Fusion protein comprising glutaryl COA dehydrogenase and thioredoxin

Abstract

The present invention provides a novel form of glutaryl CoA dehydrogenase, and its use in the prevention and / or treatment of glutaric aciduria type I.

Description

[0001] FUSION PROTEIN COMPRISING GLUTARYL COA DEHYDROGENASE AND THIOREDOXIN

[0002] Introduction

[0003] The present invention relates to a novel fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin, and the use of the novel fusion protein in the treatment of glutaric aciduria type I. Also provided is a nucleic acid molecule encoding a fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin. The present invention also provides a composition comprising a fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin or a nucleic acid molecule encoding the same; and a host cell comprising a fusion protein comprising glutaryl CoA 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] Glutaryl CoA dehydrogenase (“GCDH”; EC 1.3.99.7) catalyzes the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA and carbon dioxide in the degradative pathway of L-lysine, L-hydroxylysine, and L-tryptophan metabolism.

[0007] Mutations or deficiencies in the GCDH gene can lead to defects in the enzyme encoded by it, which lead to the formation and accumulation of lysine, tryptophan, hyroxylysine, the metabolites glutaryl CoA, glutaric acid and 3-hydroxyglutaric acid as well as glutarylcarnitine in body fluids. The accumulation of any of these may lead to glutaric aciduria type I, a rare autosomal recessive metabolic disorder. Symptoms of this disease include macrocephaly, acute encephalitis-like crises, spasticity, dystonia, choreoathetosis, ataxia, dyskinesia and seizure. The disorder typically develops in early childhood, at an instance of approx. 1 : 40,000. Prevention of metabolic acidosis appears key to long term outcomes, and as such GA1 is included in newborn screening in an increasing number of countries. Without treatment, most affected children develop an acute encephalopathic crisis (AEC) following episodes of fever or colds. Such children may be hospitalised, placed on protein restriction (for 12-24 hours) with the provision of calories as IV glucose at high concentrations with IV intralipids, insulin, carnitine and if necessary supportive neurological care. An AEC crisis may result in bilateral striatal injury and consequently, dystonic movement disorder (MD), within the first three years of life. Typically, GCDH enzyme activity in a patient with GA1 is 0-10% compared to the enzyme activity in tissue of a healthy subject.

[0008] Currently, chronic GA1 is treated by putting a patient on a low protein diet, balanced with supplementation of a lysine-free, tryptophan-reduced, and arginine-containing formula, carnitine and riboflavin.

[0009] Despite early identification via newborn screening programs and early dietary treatment, the longer term outcomes are still poor, including death (11% at early age 6 - 10 yrs). 66% of patients were dead in a long term study by 44 yrs of age. Many suffer epilepsy to 6 yrs of age, and around a quarter of patients develop a movement disorder, primarily dsystonia. Patients may still develop (progressive) neurologic disease in the absence of any reported acute crisis.

[0010] CN 1331304 describes the use of a novel polypeptide for use in treating glutaric aciduria. The novel polypeptide is described therein as “glutaryl CoA dehydrogenase 16.17” based on the expression profile being similar to GCDH. Based on a BLAST alignment, the polypeptide described in CN 1331304 has no sequence similarity to that of GCDH of EC 1.3.99.7.

[0011] WO2021 / 256579 describes a composition comprising human glutaryl CoA dehydrogenase, for treating glutaric aciduria. The human enzyme described in the application is produced in CHO cells.

[0012] There remains a need for a commercially available and cost effective treatment for GA1. The present invention seeks to address this need and is based on the concept of using a novel form of glutaryl CoA dehydrogenase for enzyme replacement therapy.

[0013] Summary of the invention

[0014] In a first aspect, there is provided a fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin. Suitably, the fusion protein is recombinant. In a second aspect, there is provided a method for treatment of glutaric acidura type 1 (GA1) in a subject, wherein the method comprises administering a therapeutically effective amount of a fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin to the subject in need thereof. Also provided is a fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin for use in the treatment of glutaric acidura type 1 (GA1) in a subject.

[0015] In a third aspect, the present invention provides a nucleic acid molecule encoding a fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin. The nucleic acid molecule may be provided in an expression vector. Also provided is a host cell or a cell free system comprising a nucleic acid molecule or expression vector of the invention. A host cell may be a microbial cell, suitably a bacterial cell, suitably E. coli. The nucleic acid molecule, expression vector and / or host cell or cell free system may be used in the recombinant manufacture of a fusion protein of the present invention.

[0016] In a fourth 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. A composition may be a pharmaceutical or nutritional composition.

[0017] In a fifth 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.

[0018] Detailed description of the invention

[0019] The present invention is based upon the development of a novel form of glutaryl CoA dehydrogenase, which is suitable for recombinant manufacture and which has been shown to be effective in vivo at reducing glutaryl CoA levels. Whilst enzyme replacement therapy has been suggested for treatment of glutaryl CoA dehydrogenase deficiency, there are difficulties in producing the wild type enzyme recombinantly for therapeutic purposes. Thus, an enzyme which is suitable for large scale manufacture and also suitable for therapeutic use has not, to date, been available.

[0020] The present invention is based upon the generation of a novel fusion protein which comprises the enzyme, glutaryl CoA dehydrogenase, and thioredoxin. This fusion protein has been shown to be capable of being manufactured recombinantly, and allows the enzyme to be manufactured on a commercial scale. The fusion protein has been shown to be stable, and can be recombinantly produced in E. coli. In contrast, the wild type enzyme is poorly expressed in E. coli, and difficult to manufacture.

[0021] A recombinant fusion protein comprising glutaryl CoA dehydrogenase which is capable of being manufactured and reducing glutaryl CoA levels in vivo, provides for the first time an effective treatment of glutaric aciduria type 1.

[0022] Definitions

[0023] 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.

[0024] 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.

[0025] An enzyme is a biological catalyst, capable of accelerating a reaction between one or more substrates to produce a product.

[0026] 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.

[0027] “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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] A “non-conservative substitution” is a substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] “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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 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.

[0043] The term “subject” and “patient” are used interchangeably herein and refer to both human and non-human animals, for example as described herein.

[0044] The term “enzyme replacement therapy (ERT)” refers to medical treatment which replaces an enzyme that is deficient or absent in the body.

[0045] 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.

[0046] The N-terminus (also known as the amino-terminus, NH2-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.

[0047] 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.

[0048] The term “glutaryl CoA dehydrogenase” as used herein includes any protein having glutaryl CoA dehydrogenase activity. Suitably, the protein may be “a protein having Glutaryl CoA Dehydrogenase (GCDH) activity”.

[0049] 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.

[0050] Glutaryl CoA dehydrogenase (GCDH)

[0051] Glutaryl CoA dehydrogenase is a mitochondrial, non-glycosylated protein, formed of four identical monomer subunits in a dimer-dimer configuration to form a tetramer of about 4.5kDa. The substrate-binding pocket is filled with a string of three water molecules, which gets displaced when the substrate binds to the enzyme.

[0052] GCDH is responsible for the oxidative decarboxylation of glutaryl-CoA to crotonyl- CoA and carbon dioxide, which is part of the process of mitochondrial oxidation of the amino acids lysine, tryptophan, and hydroxylysine. The enzyme mediates the conversion of glutaryl-CoA to crotonyl-CoA through a series of physical, chemical, and electron-transfer steps, via the intermediate glutaconyl CoA. Therefore, GCDH may be referred to as an enzyme which has the ability to oxidise glutaryl CoA to glutaconyl CoA and / or crotonyl-CoA, for example as shown in Figure 1 . Suitably, such an enzyme may fall within enzyme classification number EC 1.3.8.6 (previously EC 1 .3.99.7). Glutaryl CoA dehydrogenase activity may be determined or measured using any suitable assay available to a person skilled in the art. For example, a direct measure of enzyme activity may be to measure the reduction of a DCIP (Dichloroindophenol sodium), which causes a color change that can be read at approximately absorbance OD 600 - OD 620. Glutaryl CoA dehydrogenase may also be referred to as ACAD5 and GCD.

