (italics) - linker and his runs (underline) - SORD protein

[0195] M PTLKGGGGSMVKQIESKTA FQEALDAAGD KLVVVDFSAT WCGPCKMIKP FFHSLSEKYS NVIFLEVDVDDCQDVASECE VKCMPTFQFF KKGQKVGEFS GANKEKLEA TIN EL \ / GGGGSGGGGSM AH H H H HGGGGSM AAAAKPN N LSLVVHG PGDLRLENYPIPEPGPNEVLLRMHSVGICGSDVHYWEYGRIGNFIVKKPMVLGHE ASGTVEKVGSSVKHLKPGDRVAIEPGAPRENDEFCKMGRYNLSPSIFFCATPPDD GNLCRFYKHNAAFCYKLPDNVTFEEGALIEPLSVGIHACRRGGVTLGHKVLVCGA GPIGMVTLLVAKAMGAAQVWTDLSATRLSKAKEIGADLVLQISKESPQEIARKVEG QLCKPEVTIECTGAEASIQAGIYATRSGGNLVLVGLGSEMTTVPLLHAAIREVDIKG VFRYCNTWPVAISMLASKSVNVKPLVTHRFPLEKALEAFETFKKGLGLKIMLKCDP SDQNP**

[0196] Example 1

[0197] The protein was produced as described below (‘Production and purification of rhSord.8, rhSord.15 and rhSord.17’). For the SDS Page analysis, 2 ug of purified protein was applied per well with 2 ug of BSA protein under reducing conditions. The molecular weight marker used was Protein Marker, Bio-rad, Cat. No. 1610374S.

[0198] Production and purification of rhSord.8, rhSord 15 and rhSord 17

[0199] Fermentation: pET 30a+ vector in BL21 Star (DE3) cells were used. They were transformed with recombinant pET 30a vector containing gene of interest. A single colony was inoculated in to LB medium containing kanamycin; cultures were incubated at 37 degrees Celsius at 200 rpm. Once cell density reached OD = 0.6 - 0.8, 0.5mM IPTG was introduced for for 16 h at 15 degrees Celsius OR 4 h at 37 degrees Celsius. Cells were spun down and following centrifugation, cells were lysed and subsequently sonicated with lysis buffer (50 mM Tris-HCI, 500mM NaCI, 1mM TCEP, 1% Triton X-100, 0.1 % TX-114, pH 8.0)

[0200] Purification: 1ststep Nickel column purification, with 20mM Imidazole wash and elution with 50 - 250 mM imidazole. Further steps following include buffer exchanging and concentration with MW cut off columns to further improve purity. These were performed with PD-10 columns and Millipore concentration columns with a 30 kDa MW cutoff. Dialysis with MW cut off may also be performed.

[0201] Final buffer formulation is: 0.1 M NasPCU, 10% Trehalose, 5% trehalose, 0.1 % tween 80, 0.6M L-Arginine, 200 uM ZnSO4 (pH 7.1 to pH 8))

[0202] Example 2 in Vitro activity

[0203] Assay principle: This assay measures the ability of the enzyme candidates to utilise sorbitol to form NADH from NAD+ rhSord8, rhSord15 and rhSord17 were tested and compared to wt Sord.

[0204] Assay method: The Assay reaction buffer was composed of pH7.1 Tris HCI containing 10 % trehalose, 20 mM NAD+, 200 uM methoxy-PMS, 1mM WST-8, 500 mM sorbitol. Each candidate was diluted 1 / 2 from 7.5 ug / well at the top of the curve. 30 ul of enzyme + 70 ul of reaction buffer used in this assay. Incubated for 10 mins at 37 degrees Celsius and read at OD 450 nm.

[0205] Results

[0206] The fusions (rhSord.8, rhSord.17 and rhSord.15) were clearly more active and far superior to the wild type Sord enzyme in this competitive in vitro sorbitol assay.

