AAV-mediated gene therapy of diseases caused by impaired function of gldc

Abstract

The present invention relates to a nucleotide sequence comprising a codon-optimised nucleotide sequence encoding glycine decarboxylase (GLDC), an expression vector cassette encoding GLDC, an expression vector comprising the expression vector cassette, a pharmaceutical composition, use in therapy thereof, use in a method of treating a disease caused by impaired GLDC function, method of treating a disease caused by impaired GLDC function and a method of obtaining a codon-optimised GLDC sequence for gene therapy.

Description

[0001] AAV-mediated gene therapy of diseases caused by impaired function of GLDC Background Non-Ketotic Hyperglycinemia (NKH) is a life-limiting neurometabolic disease. NKH has an incidence of 1 / 60,000 worldwide, with higher frequency in certain populations. Affected individuals present as neonates with respiratory distress, muscle hypotonia and seizures. Babies that survive the neonatal period suffer profound developmental delay, complex epilepsy, and premature lethality (median age of death is 8 years). NKH is caused by mutation in genes encoding the glycine cleavage system (GCS); the majority of cases being caused by mutations in GLDC, the gene encoding glycine decarboxylase (GLDC). NKH is characterised by accumulation of excess glycine in body fluids and tissues. The GCS acts to cleave glycine, with donation of one carbon to folate one-carbon metabolism (FOCM). Hence, in addition to accumulation of glycine, loss of GCS function results in suppression of folate one-carbon metabolism. Presently, there is no cure for NKH. Current treatments involve administration of sodium benzoate to lower glycine and the use of multiple anti-epileptic medications. While these treatments can help control epilepsy they are not curative and do not improve developmental outcome. In addition, a limitation of this approach is that benzoate causes severe gastrointestinal distress and vomiting, may lead to carnitine deficiency, and can be toxic. Furthermore, while benzoate can lower systemic glycine, this does not overcome the suppression of folate one-carbon metabolism that results from loss of glycine cleavage system activity. There is therefore a need for novel treatments for diseases caused by impaired function of GLDC, such as NKH, that lower systemic glycine level while overcoming the suppression of the folate one-carbon metabolism. Summary of Invention The present inventors have provided a novel gene therapy for diseases caused by impaired function of GLDC, such as NKH. As demonstrated in the data below, the inventors’ novel gene therapy is able to target sites of GCS activity (such as the brain or liver) and provide genetic rescue which restores GLDC and / or lowers circulating glycine and / or can overcome the suppression of the folate one-carbon metabolism. The invention involved designing an expression vector cassette encoding GLDC, which required reduction in the length of particular sequences used in the expression vector cassette (such as reduction of the 5’UTR of GLDC). This helped overcome the risk of truncation of the transgene or the transcribed or expressed GLDC. Using expression vector cassettes of the present invention enables expression of functional GLDC. Further developments include an expression vector comprising the specifically designed expression vector cassette encoding GLDC, confirming its value as a therapy, as well as providing certain optimized sequences encoding GLDC. In a first aspect of the invention, there is provided a nucleotide sequence comprising a codon- optimised nucleotide sequence encoding glycine decarboxylase (GLDC). The codon-optimised nucleotide sequence may comprise or be a sequence at least 90% identical to SEQ ID No:2 (Optz 1), SEQ ID No:3(Optz 2), or SEQ ID No:4 (Optz 3). The codon-optimised nucleotide sequence may comprise or be SEQ ID No:3 (Optz 2). In a second aspect of the invention, there is provided an expression vector cassette encoding GLDC, wherein the expression vector cassette comprises: a 5’ inverted terminal repeat (ITR); a promoter; a sequence selected from a wild-type GLDC sequence, a human GLDC sequence, a consensus GLDC sequence at least 70% identical to SEQ ID No:1, or a codon-optimised GLDC sequence; a termination sequence; and a 3’ inverted terminal repeat (ITR),wherein the distance between the end of the promoter and the start codon of the GLDC sequence is 201 bp or less. The expression vector cassette may comprise the nucleotide