[0053] Glutaryl CoA dehydrogenase is expressed and found in the mitochondria. Its main expression is in somatic tissues, such as liver, kidney and fat. Herein, reference to the glutaryl CoA dehydrogenase of the fusion protein of the invention may include any glutaryl CoA dehydrogenase enzyme from any organism, including plant, microbial, mammal or non-mammal. The glutaryl CoA 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 glutaryl CoA dehydrogenase is a mammalian enzyme, most suitably a human glutaryl CoA dehydrogenase. The degree of structural and functional homology between glutaryl CoA dehydrogenase from different sources is high, for example the human enzyme shares 99.8% homology with the pygmy chimpanzee, 99.3% with the Gorilla, 90% with the pig, 89.7% with the dog, 87.7% with the cow, 86.4% with the mouse, 79% with the mainland tiger snake, 74.7% with the gilthead sea bream fish, around 58% homology with the quercus suber (cork oak) plant, 73% homology with the fungi Zopfochytrium polystomum, and 73.85% from the bacteria Dongi deserti. Therefore, non-human enzymes, such as other animal, plant, bacteria or fungi may, in certain circumstances, be used interchangeably with a human or mammalian glutaryl CoA dehydrogenase. Nucleic acid and amino acid sequences of glutaryl CoA 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 glutaryl CoA dehydrogenase may be any enzyme falling within classification EC 1.3.8.6 (previously EC 1.3.99.7). The glutaryl CoA dehydrogenase may be the human sequence, as set forth in SEQ ID NO. 1. The glutaryl CoA dehydrogenase may be encoded by the human glutaryl CoA dehydrogenase gene of SEQ ID NO. 2. Non-human examples of glutaryl CoA dehydrogenase include, as described above, include non-human animals such as monkey, pig, bat, cow, sheep; alternatively plant, fungi, bacterial or microbial.

[0054] Also included are variants of a native glutaryl CoA dehydrogenase enzyme. The term “variant” as used herein refers to a polypeptide comprising an alteration of the primary structure of the polypeptide of glutaryl CoA dehydrogenase. Therefore, the glutaryl CoA dehydrogenase enzyme of the fusion protein of the invention may include sequence variants and fragments of the native glutaryl CoA dehydrogenase sequence. A variant glutaryl CoA dehydrogenase protein may comprise one or more amino acid substitutions compared to the native glutaryl CoA dehydrogenase protein sequence. Therefore, the glutaryl CoA 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 glutaryl CoA dehydrogenase protein. Suitably, the glutaryl CoA 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 glutaryl CoA dehydrogenase of SEQ ID NO. 1. Suitably, a sequence variant of glutaryl CoA 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 reduction of DCIP (Dichloroindophenol sodium) in the presence of a GCDH substrate.

[0055] 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, 350, 360, 370, 380 or 390 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 glutaryl CoA dehydrogenase of the fusion protein of the present invention may be a fragment of a full length glutaryl CoA dehydrogenase protein, for example the human glutaryl CoA dehydrogenase of SEQ ID NO. 1. Such a fragment may comprise one or more amino acid deletions compared to the native glutaryl CoA 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 oxidise glutaryl CoA to glutaconyl CoA and / or crotonyl-CoA. Suitably, a fragment of glutaryl CoA 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 reduction of DCIP (Dichloroindophenol sodium) in the presence of a GCDH substrate.

[0056] “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.

[0057] 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.

[0058] A fragment of a glutaryl CoA dehydrogenase enzyme may be a mature form a of native glutaryl CoA 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 glutaryl CoA dehydrogenase sequence may comprise a sequence which is the protein of SEQ ID NO. 1 , or which is the glutaryl CoA dehydrogenase of SEQ ID NO. 1 which lacks the N terminal methionine residue.

[0059] It is well within the capabilities of a skilled person to modify a glutaryl CoA 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 glutaryl CoA 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 glutaryl CoA dehydrogenase enzyme to oxidise glutaryl CoA to glutaconyl CoA and / or crotonyl- CoA. As described above, glutaryl CoA dehydrogenase in its active form is a tetramer comprising four identical monomer units. The glutaryl CoA dehydrogenase of SEQ ID NO. 1 represents a monomer subunit.

[0060] Reference to a glutaryl CoA dehydrogenase enzyme includes any form of enzymatically-active glutaryl CoA dehydrogenase, including human and non-human forms. The glutaryl CoA dehydrogenase is suitably recombinant. In some embodiments, the glutaryl CoA dehydrogenase may be synthetic or may be isolated.

[0061] Thioredoxin

[0062] 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 an active site which contains dithiols in a CXXC motif. These two cysteines are the key to the ability of thioredoxin to reduce other proteins.

[0063] 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 glutaryl CoA 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. 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.

[0064] 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. Fusion protein

[0066] A fusion protein comprising thioredoxin operably linked to glutaryl CoA dehydrogenase has been shown by the present inventors to be recombinantly manufacturable in E. coli, and therefore a suitable candidate for enzyme replacement therapy in the treatment of glutaric aciduria type 1. The fusion protein of the invention has been shown by the present inventors to have both in vitro and in vivo activity.

[0067] The glutaryl CoA dehydrogenase and thioredoxin fusion protein of the present invention may be the product of a nucleic acid sequence encoding the glutaryl CoA dehydrogenase gene and a nucleic acid sequence encoding the thioredoxin protein, which are transcribed and translated as a single unit to provide glutaryl CoA dehydrogenase and thioredoxin fusion protein as single protein sequence.

[0068] The fusion protein of the present invention comprises a glutaryl CoA dehydrogenase enzyme and a thioredoxin protein. Suitably, the glutaryl CoA dehydrogenase enzyme is as described above, and most suitably is the human glutaryl CoA 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 glutaryl CoA 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 glutaryl CoA 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 glutaryl CoA 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.

[0069] The thioredoxin sequence may be provided at the N terminal or at the C terminal of the glutaryl CoA dehydrogenase enzyme. Most suitably, the thioredoxin is provided at the N terminal end of the glutaryl CoA dehydrogenase enzyme.

[0070] 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.

[0071] A fusion protein of the present invention may consist of glutaryl CoA dehydrogenase and thioredoxin. Alternatively, a fusion protein of the present invention may comprise glutaryl CoA 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 glutaryl CoA dehydrogenase enzyme and the thioredoxin protein.

[0072] Suitably, an “inter protein” linker sequence may be provided between the glutaryl CoA 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 glutaryl CoA 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 glutaryl CoA dehydrogenase enzyme and the thioredoxin protein as shown herein may be 22 amino acids in length.

[0073] An “inter protein” linker sequence between the glutaryl CoA dehydrogenase enzyme and the thioredoxin protein may have any suitable sequence. An inter-protein 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. 9) or GGGGG (SEQ ID NO. 10) sequence. Suitably, such a linker may comprise a GGGG (SEQ ID NO. 9) sequence. Suitably, such a linker may comprise two or more, suitably three GGGG (SEQ ID NO. 9) 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. 11). Suitably, a linker may comprise two or more, suitably three GGGGS sequences (SEQ ID NO. 11). An inter-protein linker sequence between the glutaryl CoA dehydrogenase enzyme and the thioredoxin protein may comprise a multi-H sequence, for example a HH, HHH, HHHH (SEQ ID NO. 12), HHHHH (SEQ ID NO. 13) or HHHHHH (SEQ ID NO. 14) sequence. Suitably, such a linker may comprise a HHHH (SEQ ID NO. 12) or HHHHH (SEQ ID NO. 13) sequence. Suitably, such a linker may comprise one HHHH (SEQ ID NO. 12) and one HHHHH (SEQ ID NO. 13) sequence. In a suitable embodiment, a linker between the glutaryl CoA dehydrogenase enzyme and the thioredoxin protein may comprise a combination of multi H sequence (e.g. HHHHH (SEQ ID NO. 13)) and multi-G sequences (e.g. GGGG (SEQ ID NO. 9)). The GGGG (SEQ ID NO. 9) and HHHHH (SEQ ID NO. 13) sequence may be contiguous or may be separated by other non-H or non-G amino acids, such as S, M and / or A.

[0074] Suitably, an interprotein may comprise the sequence GGGGXHHHHHXGGGG (SEQ ID NO. 15) where X may be 0, 1 , 2 or 3 amino acids, and each amino acid may be S, M or A.