[0207] EC50 formula used and analysis

[0208] Example 3 in vivo efficacy study

[0209] Aims: to test the in vivo efficacy and safety of the rhSord.8. Methods: BI / 6 male mice were given 10% sorbitol in drinking water (ad libitum). They were split in to the following groups: vehicle, 30 mg / kg intravenous rhSord.8 dosed, 30 mg / kg subcutaneous rhSord.8 dosed. Mice were dosed on days 3, 5, 7, 9, 11. There were 6 animals in the vehicle group, 5 animals in the i.p. injected group and 3 animals in the s.c. injected group. Urines for sorbitol analysis were taken one days 10 (24 hr post-dose), day 11 (4 hours post dose) and day 12 (24 hours post dose). Method of urinary detection in urine: Urines were taken at timepoints indicated (day 0, day 10, day 11 , day 12), spun down for 5 mins at 10,000 rpm on a benchtop centrifuge, with the supernatant removed. Urines were assayed with an in-house sorbitol assay. The assay for sorbitol was conducted as follows.

[0210] The reaction buffer was composed of: 0.1 M Tris-HCI, pH 7.1 , 10 mM NAD+, 1 mM ZnS04, 0.5 ug / ul rhSord enzyme, 1 mM WST-8, and 200 uM Methoxy-PMS.

[0211] Urines, aftering being spun down for 5 minutes at 10,000 rpm, were diluted between 1 / 8 and 1 / 10 in PBS, and 20 ul of diluted urine was added to 80 ul of Reaction buffer, and the plates placed in an incubator at 37 degrees Celsius. A sorbitol standard curve was performed on every plate. The reactions were read between 45 - 60 mins post the start of incubation. Plates were read at OD 450 on a Byony absorbance plate reader.

[0212] Results: rhSord.8 was highly effective at lowering sorbitol in both the i.p. and s.c. dosed groups at all timepoints measured. No safety issues were noted in any group

[0213] Example 4 in vivo efficacy study

[0214] Aim: To investigate the efficacy in lowering sorbitol blood levels of the recombinant human SORD enzyme candidates, rhSord.8 and rhSord.17 in a Sorbitol overload mouse model with both i.p. and s.c. routes Methods:

[0215] BI / 6 male mice 2 - 4 months of age were given 10% sorbitol in drinking water (ad libitum) at the start of the study. They were on normal mouse chow (Altromin 1311) for the duration of the study. The mice were split in to the following groups (5 animals per group): vehicle, rhSord.8 i.p, rhSord.8 s.c., rhSord.17 i.p., rhSord.17 s.c. Mice were dosed on day 12 with 80 mg / kg or vehicle control following introduction of the 10% sorbitol in water. 24 hours post-dose, blood was taken and mice culled and examined for sorbitol levels, gross pathology and blood biochemistry.

[0216] Blood sorbitol assay: Whole blood was taken at 24 hours post-dose, spun down for 5 mins at 10,000 rpm on a benchtop centrifuge and blood plasma taken. The assay for sorbitol was conducted as follows.

[0217] The reaction buffer was composed of: 0.1 M Tris-HCI, pH 7.1 , 20 mM NAD+, 200 uM ZnSo4, 0.6 ug / ul rhSord enzyme, 1 mM WST-8, and 200 uM Methoxy-PMS

[0218] 20 ul of plasma or standard + 80 ul of Reaction buffer was added to each well. Plates were placed in an incubator at 37 degrees Celsius. A sorbitol standard curve was performed. The reactions were read at 90 mins post the start of incubation. Plates were read at OD 450 on a Byony absorbance plate reader.

[0219] Results:

[0220] The rhSord.8 and rhSord.17 fusion enzyme therapeutic candidates were both highly effective when injected at 80 mg / kg either subcutaneously or intraperitoneal at 24 hours post-dose in a sorbitol overload mouse model. Both candidates were found to be highly effective in terms of significantly lowering sorbitol blood levels compared to vehicle control animals.