sequence according to the first aspect. The promoter may comprise or be a promoter comprising from about 0.6 kb to about 1.0 kb. The promoter may comprise or be a truncated chicken beta-actin hybrid promoter (CBh) or a truncated human glial fibrillary acidic protein (GFAP) promoter. The termination sequence may comprise or be bovine growth hormone polyadenylation sequence (BGH-PolyA). The 5’ ITR and 3’ ITR may be derived from adeno-associated virus 2 (AAV2). The expression vector cassette may comprise a promoter comprising from about 0.6 kb to about 1.0 kb, for example a truncated chicken beta-actin hybrid promoter (CBh) or a truncated human glial fibrillary acidic protein (GFAP) promoter, and bovine growth hormone polyadenylation sequence (BGH-PolyA). The expression vector cassette may comprise a promoter comprising from about 0.6 kb to about 1.0 kb, for example a truncated chicken beta-actin hybrid promoter (CBh) or a truncated human glial fibrillary acidic protein (GFAP) promoter, and 5’ ITR and 3’ ITR derived from adeno-associated virus 2 (AAV2). The expression vector cassette may comprise bovine growth hormone polyadenylation sequence (BGH-PolyA) and 5’ ITR and 3’ ITR derived from adeno-associated virus 2 (AAV2). The expression vector cassette may comprise a promoter comprising from about 0.6 kb to about 1.0 kb, for example a truncated chicken beta-actin hybrid promoter (CBh) or a truncated human glial fibrillary acidic protein (GFAP) promoter, bovine growth hormone polyadenylation sequence (BGH-PolyA), and 5’ ITR and 3’ ITR derived from adeno-associated virus 2 (AAV2). The distance between the end of the promoter and the start codon of the GLDC sequence may be 15 bp or less. The GLDC sequence may comprise a truncated 5’ untranslated region (UTR) of 60 bp or less. The 5’ UTR of the GLDC sequence may be absent. In a third aspect of the present invention, there is provided an expression vector capable of expressing GLDC comprising the expression vector cassette according to the second aspect. The expression vector cassette may be packaged into an adeno-associated virus (AAV) capsid. The AAV may be an AAV9. The AAV capsid may target the central nervous system (CNS). The AAV may target the brain. The AAV may target the liver. The AAV may target the CNS and brain. The AAV may target the brain and liver. The AAV may target the CNS and liver. The AAV may target the CNS, brain and liver. In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the expression vector according to the third aspect and a pharmaceutically acceptable carrier. In a fifth aspect of the present invention, there is provided an expression vector according to the third aspect, or the pharmaceutical composition according to fourth aspect, for use in therapy. In a sixth aspect of the present invention, there is provided an expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use in a method of treating a disease caused by impaired function of GLDC. The disease may be non-ketotic hyperglycinemia (NKH). The disease may be severe NKH. The disease may be attenuated NKH. The expression vector may comprise the expression vector cassette according to the second aspect. The expression vector cassette may be packaged into an adeno-associated virus (AAV) capsid. The AAV may be an AAV9. The AAV capsid may target the central nervous system (CNS). The AAV may target the brain. The AAV may target the liver. The AAV may target the CNS and brain. The AAV may target the brain and liver. The AAV may target the CNS and liver. The AAV may target the CNS, brain and liver. The method may comprise administering the expression vector to the blood stream. The method may comprise administering the expression vector intra-venously. The method may comprise administering the expression vector to the central nervous system (CNS). The method may comprise administering the expression vector intra-cisternally, intra-thecally or intra-cerebroventricularly. The method may comprise administering the expression vector to the blood stream and the CNS, for example, the method may comprise administering the expression vector intra-venously and intra-cisternally, intra-thecally or intra- cerebroventricularly. In a seventh aspect of the present invention, there is provided a method of treating a disease caused by impaired function of GLDC, wherein the method comprises administering an expression vector comprising a sequence encoding glycine decarboxylase (GLDC) to a subject in need thereof. The disease caused by impaired function of GLDC may be non-ketotic hyperglycinemia (NKH). The disease caused by impaired function of GLDC may be severe NKH. The disease caused by impaired function of GLDC may be attenuated NKH. The expression vector may comprise the expression vector cassette according to the second aspect. The expression vector cassette may be packaged into an adeno-associated virus (AAV) capsid. The AAV may be an AAV9. The AAV capsid may target the central nervous system (CNS). The AAV may target the brain. The AAV may target the liver. The AAV may target the CNS and brain. The AAV may target the brain and liver. The AAV may target the CNS and liver. The AAV may target the CNS, brain and liver. The method may comprise administering the expression vector to the blood stream. The method may comprise administering the expression vector intra-venously. The method may comprise administering the expression vector to the central nervous system (CNS). The method may comprise administering the expression vector intra-cisternally, intra-thecally, or intra-cerebroventricularly. The method may comprise administering the expression vector to the blood stream and the CNS, for example, the method may comprise administering the expression vector intra-venously and intra-cisternally, intra-thecally or intra- cerebroventricularly. In an eighth aspect of the present invention, there is provided a method for obtaining a codon- optimised GLDC sequence for gene therapy the method comprising: applying a codon optimisation algorithm to a wild-type GLDC sequence, to obtain a codon-optimised GLDC sequence, optionally, translating, in silico, the codon-optimised GLDC sequence to confirm identity of the encoded protein with wild-type GLDC protein, administering an expression vector comprising an expression vector cassette comprising the codon-optimised GLDC sequence from step a) or b) to a Gldc-deficient disease model, and determining rescue of the disease phenotype in the Gldc-deficient disease model, wherein rescue of the disease phenotype indicates that the sequence is a codon-optimised GLDC sequence for gene therapy. In a ninth aspect of the present invention, there is provided a codon-optimized GLDC sequence for gene therapy obtainable by the method according to the eighth aspect.

[0002] Brief Description of Figures Figure 1 shows GLDC protein expression in cells treated with expression vectors comprising codon-optimised GLDC sequences, assessed by immunoblot analysis Figure 2 shows relative GLDC protein abundance in cells treated with expression vectors comprising codon-optimised GLDC sequences Figure 3 shows mGldc mRNA expression, mediated by endogenous GLDC allele and AAV9- mGLDC, in brain and liver tissue, assessed by qRT-PCR Figure 4 shows hGLDC mRNA expression mediated by AAV9-hGLDC-Optz2, in brain tissue, assessed by qRT-PCR (normalised to GAPDH). Untreated + / + and GldcGT1 / GT1samples show mGldc mRNA expression (normalised to Gapdh) Figure 5 shows GLDC protein expression in brain tissue of AAV-hGLDC-Optz2 treated mice, assessed by immunoblot analysis Figure 6 shows GLDC protein expression in brain tissue of GLDC protein in brain of AAV9- hGLDC-Optz2 treated mice, assessed by immunohistochemistry analysis Figure 7 shows GLDC protein expression in liver tissue of AAV9-hGLDC-Optz2 treated mice, assessed by immunoblot analysis Figure 8 shows plasma glycine concentration in untreated wild-type mice (+ / +), untreated Gldc-deficient mice (NKH mouse model, GldcGT1 / GT1), iv+ic AAV9-mGldc treated Gldc- deficient mice, and ic only AAV9-mGldc treated Gldc-deficient mice Figure 9 shows plasma glycine concentration in untreated wild-type mice(+ / +), untreated Gldc- deficient mice (NKH mouse model, GldcGT1 / GT1), AAV9-mGldc treated Gldc-deficient mice, AAV9-hGLDC treated Gldc-deficient mice, AAV9-hGLDC-Optz1 treated Gldc-deficient mice and AAV9-hGLDC-Optz2 treated Gldc-deficient mice Figure 10 shows brain tissue glycine concentration in untreated wild-type mice (+ / +), untreated Gldc-deficient mice (NKH mouse model, GldcGT1 / GT1), iv+ic AAV9-mGldc treated Gldc- deficient mice, ic only AAV9-mGldc treated Gldc-deficient mice and iv+ic AAV9-hGLDC- Optz2 vector treated mice Figure 11 shows THF and 5methyl-THF relative amounts in brain tissue of untreated wild-type mice (+ / +), untreated Gldc-deficient mice (NKH mouse model, GldcGT1 / GT1), iv+ic AAV9- mGldc treated Gldc-deficient mice, ic only AAV9-mGldc treated Gldc-deficient mice and iv+ic AAV9-hGLDC-Optz2 vector treated Gldc-deficient mice Figure 12 shows betaine and choline relative amounts in brain tissue of