[0075] Suitably, an interprotein linker may comprise or consist of the sequence GGGGX1GGGGX2HHHHHX3GGGGX4(SEQ ID NO. 16) where X1, X2, X3, and X4are each independently is 0, 1 , 2, 3, 4, 5, or 6 amino acids in length. Suitably, X1, X2, X3, and X4are each independently is 0, 1 , 2, or 3, amino acids in length. Suitably, X1is 0 or 1 amino acids in length; X2is 2, 3 or 4 amino acids in length; X3is suitably 0 or 1 amino acids in length; and X4is suitably 0 or 1 amino acids in length.

[0076] Suitably, X1is S; X2is SMA; X3is suitably 0 amino acid in length; and X4is suitably S.

[0077] Suitably, an interprotein linker may comprise or consist of the sequence GGGGX1GGGGX2HHHHHX3GGGGX4HHHHX5(SEQ ID NO. 17) where X1, X2, X3, X4and X5are each independently is 0, 1 , 2, 3, 4, 5, or 6 amino acids in length. Suitably, X1, X2, X3, X4and X5are each independently is 0, 1 , 2, or 3, amino acids in length. Suitably, X1is 0 or 1 amino acids in length; X2is 2, 3 or 4 amino acids in length; X3is suitably 0 or 1 amino acids in length; X4is suitably 0 or 1 amino acids in length; and X5is suitably 0 or 1 amino acids in length.

[0078] Suitably, X1is S; X2is SMA; X3is suitably 0 amino acid in length; X4is suitably S; and X5is suitably 0 amino acids.

[0079] Suitably, an interprotein linker is GGGGSGGGGX2HHHHHGGGGSHHHH (SEQ ID NO. 18), where X2is a sequence of 2, 3 or 4 amino acids in length, and comprises or consists of the amino acids M, A, S or any combination thereof.

[0080] Suitably, an interprotein linker is GGGGSGGGGX2HHHHHGGGGSHHHH (SEQ ID NO. 19), where X2is a sequence of 2, 3 or 4 amino acids in length, and comprises or consists of the amino acids M, A, S or any combination thereof.

[0081] Suitably, an interprotein linker is GGGGSGGGGSMAHHHHHGGGGSHHHH (SEQ ID NO. 20) or GGGGSGGGGSMAHHHHHGGGGS (SEQ ID NO. 21).

[0082] Suitably a fusion protein of the invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: Thioredoxin (e.g. SEQ ID NO. 3)- GGGGX1GGGGX2HHHHHX3GGGGX4HHHHX5(SEQ ID NO. 17) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X1, X2, X3, X4and X5are each independently a non-H or non-G amino acids. X1, X2, X3, X4and X5are each independently M, A, S or any combination thereof. X1, X2, X3, X4and X5may be as defined above.

[0083] Suitably a fusion protein of the invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: Thioredoxin (e.g. SEQ ID NO. 3)- GGGGX1GGGGX2HHHHHX3GGGGX4(SEQ ID NO. 16) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X1, X2, X3, and X4are each independently are each independently a non-H or non-G amino acids. X1, X2, X3, and X4are each independently M, A, S or any combination thereof. X1, X2, X3, and X4may be as defined above.

[0084] More suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGX2HHHHHGGGGSHHHH (SEQ ID NO. 18) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1), where X2is a sequence of 2, 3 or 4 amino acids in length, and comprises or consists of the amino acids M, A, S or any combination thereof.

[0085] More suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGX2HHHHHGGGGS (SEQ ID NO. 19) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1), where X2is a sequence of 2, 3 or 4 amino acids in length, and comprises or consists of the amino acids M, A, S or any combination thereof.

[0086] More suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGSHHHH (SEQ ID NO. 20) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1).

[0087] More suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGS (SEQ ID NO. 21) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1).

[0088] 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).

[0089] 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. 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 glutaryl CoA dehydrogenase, as described above.

[0090] The N terminal sequence may additonally comprise a multi H and / or a multi G sequence, for example a GGGG (SEQ ID NO. 9) and / or a HHHH (SEQ ID NO. 12) sequence. Suitably, an N terminal sequence may additonally comprise a GGGGX (SEQ ID NO. 22) or HHHHXGGGGX (SEQ ID NO. 23) sequence, where X may be 0, 1 or 2 amino acids in length. Therefore, suitably an N terminal sequence may comprise or consist of the sequence MACXGGGGX (SEQ ID NO. 24) or MACX1HHHHX2GGGGX3(SEQ ID NO. 25) where X is 0, 1 or 2 and is selected from S, M and / or A.

[0091] Suitably, an N terminal sequence is MACHHHHGGGGS (SEQ ID NO. 26) or MACGGGGS (SEQ ID NO. 27),

[0092] Therefore, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: N-terminal extension sequence - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGX1GGGGX2HHHHHX3GGGGX4HHHHX5(SEQ ID NO. 17) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X1is 0 or 1 amino acids in length; X2is 2, 3 or 4 amino acids in length; X3is suitably 0 or 1 amino acids in length; X4is suitably 0 or 1 amino acids in length; and X5is suitably 0 or 1 amino acids in length; and wherein X1, X2, X3, X4and X5are each independently M, A, S or any combination thereof. Suitably, the N terminal is MACX1HHHHX2GGGGX3(SEQ ID NO. 25) where X is 0, 1 or 2 and is selected from S, M or A. Suitably, an N terminal sequence is MACHHHHGGGGS (SEQ ID NO. 26).

[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 glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: N-terminal extension sequence - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGX1GGGGX2HHHHHX3GGGGX4(SEQ ID NO. 16) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X1is 0 or 1 amino acids in length; X2is 2, 3 or 4 amino acids in length; X3is suitably 0 or 1 amino acids in length; and X4is suitably 0 or 1 amino acids in length; and wherein X1, X2, X3, and X4are each independently M, A, S or any combination thereof. Suitably, the N terminal is MACXGGGGX (SEQ ID NO. 24), where X may be 0, 1 or 2 amino acids in length and is selected from S, M or A. Suitably, an N terminal sequence is MACGGGGS (SEQ ID NO. 27).

[0094] Suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: N-terminal extension sequence - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGX2HHHHHGGGGSHHHH (SEQ ID NO. 18) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X2is a sequence of 2, 3 or 4 amino acids in length, and comprises or consists of the amino acids M, A, S or any combination thereof. Suitably, the N terminal sequence is MACX1HHHHX2GGGGX3(SEQ ID NO. 25) where X is 0, 1 or 2 and is selected from S, M or A. Suitably, the N terminal sequence is MACHHHHGGGGS (SEQ ID NO. 26).

[0095] Suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: N-terminal extension sequence - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGX2HHHHHGGGGS (SEQ ID NO. 19) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X2is a sequence of 2, 3 or 4 amino acids in length, and comprises or consists of the amino acids M, A, S or any combination thereof. Suitably, the N terminal sequence is MACXGGGGX (SEQ ID NO. 24) where X is 0, 1 or 2 and is selected from S, M or A. Suitably, the N terminal sequence is MACGGGGS (SEQ ID NO. 27).

[0096] Suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: N-terminal extension sequence (e.g. MAC) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGSHHHH (SEQ ID NO. 20) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1). Suitably, the N terminal sequence is MACX1HHHHX2GGGGX3(SEQ ID NO. 27) where X is 0, 1 or 2 and is selected from S, M or A. Suitably, the N terminal sequence is MACHHHHGGGGS (SEQ ID NO. 26).

[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 glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: N-terminal extension sequence (e.g. MAC) - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGS (SEQ ID NO. 21) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1). Suitably, the N terminal sequence is MACXGGGGX (SEQ ID NO. 24) where X is 0, 1 or 2 and is selected from S, M or A. Suitably, the N terminal sequence is MACGGGGS (SEQ ID NO. 27).

[0098] Most suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: MACHHHHGGGGS (SEQ ID NO. 26)-thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGSHHHH (SEQ ID NO. 20) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1).

[0099] Most suitably, a fusion protein of the present invention may comprise or may consist of a sequence as follows, where the thioredoxin and / or the glutaryl CoA dehydrogenase may each independently be the native sequence as described herein or a variant or fragment of the native sequence, as described herein: MACGGGGS (SEQ ID NO. 27)-thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGS (SEQ ID NO. 21) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1).

[0100] Most suitably, a fusion protein of the present invention may comprise or may consist of SEQ ID NO. 5 or SEQ ID NO. 8.