[0221] SEQ ID NO. 1

[0222] GANKEKLEATINELVAHHHHHGGGGSMAAAAKPNNLSLWHGPGDLRLENYPIPE PGPNEVLLRMHSVGICGSDVHYWEYGRIGNFIVKKPMVLGHEASGTVEKVGSSVK HLKPGDRVAIEPGAPRENDEFCKMGRYNLSPSIFFCATPPDDGNLCRFYKHNAAF CYKLPDNVTFEEGALIEPLSVGIHACRRGGVTLGHKVLVCGAGPIGMVTLLVAKAM GAAQVVVTDLSATRLSKAKEIGADLVLQISKESPQEIARKVEGQLGCKPEVTIECTG AEASIQAGIYATRSGGNLVLVGLGSEMTTVPLLHAAIREVDIKGVFRYCNTWPVAIS MLASKSVNVKPLVTHRFPLEKALEAFETFKKGLGLKIMLKCDPSDQNP**

[0223] SEQ ID NO. 2 (DNA sequence encoding sorbitol dehydrogenase (stop codon underlined)

[0224] ATGGCTGCGGCTGCTAAGCCGAATAACTTGAGCCTCGTGGTGCACGGCCCAG GCGATCTCCGCCTGGAGAACTATCCGATTCCGGAACCGGGTCCAAACGAAGT TCTGCTGCGTATGCACTCCGTCGGCATCTGCGGTAGCGACGTTCATTATTGGG AATATGGTCGTATTGGCAACTTTATCGTGAAGAAGCCGATGGTTTTGGGCCAC GAAGCGAGCGGCACAGTAGAAAAGGTAGGTAGCTCCGTCAAACATCTGAAAC CGGGCGATCGTGTTGCCATCGAGCCAGGCGCTCCGCGCGAAAATGATGAATT CTGCAAGATGGGTCGTTACAACCTGTCCCCGAGCATTTTCTTCTGCGCAACTC CGCCTGACGATGGTAATCTTTGCCGCTTTTATAAACACAACGCGGCGTTTTGC

[0225] TACAAACTGCCGGATAACGTTACCTTCGAAGAAGGTGCCCTGATTGAGCCGCT

[0226] GAGCGTTGGCATCCACGCGTGTAGACGTGGTGGTGTTACCCTGGGCCATAAG

[0227] GTGTTGGTATGTGGTGCCGGTCCGATTGGCATGGTGACGTTGTTGGTGGCGA

[0228] AAGCAATGGGCGCCGCTCAAGTTGTTGTGACCGATTTATCTGCGACCCGTCTG

[0229] AGCAAAGCGAAGGAGATCGGGGCGGATCTGGTCCTACAAATTTCGAAGGAGT

[0230] CCCCGCAGGAGATCGCCCGTAAAGTCGAGGGTCAGCTGGGGTGCAAGCCGG

[0231] AGGTGACCATTGAATGCACCGGTGCGGAAGCGTCTATCCAGGCTGGTATTTAC

[0232] GCAACTCGCAGCGGTGGTAACTTGGTGCTGGTCGGTCTGGGTAGCGAAATGA

[0233] CCACCGTTCCGTTACTGCACGCCGCGATCCGTGAAGTGGACATCAAAGGTGT

[0234] TTTTCGCTACTGCAACACCTGGCCGGTTGCGATTTCCATGCTGGCATCCAAGA

[0235] GTGTTAATGTGAAACCGTTGGTGACCCATCGTTTTCCGCTGGAGAAGGCACTG

[0236] GAGGCGTTCGAGACGTTCAAAAAGGGCCTGGGTCTGAAAATTATGCTGAAGT

[0237] GCGATCCGAGCGATCAGAACCCGTAA

[0238] SEQ ID NO. 3

[0239] MVKQIESKTAFQEALDAAGDKLWVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLE

[0240] VDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV

[0241] SEQ ID NO. 4

[0242] ATGGTAAAGCAGATCGAGTCGAAAACCGCATTTCAAGAAGCTCTGGACGCGG

[0243] CGGGTGACAAACTGGTTGTTGTGGACTTCAGCGCGACGTGGTGTGGTCCGTG

[0244] TAAAATGATCAAACCGTTTTTCCATAGCCTTTCGGAGAAGTACAGCAACGTGAT

[0245] CTTTCTGGAGGTGGACGTTGACGACTGTCAGGACGTGGCTAGCGAGTGCGAA

[0246] GTTAAGTGCATGCCGACCTTCCAATTTTTCAAAAAAGGTCAAAAGGTGGGTGA

[0247] ATTCAGTGGCGCAAATAAAGAGAAGTTGGAGGCCACGATCAATGAACTGGTC

Claims

Claims:1 . A fusion protein comprising sorbitol dehydrogenase and thioredoxin.

2. A fusion protein according to claim 1 for use in the prevention or reduction of excess sorbitol in a subject.

3. A fusion protein according to claim 1 or for use according to claim 2, wherein the sorbitol dehydrogenase is human.

4. A fusion protein according to claim 3 wherein the sorbitol dehydrogenase is encoded by the nucleic acid sequence of SEQ ID NO. 2 or a sequence having 90%, 95%, 97%, 98% or 99% sequence identity with nucleic acid sequence of SEQ ID NO. 2, across the full length of the nucleic acid sequence, or over a continuous window of 300 to 350 amino acids.

5. A fusion protein according to claim 3 or 4 wherein the sorbitol dehydrogenase is the human sorbitol dehydrogenase of SEQ ID NO. 1 , or a sequence having 90%, 95%, 97%, 98% or 99% sequence identity with the sorbitol dehydrogenase protein of SEQ ID NO. 1 , across the full length of the native protein, or over a continuous window of 300, 310, 320, 330, 340, or 350 amino acids.

6. A fusion protein according to any one of claims 1 to 5 or a fusion protein for use according to any one of claims 2 to 5, wherein the sorbitol dehydrogenase enzyme lacks the N terminal methionine residue.

7. A fusion protein according to any one of the preceding claims or a fusion protein for use according to any one of claims 2 to 6 wherein the thioredoxin is the human of SEQ ID NO. 3 or a variant thereof which 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a the thioredoxin protein of SEQ ID NO. 3, over a continuous window of 80 to 100 amino acid residues.

8. A fusion protein according to any one of the preceding claims or a fusion protein for use according to any one of claims 2 to 7 wherein the thioredoxin sequence may be provided at the N terminal of the sorbitol dehydrogenase enzyme.

9. A fusion protein according to any one of the preceding claims or a fusion protein for use according to any one of claims 2 to 8 wherein a first linkersequence is present between the sorbitol dehydrogenase enzyme and the thioredoxin protein.

10. A fusion protein according claim 9 or a fusion protein for use according to claim 9 wherein the first linker sequence is 10 to 32 amino acids in length, preferably 11 , 22 or 27 amino acids in length.

11. A fusion protein, or a fusion protein for use, according to claim 9 or 10, wherein the first linker comprises one, two or three GGGG sequences.

12. A fusion protein, or a fusion protein for use, according to claims 9 to 11 wherein the first linker comprises two or more HHHHH sequences.

13. A fusion protein, or a fusion protein for use, according to any one of claims 9 to 12 wherein the first linker comprises the sequence GGGG - HHHHH - GGGG orGGGG - HHHHH.

14. A fusion protein, or a fusion protein for use, according to any one of clams 9 to 13 wherein the first linker comprises or consists of the sequence GGGGXGGGGXHHHHHXGGGGX (SEQ ID NO. 11) , orGGGGXGGGGXHHHHHXHHHHHXGGGGX (SEQ ID NO. 12) or XHHHHHGGGGX (SEQ ID NO. 13) where each X is independently 0, 1 , 2 or 3 amino acids; wherein each instance of X may be independently selected from S, or M, or A or MA, or a combination thereof.