untreated wild-type mice (+ / +), untreated Gldc-deficient mice (NKH mouse model, GldcGT1 / GT1), iv+ic AAV9- mGldc treated Gldc-deficient mice, and ic only AAV9-mGldc treated Gldc-deficient mice Figure 13 shows a generalised schematic of an exemplary expression vector cassette according to the invention Figure 14 shows expression of GLDC protein in brain of AAV-GFAP-hGLDCoptz2 treated mice. Figure 14A shows immunostaining using anti-GLDC antibody with nuclei counterstained using DAPI shows widespread expression in the brain. The location of enlarged areas shown in Figure 14B-D are indicated by boxes in Figure 14A. Regions of the brain shown at higher magnification correspond to olfactory bulb (Figure 14B), cortex (Figure 14C) and hippocampus (Figure 14D). Figure 15 shows normalisation of metabolite biomarkers and therapeutic targets in brain of GLDC-deficient mice at 12 weeks of age following neonatal treatment with AAV9-GFAP- hGLDCoptz2. Figure 15A shows glycine is significantly more abundant in brain tissue of GLDC-deficient (GldcGT1 / GT1) mice than in wild-type controls (* p<0.05). In Gldc GT1 / GT1 mice treated with AAV9-GFAP-GLDC at neonatal stage, glycine shows a trend towards lower abundance compared with vehicle treated GldcGT1 / GT1 mice (p = 0.06; n = 6 per group). Figure 15B shows betaine is significantly less abundant in brain tissue of GldcGT1 / GT1 mice than in wild-type controls (p<0.05). In Gldc GT1 / GT1 mice treated with AAV9-GFAP-GLDC at neonatal stage, betaine abundance is significantly increased compared with vehicle treated GldcGT1 / GT1 mice (** p < 0.01; n = 6 per group). Figure 15C shows choline is significantly less abundant in brain tissue of GldcGT1 / GT1 mice than in wild-type controls (* p<0.05). In Gldc GT1 / GT1 mice treated with AAV9-GFAP-GLDC at neonatal stage, choline abundance does not differ from wild-type controls. Detailed Description The terms "treatment" and "treating" herein refer to an approach for obtaining beneficial or desired results in a subject, which includes a therapeutic benefit. “Therapeutic benefit” refers to eradication, amelioration or slowing the progression of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the patient may still be afflicted with the underlying disorder. The term "effective amount" or "therapeutically effective amount" refers to the amount of the nucleotide sequence, expression vector cassette, expression vector, or pharmaceutical composition needed to bring about an acceptable outcome of the therapy as determined by reducing the likelihood of disease as measurable by clinical, biochemical or other indicators that are familiar to those trained in the art. The therapeutically effective amount may vary depending upon the condition, the severity of the condition, the subject, e.g., the weight and age of the subject and the mode of administration and the like, which can readily be determined by one of ordinary skill in the art. The term "subject" refers to any suitable subject, including any animal, such as a mammal. In preferred embodiments described herein, the subject is a human. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes those embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, that "consist of” or "consist essentially of” the described features. The term “comprises” or “comprising” can be used interchangeably with “includes”. “Sequence identity” or “% sequence identical” as described herein refers to the % degree of similarity between two nucleotide sequences of the same length. “GLDC” as defined herein is a gene that encodes for the glycine decarboxylase (GLDC) protein (SEQ ID NO: 5). The GLDC protein is an enzyme that plays an important role in the glycine cleavage system (GCS) and folate one carbon metabolism (FOCM). A “nucleotide sequence” described herein has its normal meaning in the art and refers to nucleic acid sequence, for example a naturally occurring or synthetic nucleic acid sequence, that may be used to incorporate genetic material into a target cell or tissue. “Nucleotides” described herein describe the constituent parts of a nucleic acid sequence. Nucleotides comprise a nucleobase (e.g., A, G, T and C in DNA, or A, G, U and C in RNA, however other nucleobases may be used), linked to a sugar (e.g., deoxyribose in DNA, and ribose in RNA, however, other sugars may be used). In DNA and RNA, the sugars are linked by a phosphodiester backbone to form a nucleic acid sequence, however other backbones may be used. “Codon-optimisation” defined herein describes a gene engineering approach