[0101] 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.

[0102] 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.

[0103] Nucleic acid sequence

[0104] The present invention provides a nucleic acid sequence which encodes a protein which consists of or comprises a fusion protein as described herein.

[0105] 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. Nucleic acid sequence for thioredoxin and glutaryl CoA 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 and 4.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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. 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.

[0110] Production

[0111] 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.

[0112] 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, e.g. 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.

[0113] 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.

[0114] Most suitably, there is provided a method for the production of a fusion protein of the invention, wherein the method comprises transforming an E. coli host cell with a nucleic acid sequence encoding a fusion protein of the invention, and maintaining the host cell under conditions for expression of the nucleic acid encoding the fusion protein. Suitably, the method may comprise isolating the expressed fusion protein from the cell. Suitably, the method may further comprise purifying the fusion protein. The nucleic acid sequence may be provided in a suitable expression vector, as described herein. 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.

[0115] 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.

[0116] 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. Following expression in a host cell, a fusion protein of the present invention may be isolated and then 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 glutaryl CoA 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).

[0117] 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.

[0118] Conditions and disorders, subjects

[0119] 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. A subject may be a newborn baby, an infant, or a child.

[0120] Suitably, a subject may have a genetic and / or metabolic disorder that alters glutaryl CoA metabolism. Suitably, a subject may have a deficiency or absence of glutaryl CoA dehydrogenase. A subject may have increased levels of Glutaryl CoA, glutaric acid (GA), 3-hydroxy glutaric acid (3-OH-GA), glutaconic acid and / or glutarylcarnitine (C5DC) in a tissue or in a body fluid sample, compared to a healthy subject. This is known as glutaric aciduria type 1 (GA-1) or glutaric acidemia type 1 . Most suitably, the subject has a deficiency or absence of glutaryl CoA dehydrogenase, for example due to a genetic abnormality. Presence of C5-DC in urine is a suitable biochemical marker of GA-1 , and may be elevated even in low excretors (i.e. patients with normal levels of glutaric acid in urine).

[0121] Any suitable assay may be employed to determine the level of GA or C5-DC in a blood or urine sample from a subject. Such an assay may include an indication of a healthy or unhealthy level of GA, 3-OH-GA or C5DC. By way of non-limiting example, the Mayo clinic (http: / / www.mayocliniclabs.com / test- cataloq / overview / 88831#Clinical-and-lnterpretive) suggests as normal / healthy, less than 1.54 mmol / mole creatine in urine for C5-DC, and / or 4.5nmol / ml or less 2-OH glutaric acid in blood, 0.7nmol / ml or less 3-OH glutaric acid in blood, or 0.8 nmol / ml or less glutaric acid in blood. Anything above these levels for a given parameter may be considered to be high / unhealthy / indicative of a condition as described herein. A high or excess level of GA, 3-OH-GA or C5DC indicate that the subject has glutaric aciduria type I. Glutaryl CoA levels in a subject may be determined using any method as described herein or available in the art.

[0122] Symptoms of glutaric aciduria type I (or excess GA, 3-OH-GA or C5DC) in a subject may include muscle weakness, jerking, rigidity, decreased muscle tone, floppiness, poor feeding, decreased activity, vomiting, loss of visual activity, bleeding in the brain or eyes, an odor of smelly feet. A subject with glutaric aciduria may have physical abnormalities such as brain malformation, enlarged liver, weakened and / or enlarged heart, fluid filled cysts, malformation of kidney, unusual facial features, and / or genital abnormalities.

[0123] GA, 3-OH-GA or C5DC levels in a subject may be measured or determined using any suitable method. Appropriate samples for determination of levels of GA, 3-OH- GA or C5DC include any appropriate or desired sample in which the excess glutaryl CoA may occur. These may be any appropriate or desired tissue or body fluid sample. An example of a suitable tissue is kidney, liver, and / or fat tissue. Conveniently, a sample may be a body fluid sample for example urine or blood. Other samples include plasma, serum, cerebrospinal fluid, or a stool or tissue sample, biopsy sample, a lavage or washing fluid sample, or saliva, or suitably may be a blood or any blood-derived sample e.g. plasma or serum etc. In a screening test, C5DC may be tested in a blood sample.

[0124] Suitable methods for measurement of GA, 3-OH-GA or C5DC in a sample may include a microplate reader, high performance liquid chromatography (HPLC), and Gas Chromatography - Mass spectrometry (GC / MS). A simple measurement method of gl utary I CoA is the F-kit, which is enzyme colorimetric assay and quantifies glutaryl CoA 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 GA, 3-OH-GA or C5DC measurement.

[0125] Methods of treatment

[0126] The present invention provides a method for the prophylaxis or treatment of a disorder which results in excess GA, 3-OH-GA and / or C5DC in a subject, wherein the method comprises administering a therapeutically effective amount of a fusion protein comprising a glutaryl CoA dehydrogenase enzyme and a thioredoxin protein to a subject in need thereof. Suitably, the fusion protein is as described herein.

[0127] The present invention also provides a fusion protein comprising a glutaryl CoA dehydrogenase enzyme and a thioredoxin protein for use in the prophylaxis or treatment of a disorder which results in excess GA, 3-OH-GA and / or C5DC in a subject.

[0128] A method of the first aspect may result in a decrease in accumulation of GA, 3-OH- GA and / or C5DC in a tissue of a subject.

[0129] Therefore, the present invention also provides a method for the prevention or reduction of excess GA, 3-OH-GA and / or C5DC in a subject, wherein the method comprises administering a therapeutically effective amount of a fusion protein comprising a glutaryl CoA 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 glutaryl CoA dehydrogenase enzyme and a thioredoxin protein for use in the prevention or reduction of excess glutaryl CoA in a subject. Suitably, the fusion protein is as described herein.

[0130] The fusion protein may be provided in the form of a composition, as described herein. In the case of a glutaryl CoA dehydrogenase deficiency, treatment according to the present invention may not treat the underlying genetic disorder, but rather the resulting clinical condition of excess GA, 3-OH-GA and / or C5DC.

[0131] In a suitable embodiment, an effective therapy according to the present invention is one which provides an effective relief from one or more symptoms selected from one or more of muscle weakness, jerking, rigidity, decreased muscle tone, floppiness, poor feeding, decreased activity, vomiting, loss of visual activity, bleeding in the brain or eyes, an odor of smelly feet, and improvement of, or delayed or reduced progression of symptoms arising from physical abnormalities such as brain malformation, enlarged liver, weakened and / or enlarged heart, fluid filled cysts, malformation of kidney, unusual facial features, and / or genital abnormalities.

[0132] An effective treatment may reduce GA, 3-OH-GA and / or C5DC in a selected tissue or body fluid sample (such as blood or urine) to a normal or healthy level, according to a chosen test available to a clinician, for example as described above. The level of GA, 3-OH-GA and / or C5DC in a subject may be measured as described herein, for example by assaying a biological sample of the subject.

[0133] The present methods and uses as described herein may be useful in maintenance of GA, 3-OH-GA and / or C5DC levels at any normal or healthy range, for example to a normal or healthy level, according to a chosen test available to a clinician, for example as described above.

[0134] Treatment, according to the present invention, may provide an increase in the amount of active glutaryl CoA 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, fat tissue. The increased level of glutaryl CoA dehydrogenase may be sufficient to reduce GA, 3-OH-GA and / or C5DC in a sample of the subject to a normal or healthy range. It will also be appreciated that normal or healthy levels of GA, 3- OH-GA and / or C5DC may be subject specific, and depend on factors such as the subject’s weight, diet, sex and age. Normal or healthy levels of GA, 3-OH-GA and / or C5DC in a sample from a subject may depend on the chosen test. Exemplary levels using a test available from the Mayo Clinic are provided above. An "effective amount” of a fusion protein for the reduction of excess GA, 3-OH-GA and / or C5DC 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 GA, 3-OH-GA and / or C5DC 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 GA, 3-OH-GA and / or C5DC levels in a subject with a genetic and / or metabolic disorder that alters glutaryl CoA metabolism or causes increased levels of GA, 3-OH-GA and / or C5DC, such as glutaric aciduria type I.