15. A fusion protein, or a fusion protein, for use according to claim 14 wherein the first linker comprises or consists of the sequence GGGGSGGGGSXHHHHHXGGGGS (SEQ ID NO. 14), GGGGSGGGGSHHHHHXHHHHHXGGGGS (SEQ ID NO. 15) or XHHHHHGGGGS (SEQ ID NO. 16) where each X is independently 0, 1 , 2 or 3 amino acids.

16. A fusion protein, or a fusion protein for use, according to claim 15 wherein the first linker comprises or consists of the sequence GGGGSGGGGSMAHHHHHGGGGS (Sord 15; SEQ ID NO. 8), GGGGSGGGGSHHHHHMAHHHHHGGGGS (Sord 17) SEQ ID NO. 9), AHHHHHGGGGS (Sord 8) SEQ ID NO. 10).

17. A fusion protein, or a fusion protein for use, according to any one of clams 8 to 16, wherein the fusion protein further comprises an N terminal extension sequence.

18. A fusion protein, or a fusion protein for use, according to claim 17 wherein theN terminal extension sequence is a His tag, an uptake tag, a signal peptide, an N terminal methionine, an affinity tag for purification, a leader sequence, a sequence for directing protein secretion or subcellular targeting or organ / tissue targeting, or a protein or sequence for stabilising and / or solubilising the fusion protein, or a protein for increasing in vivo half-life.

19. A fusion protein, or a fusion protein for use, according to claim 18 wherein the tag is an uptake tag, and is selected from MAC or MPTLK.

20. A fusion protein, or a fusion protein for use, according to any one of claims 17 to 19 wherein the N terminal extension is an N terminal methionine residue, optionally with a cysteine residue.21 . A fusion protein, or a fusion protein for use, according to claim 20 wherein the N terminal extension sequence is MAC.

22. A fusion protein, or a fusion protein for use, according to any one of claims 17 to 21 , wherein the fusion protein comprises or consists of the sequence MAC -Thioredoxin-SEQ ID 10-sorbitol dehydrogenase, MPTLK - Thioredoxin - SEQ ID 8 or 9- sorbitol dehydrogenase.

23. A fusion protein, or a fusion protein for use, according to any one of claims 17 to 22, wherein the fusion protein comprises a second linker sequence comprising GGGG, wherein the second linker is provided between the thioredoxin protein and the N terminal extension sequence.

24. A fusion protein, or a fusion protein for use, according to claim 23, wherein the second linker comprises or consists of the sequence GGGGS.

25. A fusion protein, or a fusion protein for use, according to claim 23 or 24 wherein the second linker comprises or consists of the sequence GGGGHHHH or GGGGSHHHH.

26. A fusion protein, or a fusion protein for use, according to any one of claims 17 to 25, wherein the fusion protein comprises or consists of the sequence of SEQ ID NO. 5, 6 or 7.

27. A fusion protein according to any one of claims 1 to 26 or a fusion protein for use according to any one of claims 2 to 26, wherein the fusion protein is pegylated.

28. A fusion protein according to any one of claims 1 to 27 or a fusion protein for use according to any one of claims 2 to 27, wherein the fusion protein is a tetramer, preferably comprising four identical monomer units29. A fusion protein according to any one of the preceding claims or a fusion protein for use according to any one of claims 2 to 28, wherein the fusion protein is recombinant.

30. A pharmaceutical or nutritional composition comprising sorbitol dehydrogenase, wherein the composition comprises one or more physiologically or pharmaceutically acceptable carriers or excipients.

31. A composition according to claim 30, wherein the sorbitol dehydrogenase is a fusion protein as defined in any one of claims 1 to 29.

32. A composition according to claim 31 , wherein the composition is provided in a unit dose form selected from a tablet, capsule or single-dose vial.