that utilises synonymous codon changes in order to try and increase encoded protein production. Publicly available algorithms may be used to undertake codon-optimisation, for example, GenSmart Codon Optimisation tool, GeneArt GeneOptimizer, or Genewiz algorithm. A “codon-optimised nucleotide sequence” defined herein describes a nucleotide sequence that has undergone codon-optimisation. “Adeno-associated virus” or “AAV” defined herein describes a non-enveloped virus that can be engineered to deliver DNA to target cells. AAV are small and can infect humans. “Inverted terminal repeat” or “ITR” defined herein describes a single stranded nucleotide sequence followed downstream by its reverse complement. In an adeno-associated virus (AAV), ITR serve as the origin of replication and are required for replication, packaging and vector persistence. “Promoter” defined herein describes a region of DNA upstream of the gene sequence of interest, to which proteins bind to initiate transcription of an RNA transcript from the DNA downstream of the promoter. “Start codon” defined herein describes the site from which translation of the desired protein sequence is initiated. “Wild-type sequence” defined herein describes a sequence, e.g., DNA, RNA or protein, as it occurs in nature. “Consensus sequence” defined herein describes the calculated sequence of most frequent residues, nucleotide or amino acid, found at each position in a sequence alignment. “Termination sequence” defined herein describes a sequence of DNA which marks the end of the gene transcription. “Stop codon” defined herein describes the site at which translation of the desired protein sequence is terminated. “Distance between the end of the promoter and the start codon” defined herein describes the number of base pairs (bp) between the end of the promoter sequence and the start of the protein encoding gene sequence. “Untranslated region” or “UTR” defined herein describes sequences that may be present at either the 5’ or 3’ends or both the 5’ and 3’ ends of the protein encoding gene sequence that are not translated. “Complementary DNA” or “cDNA” refers to DNA which contains only the coding sequences (i.e., the non-coding sections of the DNA sequence have been removed). “Gene therapy” defined herein describes the introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders. “Gldc-deficient mouse model” defined herein describes a mouse model in which the mice carry a gene-trap allele of Gldc (denoted GldcGT1). This mouse model is also referred to as an NKH mouse model and denoted GldcGT1 / GT1. Further details of Gldc-deficient mouse model can be found in Pai et al. Nat Commun 6:6388 (2015); Leung et al.. J. Inherit.. Metab. Dis. 43: 1186- 1198 (2020). “Kozak sequence” defined herein describes a nucleic acid motif located upstream from the coding gene sequence and containing the start codon, which plays a role in the initiation of the translation process. “Disease phenotype” defined herein describes any observable characteristic or trait of a disease, such as morphology, development, biochemical or physiological properties, or behaviour, without any implication of a mechanism. Unless context explicitly states otherwise, it is envisaged that any embodiment described herein may be combined with any other embodiment described herein. Similarly, the features of any dependent claim (i.e., representing preferred embodiments of the present invention) may be readily combined with the features of any of the independent claims or other dependent claim or embodiments, unless context clearly dictates otherwise. When ranges are used herein, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included. The term "about" or “~”when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary. Typical experimental variabilities may stem from, for example, changes and adjustments necessary during scale-up from laboratory experimental and manufacturing settings to large scale. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and “the” include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used herein have their conventional meaning within the chemical and biological arts, unless otherwise indicated. Nucleotide Sequence The first aspect of the invention provides a nucleotide sequence comprising a codon-optimised nucleotide sequence encoding glycine decarboxylase (GLDC). Advantages of a nucleotide sequence comprising a codon-optimised nucleotide sequence encoding glycine decarboxylase (GLDC) include increased translational efficiency of glycine decarboxylase (GLDC) protein and higher abundance of glycine decarboxylase (GLDC) protein in comparison with wild-type consensus GLDC. Therefore, the use of the codon-optimised nucleotide sequences encoding glycine decarboxylase (GLDC) of the present invention in expression vectors leads to improved protein expression in comparison with a wild- type sequence encoding GLDC. It was found that codon-optimised nucleotide sequences 1, 2 and 3 (SEQ ID No:2 and 3 and 4, Optz 1 and 2 and 3), when included in expression vectors and tested in vitro, provided good translational efficiency and GLDC protein. It was surprisingly found that codon-optimised nucleotide sequences 1 to 2 (SEQ ID No:2 and 3, Optz 1 and 2), when included in expression vectors and tested in vitro, provided better translational efficiency and higher quantities of GLDC protein than comparative vectors comprising wild-type GLDC. In particular, it was surprisingly found that codon-optimised nucleotide sequence 2 (SEQ ID No:3, Optz2) provided the best translational efficiency and glycine decarboxylase abundance of the codon-optimised sequences developed and tested, (see, for example, section “Design and cloning of AAV-GLDC plasmid DNA” of the examples and Figures 1 and 2) The nucleotide sequence may consist essentially of or be a codon-optimised nucleotide sequence encoding glycine decarboxylase (GLDC). A codon-optimised nucleotide sequence encoding glycine decarboxylase (GLDC) may be obtained by using publicly available software or algorithms, for codon-optimisation of the GLDC sequence. The codon-optimised nucleotide sequence encoding GLDC may have a sequence identity of at least 70%, for example at least 75%, at least 77 %, at least 79%, at least 80%, at least 85%, at least 90%, at least 95% identical to the wild-type consensus sequence. The codon-optimised nucleotide sequence encoding GLDC may have a sequence identity 77% identical to the wild- type consensus sequence. The codon-optimised nucleotide sequence encoding GLDC may have a sequence identity 79% identical to the wild-type consensus sequence. The wild-type consensus sequence may be a cDNA sequence. The codon-optimised nucleotide sequence encoding GLDC may be a cDNA sequence. The codon-optimised nucleotide sequence encoding GLDC may differ from the wild-type consensus sequence at at least 400 bp. The codon-optimised nucleotide sequence encoding GLDC may differ from the wild-type consensus sequence at at least 450 bp. The codon- optimised nucleotide sequence encoding GLDC may differ from the wild-type consensus sequence at at least 500 bp. The codon-optimised nucleotide sequence encoding GLDC may differ from the wild-type consensus sequence at at least 550 bp. The codon-optimised nucleotide sequence encoding GLDC may differ from the wild-type consensus sequence at at least 600 bp. The codon-optimised nucleotide sequence encoding GLDC may differ from the wild-type consensus sequence at at least 650 bp. The codon-optimised nucleotide sequence encoding GLDC may differ from the wild-type consensus sequence at at least 700 bp. The wild- type consensus cDNA sequence encoding GLDC is 3063 bp. The codon-optimised nucleotide sequence encoding GLDC may comprise or be a sequence at least 90% identical, for example at least 92 % identical, or at least 95 % identical, or at least 97 % identical, or at least 99 % identical, or at least 100% identical to SEQ ID No:2 (Optz1), SEQ ID No:3 (Optz2), or SEQ ID No:4 (Optz3). SEQ ID No:2 refers to codon-optimisation GLDC sequence 1, also denoted Optz1. SEQ ID No:3 refers to codon-optimisation GLDC sequence 2, also denoted Optz2. SEQ ID No:4 refers to codon-optimisation GLDC sequence 3, also denoted Optz3. The codon-optimised nucleotide sequence encoding GLDC may comprise or be SEQ ID No:2 (Optz1). The codon-optimised nucleotide sequence encoding GLDC may comprise or be SEQ ID No:3 (Optz2). The codon-optimised nucleotide sequence encoding GLDC may comprise or be SEQ ID No:4 (Optz3). Preferably, the codon-optimised nucleotide sequence encoding GLDC may be SEQ ID No:3 (Optz2). The codon-optimised nucleotide sequence encoding GLDC may further comprise restriction enzyme sites. The codon-optimised nucleotide sequence encoding GLDC may further comprise a Kozak sequence. The codon-optimised nucleotide sequence encoding GLDC may further comprise a restriction enzyme site and Kozak sequence at the 5’ end of the sequence, prior to and including at least the start codon. The codon-optimised nucleotide sequence encoding GLDC may further comprise an Nhe1 restriction enzyme