[0135] Methods for measuring GA, 3-OH-GA and / or C5DC are described herein, and may be used in the diagnosis of a condition which includes excess GA, 3-OH-GA and / or C5DC as a symptom; and / or may be used in the monitoring of a disorder associated with excess GA, 3-OH-GA and / or C5DC; or in the prevention of a disorder associated with excess GA, 3-OH-GA and / or C5DC. A method of measuring GA, 3-OH-GA and / or C5DC 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, 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.

[0136] Confirmatory testing can be done by enzymatic analysis using14C-labeled glutaryl- CoA and measuring the release of14CO2.29Genetic testing for abnormalities in the GCDH gene or expression thereof may also be performed on a sample from a subject. Measuring the amount of GA, 3-OH-GA and / or C5DC 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.

[0137] Composition

[0138] In a further aspect the present invention provides a composition comprising a fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin, as described herein. 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.

[0139] 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.

[0140] 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. 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, histidine, alanine, valine, leucine, isoleucine and phenylalanine, Kreb's-Ringer buffer, TRIS, Sodium phosphate-based buffers, 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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. 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.

[0145] 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, glutaryl CoA 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.

[0146] 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.

[0147] 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).

[0148] Suitably, a composition is sterile.

[0149] 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.

[0150] 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.

[0151] The precise amount or dose of the fusion protein administered to the subject depends on the activity of the glutaryl CoA 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 per kg body weight per day.

[0152] Suitably, the fusion protein is administered intravenously or subcutaneously, at weekly or biweekly intervals.

[0153] Administration

[0154] 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.

[0155] 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.

[0156] Combinations

[0157] 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 Glutaryl CoA, GA, 3-OH-GA and / or C5DC, such as glutaric aciduria type I. 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.

[0158] 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 GA, 3-OH-GA and / or C5DC. The second or further agent may be administered by the same administration route or by a different administration route.

[0159] Kits

[0160] 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 Glutaryl CoA, GA, 3-OH-GA and / or C5DC.

[0161] Such a kit may be provided for use in treating or preventing such a disorder, which is associated with excess Glutaryl CoA, GA, 3-OH-GA and / or C5DC. 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] The present invention will now be further described with reference to the following non-limiting Examples and Figures in which:

[0166] Figure 1 shows the enzymatic pathway for the breakdown of the amino acids lysine, hydrolysine and tryptophan.

[0167] Figure 2 is a schematic showing the build up of GlutarylCoA and subsequent toxic metabolites.

[0168] Figure 3 shows SDS (called sodium dodecyl sulfate) Page gel electrophoresis in reducing conditions for GCDH21 and GCDH18. The left hand side of each gel shows MW (Molecular weight) ladder, BSA as a control protein. The GCDH enzyme is indicated at the appropriate expected size on each gel.

[0169] Figure 4_shows the results of an activity assay for the fusion protein of the invention. The ECso for rhGCDH.21 was shown to be 283nM and the EC50 for rhGCDH.18 was shown to be 432 nM in this assay.

[0170] Figure 5 shows the results of an in vivo assay.

[0171] SEQ ID NO. 1 (GCDH protein)

[0172] MSRPEFDWQDPLVLEEQLTTDEILIRDTFRTYCQERLMPRILLANRNEVFHREIISE MGELGVLGPTIKGYGCAGVSSVAYGLLARELERVDSGYRSAMSVQSSLVMHPIYA YGSEEQRQKYLPQLAKGELLGCFGLTEPNSGSDPSSMETRAHYNSSNKSYTLNG TKTWITNSPMADLFVVWARCEDGCIRGFLLEKGMRGLSAPRIQGKFSLRASATGM IIMDGVEVPEENVLPGASSLGGPFGCLNNARYGIAWGVLGASEFCLHTARQYALD RMQFGVPLARNQLIQKKLADMLTEITLGLHACLQLGRLKDQDKAAPEMVSLLKRN NCGKALDIARQARDMLGGNGISDEYHVIRHAMNLEAVNTYEGTHDIHALILGRAIT GIQAFTASK

[0173] SEQ ID NO. 2

[0174] Gene sequence of GCDH

[0175] ATGAGCCGTCCGGAGTTTGATTGGCAGGACCCGCTGGTTTTAGAGGAACAGC TGACCACCGACGAGATCCTTATCCGCGATACCTTTAGAACATACTGCCAAGAG CGCCTGATGCCGCGTATCCTCTTGGCTAATCGTAACGAAGTTTTTCATCGTGA AATCATTAGCGAGATGGGTGAATTGGGTGTGCTCGGTCCGACCATTAAAGGTT ATGGCTGCGCAGGTGTCAGCTCTGTCGCCTACGGCCTGCTCGCCCGCGAATT GGAGCGAGTTGACAGCGGCTATCGCAGCGCTATGAGCGTGCAGAGCAGCCTA GTTATGCATCCGATTTACGCGTACGGTTCCGAAGAGCAACGTCAGAAGTACCT GCCGCAGCTGGCCAAAGGCGAGTTGCTGGGCTGCTTCGGTCTGACGGAACC GAATTCCGGTTCCGATCCGAGCTCTATGGAAACCCGTGCGCACTATAACAGCA GCAATAAAAGCTACACTCTGAATGGTACCAAAACCTGGATCACCAACAGTCCG ATGGCCGATCTGTTTGTAGTTTGGGCACGTTGTGAAGATGGCTGCATTCGTGG TTTTCTGTTGGAGAAAGGTATGCGTGGTCTGTCTGCGCCACGTATTCAAGGTA

[0176] AGTTTAGCTTGCGCGCGTCTGCCACCGGCATGATCATCATGGATGGTGTTGAA

[0177] GTCCCGGAGGAGAACGTCCTGCCGGGTGCGTCTTCTCTGGGCGGCCCGTTC

[0178] GGCTGCCTGAACAACGCACGCTACGGCATCGCGTGGGGTGTCTTGGGTGCGT

[0179] CGGAGTTCTGCTTGCATACCGCGCGCCAATATGCACTGGATCGTATGCAGTTT

[0180] GGCGTGCCGCTGGCGCGCAACCAGCTGATCCAAAAGAAGCTGGCTGACATGC

[0181] TGACCGAGATCACGCTGGGCCTGCACGCGTGTTTGCAGCTGGGACGTCTGAA

[0182] GGATCAGGATAAAGCTGCTCCGGAAATGGTGAGCTTATTAAAGCGTAATAACT

[0183] GCGGCAAAGCACTCGACATCGCCCGTCAGGCGCGCGATATGCTGGGGGGAA

[0184] ACGGCATATCCGACGAGTATCACGTGATTCGTCATGCTATGAACCTGGAAGCG

[0185] GTGAACACCTACGAAGGCACGCATGATATTCATGCCCTGATCCTGGGCCGTG

[0186] CGATTACTGGTATTCAAGCGTTTACCGCTAGCAAG

[0187] SEQ ID NO. 3

[0188] Thioredoxin sequence

[0189] MVKQIESKTAFQEALDAAGDKLWVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLE

[0190] VDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV

[0191] SEQ ID NO. 4.

[0192] Gene sequence of Thioredoxin

[0193] ATGGTTAAACAAATTGAAAGCAAGACTGCGTTTCAAGAAGCGCTGGACGCTGC

[0194] AGGTGACAAACTGGTGGTGGTTGACTTCAGCGCGACGTGGTGTGGTCCTTGT

[0195] AAAATGATTAAGCCGTTCTTCCACAGCTTGTCAGAAAAGTATAGCAATGTTATC

[0196] TTCCTGGAGGTGGACGTGGACGACTGTCAGGACGTTGCATCCGAGTGCGAAG

[0197] TTAAGTGCATGCCAACCTTCCAATTCTTCAAAAAGGGTCAGAAAGTGGGTGAG

[0198] TTCAGCGGTGCGAACAAAGAAAAGTTGGAGGCGACGATCAACGAATTGGTT

[0199] SEQ ID NO. 5

[0200] Fusion protein rhGCDH21

[0201] MACHHHHGGGGSMVKQIESKTAFQEALDAAGDKLWVDFSATWCGPCKMIKPFF

[0202] HSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKL

[0203] EATINELVGGGGSGGGGSMAHHHHHGGGGSHHHHMSRPEFDWQDPLVLEEQL

[0204] TTDEILIRDTFRTYCQERLMPRILLANRNEVFHREIISEMGELGVLGPTIKGYGCAG

[0205] VSSVAYGLLARELERVDSGYRSAMSVQSSLVMHPIYAYGSEEQRQKYLPQLAKG

[0206] ELLGCFGLTEPNSGSDPSSMETRAHYNSSNKSYTLNGTKTWITNSPMADLFVVW ARCEDGCIRGFLLEKGMRGLSAPRIQGKFSLRASATGMIIMDGVEVPEENVLPGA

[0207] SSLGGPFGCLNNARYGIAWGVLGASEFCLHTARQYALDRMQFGVPLARNQLIQK

[0208] KLADMLTEITLGLHACLQLGRLKDQDKAAPEMVSLLKRNNCGKALDIARQARDML

[0209] GGNGISDEYHVIRHAMNLEAVNTYEGTHDIHALILGRAITGIQAFTASK***

[0210] SEQ ID NO. 6

[0211] Gene sequence of Fusion protein rhGCDH21

[0212] N-terminal

[0213] ATGGCTTGTCATCACCACCACGGAGGTGGGGGCTCT

[0214] Thioredoxin:

[0215] ATGGTTAAACAAATTGAAAGCAAGACTGCGTTTCAAGAAGCGCTGGACGCTGC

[0216] AGGTGACAAACTGGTGGTGGTTGACTTCAGCGCGACGTGGTGTGGTCCTTGT

[0217] AAAATGATTAAGCCGTTCTTCCACAGCTTGTCAGAAAAGTATAGCAATGTTATC

[0218] TTCCTGGAGGTGGACGTGGACGACTGTCAGGACGTTGCATCCGAGTGCGAAG

[0219] TTAAGTGCATGCCAACCTTCCAATTCTTCAAAAAGGGTCAGAAAGTGGGTGAG

[0220] TTCAGCGGTGCGAACAAAGAAAAGTTGGAGGCGACGATCAACGAATTGGTT

[0221] Middle linker

[0222] GGTGGGGGAGGTTCGGGCGGTGGCGGCTCCATGGCACACCACCACCACCAC

[0223] GGTGGTGGCGGCTCCCATCATCACCAC

[0224] GCDH gene

[0225] ATGAGCCGTCCGGAGTTTGATTGGCAGGACCCGCTGGTTTTAGAGGAACAGC

[0226] TGACCACCGACGAGATCCTTATCCGCGATACCTTTAGAACATACTGCCAAGAG

[0227] CGCCTGATGCCGCGTATCCTCTTGGCTAATCGTAACGAAGTTTTTCATCGTGA

[0228] AATCATTAGCGAGATGGGTGAATTGGGTGTGCTCGGTCCGACCATTAAAGGTT

[0229] ATGGCTGCGCAGGTGTCAGCTCTGTCGCCTACGGCCTGCTCGCCCGCGAATT

[0230] GGAGCGAGTTGACAGCGGCTATCGCAGCGCTATGAGCGTGCAGAGCAGCCTA

[0231] GTTATGCATCCGATTTACGCGTACGGTTCCGAAGAGCAACGTCAGAAGTACCT

[0232] GCCGCAGCTGGCCAAAGGCGAGTTGCTGGGCTGCTTCGGTCTGACGGAACC

[0233] GAATTCCGGTTCCGATCCGAGCTCTATGGAAACCCGTGCGCACTATAACAGCA

[0234] GCAATAAAAGCTACACTCTGAATGGTACCAAAACCTGGATCACCAACAGTCCG

[0235] ATGGCCGATCTGTTTGTAGTTTGGGCACGTTGTGAAGATGGCTGCATTCGTGG

[0236] TTTTCTGTTGGAGAAAGGTATGCGTGGTCTGTCTGCGCCACGTATTCAAGGTA

[0237] AGTTTAGCTTGCGCGCGTCTGCCACCGGCATGATCATCATGGATGGTGTTGAA

[0238] GTCCCGGAGGAGAACGTCCTGCCGGGTGCGTCTTCTCTGGGCGGCCCGTTC

[0239] GGCTGCCTGAACAACGCACGCTACGGCATCGCGTGGGGTGTCTTGGGTGCGT

[0240] CGGAGTTCTGCTTGCATACCGCGCGCCAATATGCACTGGATCGTATGCAGTTT

[0241] GGCGTGCCGCTGGCGCGCAACCAGCTGATCCAAAAGAAGCTGGCTGACATGC

[0242] TGACCGAGATCACGCTGGGCCTGCACGCGTGTTTGCAGCTGGGACGTCTGAA

[0243] GGATCAGGATAAAGCTGCTCCGGAAATGGTGAGCTTATTAAAGCGTAATAACT

[0244] GCGGCAAAGCACTCGACATCGCCCGTCAGGCGCGCGATATGCTGGGGGGAA

[0245] ACGGCATATCCGACGAGTATCACGTGATTCGTCATGCTATGAACCTGGAAGCG

[0246] GTGAACACCTACGAAGGCACGCATGATATTCATGCCCTGATCCTGGGCCGTG

[0247] CGATTACTGGTATTCAAGCGTTTACCGCTAGCAAG SEQ ID NO. 7 pET sequence including rhGCDH21 in underline

[0248] >U9531794G0-8_GCDH.21_pET-30a(+)

[0249] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGT

[0250] GGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCT

[0251] TTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT

[0252] CTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA

[0253] CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGAT

[0254] AGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCT

[0255] TGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATA

[0256] AGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAA

[0257] ATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTT

[0258] CGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAAT

[0259] ATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAA

[0260] CTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTC

[0261] TGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTG

[0262] GTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCC

[0263] CTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAAT

[0264] CCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGC

[0265] CAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATT

[0266] CGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATT

[0267] ACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACA

[0268] ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCG

[0269] GGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTT

[0270] GATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCAT

[0271] CTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGC

[0272] GCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATT

[0273] ATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCG

[0274] CGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTAT

[0275] TACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGT

[0276] GAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC

[0277] TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACC

[0278] GCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA

[0279] AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG

[0280] CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGC

[0281] TCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA

[0282] CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG

[0283] AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAA

[0284] CTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGA

[0285] GAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

[0286] CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT

[0287] TCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

[0288] AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG

[0289] CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA

[0290] CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACC

[0291] GAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTAT

[0292] TTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCA

[0293] GTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGC

[0294] TACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGC

[0295] GCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACC

[0296] GTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCG CGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGAT

[0297] GTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATG

[0298] TCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTC

[0299] ACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGAT

[0300] GAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGG

[0301] TTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACC

[0302] AGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGT

[0303] GTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGG

[0304] TGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAA

[0305] GACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTC

[0306] ACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGC

[0307] CAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGG

[0308] GCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGG

[0309] GACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAG

[0310] CGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATG

[0311] ACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAG

[0312] TCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGAC

[0313] TGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTG

[0314] AGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAA

[0315] CCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGT

[0316] TTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACA

[0317] GCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCAC

[0318] GCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGG

[0319] ATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGC

[0320] ACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCAT

[0321] CTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTT

[0322] GCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCT

[0323] ATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAG

[0324] ACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGA

[0325] CCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGA

[0326] AAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGA

[0327] ACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATA

[0328] GTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCT

[0329] TTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACC

[0330] CAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGC

[0331] AGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCA

[0332] GTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCC

[0333] ACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGG

[0334] AAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACT

[0335] GGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCAT

[0336] ACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC

[0337] CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGA

[0338] GCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTC

[0339] CCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGA

[0340] GCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGC

[0341] GCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGC

[0342] GTAGAGGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAG

[0343] GGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTA

[0344] AGAAGGAGATATACAT

[0345] ATGGCTTGTCATCACCACCACGGAGGTGGGGGCTCTATGGTTAAACAAATTGA

[0346] AAGCAAGACTGCGTTTCAAGAAGCGCTGGACGCTGCAGGTGACAAACTGGTG GTGGTTGACTTCAGCGCGACGTGGTGTGGTCCTTGTAAAATGATTAAGCCGTT

[0347] CTTCCACAGCTTGTCAGAAAAGTATAGCAATGTTATCTTCCTGGAGGTGGACG

[0348] TGGACGACTGTCAGGACGTTGCATCCGAGTGCGAAGTTAAGTGCATGCCAAC

[0349] CTTCCAATTCTTCAAAAAGGGTCAGAAAGTGGGTGAGTTCAGCGGTGCGAACA

[0350] AAGAAAAGTTGGAGGCGACGATCAACGAATTGGTTGGTGGGGGAGGTTCGGG

[0351] CGGTGGCGGCTCCATGGCACACCACCACCACCACGGTGGTGGCGGCTCCCA

[0352] TCATCACCACATGAGCCGTCCGGAGTTTGATTGGCAGGACCCGCTGGTTTTAG

[0353] AGGAACAGCTGACCACCGACGAGATCCTTATCCGCGATACCTTTAGAACATAC

[0354] TGCCAAGAGCGCCTGATGCCGCGTATCCTCTTGGCTAATCGTAACGAAGTTTT

[0355] TCATCGTGAAATCATTAGCGAGATGGGTGAATTGGGTGTGCTCGGTCCGACCA

[0356] TTAAAGGTTATGGCTGCGCAGGTGTCAGCTCTGTCGCCTACGGCCTGCTCGC

[0357] CCGCGAATTGGAGCGAGTTGACAGCGGCTATCGCAGCGCTATGAGCGTGCAG

[0358] AGCAGCCTAGTTATGCATCCGATTTACGCGTACGGTTCCGAAGAGCAACGTCA

[0359] GAAGTACCTGCCGCAGCTGGCCAAAGGCGAGTTGCTGGGCTGCTTCGGTCTG

[0360] ACGGAACCGAATTCCGGTTCCGATCCGAGCTCTATGGAAACCCGTGCGCACT

[0361] ATAACAGCAGCAATAAAAGCTACACTCTGAATGGTACCAAAACCTGGATCACC

[0362] AACAGTCCGATGGCCGATCTGTTTGTAGTTTGGGCACGTTGTGAAGATGGCTG

[0363] CATTCGTGGTTTTCTGTTGGAGAAAGGTATGCGTGGTCTGTCTGCGCCACGTA

[0364] TTCAAGGTAAGTTTAGCTTGCGCGCGTCTGCCACCGGCATGATCATCATGGAT

[0365] GGTGTTGAAGTCCCGGAGGAGAACGTCCTGCCGGGTGCGTCTTCTCTGGGCG

[0366] GCCCGTTCGGCTGCCTGAACAACGCACGCTACGGCATCGCGTGGGGTGTCTT

[0367] GGGTGCGTCGGAGTTCTGCTTGCATACCGCGCGCCAATATGCACTGGATCGT

[0368] ATGCAGTTTGGCGTGCCGCTGGCGCGCAACCAGCTGATCCAAAAGAAGCTGG

[0369] CTGACATGCTGACCGAGATCACGCTGGGCCTGCACGCGTGTTTGCAGCTGGG

[0370] ACGTCTGAAGGATCAGGATAAAGCTGCTCCGGAAATGGTGAGCTTATTAAAGC

[0371] GTAATAACTGCGGCAAAGCACTCGACATCGCCCGTCAGGCGCGCGATATGCT

[0372] GGGGGGAAACGGCATATCCGACGAGTATCACGTGATTCGTCATGCTATGAAC

[0373] CTGGAAGCGGTGAACACCTACGAAGGCACGCATGATATTCATGCCCTGATCCT

[0374] GGGCCGTGCGATTACTGGTATTCAAGCGTTTACCGCTAGCAAG

[0375] TAATGAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATC

[0376] CGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGA

[0377] GCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTT

[0378] TGCTGAAAGGAGGAACTATATCCGGAT

[0379] SEQ ID NO. 8

[0380] Amino acid sequence of fusion protein rhGCDH18

[0381] MACGGGGSMVKQIESKTA FQEALDAAGD KLVWDFSAT WCGPCKMIKP FFHSLSEKYS NVIFLEVDVDDCQDVASECE VKCMPTFQFF KKGQKVGEFS GANKEKLEAT

[0382] INELV GGGGSGGGGSMAHHHHHGGGGSMSRPEFDWQDPLVLEEQLTTDEILIR DTFRTYCQERLMPRILLANRNEVFHREIISEMGELGVLGPTIKGYGCAGVSSVAYG LLARELERVDSGYRSAMSVQSSLVMHPIYAYGSEEQR

[0383] QKYLPQLAKGELLGCFGLTEPNSGSDPSSMETRAHYNSSNKSYTLNGTKTWITNS PMADLFVVWARCEDGCIRGFLLEKGMRGLSAPRIQGKFSLRASATGMIIMDGVEV PEENVLPGASSLGGPFGCLN NARYGIAWGVLGASEFCLHTARQYALDRMQFGVPLARNQLIQKKLADMLTEITLGL HACLQLGRLKDQDKAAPEMVSLLKRNNCGKALDIARQARDMLGGNGISDEYHVIR HAMNLEAVNTYEGTHDIH

[0384] ALILGRAITGIQAFTASK**

[0385] Bold type - thioredoxin

[0386] Underline N terminal linker and interprotein linker Normal type - GCDH protein

[0387] ** denotes a stop codon

[0388] Example 1

[0389] The protein was produced as described below (‘Production and purification of rhGCDH.21 recombinant protein). 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.

[0390] Production and purification of rhGCDH.21 and rhGCDH.18 recombinant protein

[0391] E.Coli 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 and 100uM Riboflavin; 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 4 h at 37 C or 16h at 15 °C. Cells were spun down and following centrifugation, cells were lysed and subsequently sonicated with lysis buffer (50 mM Tris-HCI, 500mM NaCI, 1 mM TCEP, 1% Triton X- 100, 50 uM FAD, pH 8.0)

[0392] Purification: 1ststep Nickel column purification, with 20mM Imidazole wash and elution with 200 - 500 mM imidazole (containing 50 uM FAD). Further steps following include buffer exchanging and concentration with Molecular weight cut off columns to further improve purity and / or Size Exclusion chromatography and / or PD-10 columns and / or Millipore concentration columns with a 30 kDa MW cutoff. Final buffer: 0.1 M NasPOt or PBS containing 10% trehalose, 5% sucrose, 0.1% polysorbate 80, 50 uM FAD, (+ / -) 10% glycerol (+ / - 0.6M L-arginine), pH7.1 - pH 7.2.

[0393] Example 2 in Vitro activity

[0394] Assay principle: This activity assay measures the ability of the enzyme candidates rhGCDH.21 and rhGCDH.18_to reduce Dichlorophenylindophenol (DCIP) in the presence of the enzyme’s Glutaryl CoA substrate. When oxidised, the DCIP is blue in colour: in the presence of Glutaryl CoA and active enzyme - becomes colorless when reduced. This colour reaction can be measured at OD 620 nm.

[0395] Assay method: The assay reaction buffer was composed of the following: PBS, pH 7.4 + 100 uM DCIP + 200 uM Glutaryl CoA + 200 uM Methoxy-PMS + 20 uM FAD. 20 ul of enzyme + 80 ul of reaction buffer was used per well. 60 ug per well was used per well at the top of the curve, with 1 / 2 dilutions.

[0396] Results

[0397] The EC50 for rhGCDH.21 and rhGCDH.18 was shown to be 432 nM and 283 nM respectively.

[0398] EC50 formula used and analysis

[0399] Example 3 in vivo efficacy study using enzyme candidate rhGCDH.21

[0400] Aims: 3 - 4 month old Male OF1 mice. After 9 days on a 10% lysine-supplemented ad libitum diet (mixed in to Altromin 1311 powder), mice were injected with 50 mg / kg of rhGCDH.21 i.p. or 50 mg / kg s.c. or vehicle saline. Urinary Glutaryl coA was measured 4 hours post-dose in the urines of animals.

[0401] Urinary Glutaryl CoA: The Glutaryl CoA reaction buffer consisted of PBS pH7.4 + 66 uM DCIP + 200 uM Methoxy-PMS + 0.32 mg / ml of GCDH enzyme. Urine was spun down prior to use for 5 mins @ 10,000g in a microcentrifuge. Supernatant was diluted 1 / 5 and 10 ul of diluted urine was added to 90 ul of reaction buffer against a Glutaryl coA standard. Plates were left at room temperature for 10 - 20 mins until read @ 620 nm on a Byony plate reader.