33. A food comprising a fusion protein as defined in any one of claims 1 to 29.

34. A kit comprising (a) a fusion protein as defined in any one of claims 1 to 29 and (b) a further therapeutic agent.

35. A nucleic acid sequence encoding a fusion protein as defined in any one of claims 1 to 29.

36. A nucleic acid sequence according to claim 35, wherein the nucleic acid sequence comprises or is operably linked to a regulatory sequence, preferably selected from a transcriptional promoter, enhancer, translational signal, or transcriptional and translational termination signal.

37. An expression vector comprising a nucleic acid molecule as defined in claim 35 or 36.

38. A host cell comprising a nucleic acid molecule or expression vector as defined in claims 35 to 37.

39. A host cell according to claim 38 wherein the cell is a bacterial cell, preferably E. coli, or a eukaryotic cell, preferably a yeast cell or a mammalian cell.

40. A method of treatment of a disorder which results in excess sorbitol in a subject, wherein the method comprises administering a therapeutically effective amount of sorbitol dehydrogenase to a subject in seed thereof.

41. A method according to claim 40 wherein the sorbitol dehydrogenase is a fusion protein as defined in any one of claims 1 to 29, or a composition as defined in claim 30 to 32, a food according to claim 33, or a nucleic acid or expression vector according to claims 35 to 37.

42. Sorbitol dehydrogenase for use in the treatment of a disorder which results in excess sorbitol in a subject.

43. Sorbitol dehydrogenase for use according to claim 41 , wherein the sorbitol dehydrogenase is a fusion protein as defined in any one of claims 1 to 29 or a composition according to any one of claims 30 to 32 or a food according to claim 33 a nucleic acid or expression vector according to claims 35 to 37, or a host cell according to claim 38 or 39.

44. A method for treatment of excess sorbitol in a subject, wherein the method comprises administering a therapeutically effective amount of sorbitol dehydrogenase to a subject in need thereof.

45. A method according to claim 44 wherein the sorbitol dehydrogenase is a fusion protein as defined in any one of claims 1 to 29, or a composition according to any one of claims 30 to 32 or a food according to claim 33 a nucleic acid or expression vector according to claims 35 to 37, or a host cell according to claim 38 or 39.

46. Sorbitol dehydrogenase for use in the prevention or reduction of excess sorbitol in a subject.

47. Sorbitol dehydrogenase for use according to claim 46, wherein the sorbitol dehydrogenase is a fusion protein as defined in any one of claims 1 to 29 or or a composition according to any one of claims 30 to 32 or a food according to claim 33 a nucleic acid or expression vector according to claims 35 to 37, or a host cell according to claim 38 or 39.

48. A method according to claim 40, or sorbitol dehydrogenase for use according to claim 42, wherein the disorder is diabetic neuropathy or tissue damage due to sorbitol overload or sorbitol dehydrogenase deficiency resulting from a genetic abnormality.

49. A method according to claim 40 or 45, or sorbitol dehydrogenase for use according to claim 42 or 46 wherein a subject is a human child or a human adult.

50. A method according to claim 45 or sorbitol dehydrogenase for use according to claim 46 wherein the subject has one or more symptoms of excess sorbitol selected from cataracts, neuropathy including diabetic neuropathy, retinopathy including diabetic retinopathy, cardiomyopathy, nephropathy including diabetic nephropathy, microvascular complications, physical deformities (for example of limbs, such as hand, foot, arm, leg), physical disability including foot tremors, limb weakness and difficulty walking, reduced or impaired mobility, sensory impairment, atherosclerosis and othercardiovascular complications, albuminuria, hyperglycaemia and diabetes, shortness of breath and lung issues; muscle effects including muscle weakness; digestive system disorders including diarrhoea; signs of allergic reactions such as rash, itching, wheezing, throat issues; brain effects such as mood alteration, confusion and / or brain disorders associated with these symptoms.