site and Kozak sequence at the 5’ end of the sequence, prior to and including at least the start codon. The codon-optimised nucleotide sequence encoding GLDC may further comprise a restriction enzyme site at the 3’ end of the sequence, after the stop codon. The codon-optimised nucleotide sequence encoding GLDC may further comprise a Not1 restriction enzyme site at the 3’ end of the sequence, after the stop codon. Expression Vector Cassette The second aspect of the invention provides an expression vector cassette encoding GLDC, wherein the expression vector cassette comprises: a 5’ inverted terminal repeat (ITR); a promoter; a sequence selected from a wild-type GLDC sequence, a human GLDC sequence, a consensus GLDC sequence at least 70% identical to SEQ ID No:1, or a codon-optimised GLDC sequence; a termination sequence; and a 3’ inverted terminal repeat (ITR),wherein the distance between the end of the promoter and the start codon of the GLDC sequence is 201 bp or less. The expression vector cassette of the present invention has an advantageously small size. It was found that larger expression vector cassette constructs led to truncation of the GLDC at transcription and / or subsequent translation. Overcoming truncation of the transcribed and / or translated GLDC results from the design of the expression vector cassette of the present invention in which the 5’UTR is truncated, or optionally is essentially absent. Advantages of an expression vector cassette according to the invention include tailored cassette size, for inclusion in an AAV genome. This circumvents possible truncation of the GLDC sequence. It was surprisingly found that tailoring of the cassette size, optionally by reducing the size of the 5’UTR, did not negatively impact resultant transcriptional or translational efficiency of the encoded sequenced. Therefore, reducing the size of the expression vector cassette did not negatively impact protein expression or abundance stemming from treatment with an expression vector including the cassette of the invention. In particular, tailoring the distance between the end of the promoter and the start codon of the GLDC sequence to minimise the number of base pairs is shown by the present inventors to optimise the packaging and expression of GLDC (see, for example, section “Design and cloning of AAV-GLDC plasmid DNA” of the examples). It was also found to be beneficial to select a short promoter sequence, for example, a promoter sequence between 0.6 to 1.0 kb. The expression vector cassette may comprise the nucleotide sequence according to the first aspect. The wild-type GLDC sequence, a human GLDC sequence, a consensus GLDC sequence at least 70% identical to SEQ ID No:1, or a codon-optimised GLDC sequence of the expression vector cassette may be a cDNA sequence. The wild-type consensus cDNA sequence encoding GLDC is 3063 bp. The promoter may be or comprise a ubiquitous mammalian promoter. The promoter may be specific for neurons or astrocytes, or astrocytes alone. The promoter may comprise or be a truncated chicken beta-actin hybrid promoter (CBh), cytomegalovirus promoter (CMV), cytomegalovirus promoter / chicken beta-actin promoter (CMV / CBA), CAG promoter, human beta actin promoter (HBA), glial fibrillary acidic protein promoter (GFAP), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Preferably the promoter may be a truncated chicken beta-actin hybrid promoter (CBh) or a truncated glial fibrillary acidic protein promoter (GFAP). The truncated glial fibrillary acidic protein promoter (GFAP) may be a gfaABC(1)D promoter as disclosed in Lee et al. “GFA{ promoter elements required for region-sepcific and astrocyte-specific expression”, Glia. 2008 Apr;56(5):481-93. doi: 10.1002 / glia.20622. The promoter may comprise or be a promoter comprising from about 0.6 kb to about 1.0 kb, for example from about 0.65 kb to about 0.95 kb, or from about 0.7 kb to about 0.9 kb, or from about 0.75 kb to about 0.85 kb. The termination sequence may comprise or be bovine growth hormone polyadenylation sequence (BGH-PolyA), simian virus 40 polyadenylation sequence (SV40 PolyA), human growth hormone polyadenylation sequence (hGH PolyA), or rabbit beta-globin polyadenylation sequence (rbGlob Poly A). Preferably the termination sequence may be bovine growth hormone polyadenylation sequence (BGH-PolyA). The 5’ ITR and 3’ ITR may be derived from adeno-associated virus 2 (AAV2). The expression vector cassette may comprise a promoter comprising from about 0.6 kb to about 1.0 kb, for example a truncated chicken