[0402] Results: rhGCDH.21 injection successfully lowered the toxic metabolite Glutaryl CoA in the urine of animals 4 hours post dose in a lysine overload model rhGCDH.21 enzyme candidate Glutaryl CoA urinary levels (uM) 4 hours post dose (i.v. and s.c. groups) in OF1 male mice 9 days post 10% lysine overload. Dosing points indicated by blue arrows. 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 < 0.05; **p < 0.001.

Claims

Claims:1 . A fusion protein comprising glutaryl CoA dehydrogenase and thioredoxin.

2. A fusion protein according to claim 1 for use in a method of prevention or treatment of glutaric aciduria type I, wherein the method comprises administering a therapeutically effective amount of the fusion protein to a subject in need thereof.

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

4. A fusion protein, or a fusion protein for use, according to claim 3 wherein the glutaryl CoA 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 acids5. A fusion protein, or a fusion protein for use, according to claim 3 or 4 wherein the glutaryl CoA dehydrogenase is the human glutaryl CoA dehydrogenase of SEQ ID NO. 1 , or a sequence having 90%, 95%, 97%, 98% or 99% sequence identity with the glutaryl CoA 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 fusion protein 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 is provided at the N terminal of the glutaryl CoA 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 linker sequence is provided between the glutaryl CoA dehydrogenase enzyme and the thioredoxin protein.

10. A fusion protein, or a fusion protein for use, according claim 9 wherein the first linker sequence is 10 to 32 amino acids in length, preferably 22 amino acids in length.11 . A fusion protein, or a fusion protein for use, according claim 9 or 10 wherein the linker comprises 1 , 2 or 3 GGGG sequences and / or 1 or 2 HHHH sequences, preferably 2 GGGG sequences and 1 HHHH sequence, or 3 GGGG sequences and 2 HHHH sequences.

12. A fusion protein, or a fusion protein for use, according to any one of claims 9 to 11 wherein the linker comprises or consists of the sequence GGGGX1GGGGX2HHHHHX3GGGGX4HHHHX5(SEQ ID NO. 17) where X1is 0 or 1 amino acids in length; X2is 2, 3 or 4 amino acids in length; X3is suitably 0 or 1 amino acids in length; X4is suitably 0 or 1 amino acids in length; and X5is suitably 0 or 1 amino acids in length.

13. A fusion protein, or a fusion protein for use, according to claim 12 wherein X1is S; X2is SMA; X3is 0 amino acids in length; X4is S; and X5is 0 amino acids.

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 GGGGSGGGGSMAHHHHHGGGGSHHHH (SEQ ID NO. 20)15. A fusion protein, or a fusion protein for use, according to any one of clams 9 to 14, wherein the fusion protein further comprises an N terminal extension sequence.

16. A fusion protein, or a fusion protein for use, according to claim 15 wherein the N 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.

17. A fusion protein, or a fusion protein for use, according to any one of claims 15 to 16 wherein the N terminal extension is an N terminal methionine residue, optionally with a cysteine residue.

18. A fusion protein, or a fusion protein for use, according to claim 16 wherein the tag is, or comprises, the sequence MAC.

19. A fusion protein, or a fusion protein for use, according to any one of claims 15 to 18 wherein the N terminal sequence comprises a GGGG (SEQ ID NO. 9) and / or a HHHH (SEQ ID NO. 12) sequence.

20. A fusion protein, or a fusion protein for use, according to any one of claims 15 to 19 wherein the N terminal sequence comprises a a HHHHXGGGGX (SEQ ID NO. 23) sequence, where X may be 0, 1 or 2 amino acids in length.21 . A fusion protein, or a fusion protein for use, according to any one of claims 15 to 20 wherein the N terminal sequence comprises or consists of the sequence MACX1HHHHX2GGGGX3(SEQ ID NO. 25) where X is 0, 1 or 2 and is selected from S, M or A.

22. A fusion protein, or a fusion protein for use, according to any one of claims 15 to 21 wherein the N terminal sequence is MACHHHHGGGGS (SEQ ID NO. 26) or ACHHHHGGGGS (SEQ ID NO. 28).

23. A fusion protein, or a fusion protein for use, according to any one of claims 15 to 22, wherein the fusion protein comprises or consists of the sequence N- terminal extension sequence - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGX1GGGGX2HHHHHX3GGGGX4HHHHX5(SEQ ID NO. 17) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X1is 0 or 1 amino acids in length; X2is 2, 3 or 4 amino acids in length; X3is suitably 0 or 1 amino acids in length; X4is suitably 0 or 1 amino acids in length; and X5is suitably 0 or 1 amino acids in length; and wherein X1, X2, X3, X4and X5are each independently M, A, S or any combination thereof, and optionally wherein the N terminal is sequence is MACX1HHHHX2GGGGX3(SEQ ID NO. 25) where X is 0, 1 or 2 and is selected from S, M or A.

24. A fusion protein, or a fusion protein for use, according to claim 23 wherein the N terminal sequence is MACHHHHGGGGS (SEQ ID NO. 26).

25. A fusion protein, or a fusion protein for use, according to any one of claims 15 to 22, wherein the fusion protein comprises or consists of the sequence: N- terminal extension sequence - Thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGX2HHHHHGGGGSHHHH (SEQ ID NO. 18) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1) where X2is a sequence of 2, 3 or 4 amino acids in length, and comprises or consists of the amino acids M, A, S or any combination thereof, and optionally wherein the N terminal sequenceis MACX1HHHHX2GGGGX3(SEQ ID NO. 25) where X is 0, 1 or 2 and is selected from S, M or A.

26. A fusion protein, or a fusion protein for use, according to claim 25 wherein the N terminal sequence is MACHHHHGGGGS (SEQ ID NO. 26).

27. A fusion protein, or a fusion protein for use, according to any one of claims 9 to 26 wherein the fusion protein comprises or consists of the sequence MACHHHHGGGGS (SEQ ID NO. 26) -thioredoxin (e.g. SEQ ID NO. 3)- GGGGSGGGGSMAHHHHHGGGGSHHHH (SEQ ID NO. 20) - glutaryl CoA dehydrogenase (e.g. SEQ ID NO. 1).

28. A fusion protein, or a fusion protein for use, according to claim 27 wherein the fusion protein comprises or consists of the sequence of SEQ ID NO.

529. 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 28, wherein the fusion protein is pegylated.

30. A fusion protein according to any one of claims 1 to 29, or a fusion protein for use according to any one of claims 2 to 29, wherein the fusion protein is a tetramer, preferably comprising four identical monomer units31. 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 30, wherein the fusion protein is recombinant.

32. A pharmaceutical or nutritional composition comprising a fusion protein according to any one of claims 1 to 31 , wherein the composition comprises one or more physiologically or pharmaceutically acceptable carriers or excipients.

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

34. A food comprising a fusion protein as defined in any one of claims 1 to 31 .

35. A kit comprising (a) a fusion protein as defined in any one of claims 1 to 31 and (b) a further therapeutic agent.

36. A nucleic acid sequence encoding a fusion protein as defined in any one of claims 1 to 31.

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

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

39. A host cell comprising a nucleic acid molecule or expression vector as defined in claims 36 to 38.

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

41. A composition according to claim 32 or 33, a food according to claim 34, a nucleic acid molecule or expression vector according to claims 36 to 38, or a host cell according to claim 39 or 40 for use in a method of treatment of glutaric aciduria type I.

42. A fusion protein for use according to any one of claims 2 to 30, wherein the subject is a baby or a child.

43. A fusion protein for use according to any one of claims 2 to 30 or claim 42 wherein the subject has one or more symptoms selected from muscle weakness, jerking, rigidity, decreased muscle tone, floppiness, poor feeding, decreased activity, vomiting, loss of visual activity, bleeding in the brain or eyes, an odor of smelly feet, a physical abnormality such as brain malformation, enlarged liver, weakened and / or enlarged heart, fluid filled cysts, malformation of kidney, unusual facial features, and / or genital abnormality.

44. A method of producing a fusion protein as defined in any one of claims 1 to 30, wherein the method comprises providing a host cell or a cell free system comprising a nucleic acid sequence encoding the fusion protein; and maintaining the host cell or cell free system under conditions suitable for expression of the fusion protein; and optionally isolating the fusion protein.

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