beta-actin hybrid promoter (CBh) or a truncated human glial fibrillary acidic protein (GFAP) promoter, and bovine growth hormone polyadenylation sequence (BGH-PolyA). The expression vector cassette may comprise a promoter comprising from about 0.6 kb to about 1.0 kb, for example a truncated chicken beta-actin hybrid promoter (CBh) or a truncated human glial fibrillary acidic protein (GFAP) promoter, and 5’ ITR and 3’ ITR derived from adeno-associated virus 2 (AAV2). The expression vector cassette may comprise bovine growth hormone polyadenylation sequence (BGH-PolyA) and 5’ ITR and 3’ ITR derived from adeno-associated virus 2 (AAV2). The expression vector cassette may comprise a promoter comprising from about 0.6 kb to about 1.0 kb, for example a truncated chicken beta-actin hybrid promoter (CBh) or a truncated human glial fibrillary acidic protein (GFAP) promoter, bovine growth hormone polyadenylation sequence (BGH-PolyA), and 5’ ITR and 3’ ITR derived from adeno-associated virus 2 (AAV2). The expression vector cassette may further comprise one or more restriction enzyme sites. An advantageous feature of the present invention is the reduction of the length of the GLDC 5’UTR. A further advantageous feature of the present invention is that the distance between the end of the promoter and the start codon of the GLDC sequence may be less than in vivo for a GLDC gene. The distance between the end of the promoter and the start codon of the GLDC sequence may be 201 bp or less, for example, 195 bp or less, 180 bp or less, 171 bp or less, 165 bp or less, 150 bp or less, 141 bp or less, 135 bp or less, 120 bp or less, 111 bp or less, 105 bp or less, 90 bp or less, 81 bp or less, 75 bp or less, 60 bp or less, 57 bp or less, , 54 bp or less, 51 bp or less, 48 bp or less, 45 bp or less, 42 bp or less, 39 bp or less, 36 bp or less, 33 bp or less, 30 bp or less, 27 bp or less, 24 bp or less, 21 bp or less, 18 bp or less, 15 bp or less, 12 bp or less, or 9 bp or less. The distance between the end of the promoter and the start codon of the GLDC sequence may be 12 bp or less. The distance between the end of the promoter and the start codon of the GLDC sequence may be 9 bp or less. The end of the promoter and the start codon of the GLDC sequence may be separated by a sequence comprising a Kozak sequence. The Kozak sequence may comprise the start codon. The end of the promoter and the start codon of the GLDC sequence may be separated by a restriction enzyme site and include a Kozak sequence. The restriction enzyme site may be an Nhe1 restriction enzyme site. The GLDC sequence may comprise a truncated 5’ untranslated region (UTR) of 60 bp or less, for example 57 bp or less, 54 bp or less, 51 bp or less, 48 bp or less, 48 bp or less, 45 bp or less, 42 bp or less, 39 bp or less, 36 bp or less, 33 bp or less, 30 bp or less, 27 bp or less, 24 bp or less, 21 bp or less, 18 bp or less, 15 bp or less or 12 bp or less, 9 bp or less, 6 bp or less or 3 bp or less. The 5’ UTR of the GLDC sequence may be absent or essentially absent. The GLDC sequence may comprise a truncated 3’ UTR. The 3’UTR of the GLDC sequence may be absent or essentially absent. The expression vector cassette may comprise its sequence components in the following order: a 5’ inverted terminal repeat (ITR); a promoter; a sequence selected from a wild-type GLDC sequence, a human GLDC sequence, a consensus GLDC sequence at least 70% identical to SEQ ID No:1, or a codon-optimised GLDC sequence; a termination sequence; and a 3’ inverted terminal repeat (ITR). The promoter and GLDC encoding sequence may be separated by a sequence comprising a Kozak sequence. The promoter and GLDC encoding sequence may be separated by a restriction enzyme site and include a Kozak sequence. The restriction enzyme site may be an Nhe1 restriction enzyme site. The GLDC encoding sequence and termination sequence may be separated by a restriction enzyme site. The restriction enzyme site may be an Not1 restriction enzyme site. Expression Vector The third aspect of the present invention provides an expression vector capable of expressing GLDC comprising the expression vector cassette according to the second aspect. The expression vector cassette may be packaged into an adeno-associated virus (AAV) capsid. The AAV may be an optimal serotype for transduction in the central nervous system (CNS) and / or liver. The AAV may be an AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, or AAV9. Preferably, the AAV is an AAV9 capsid. The AAV capsid may target the central nervous system (CNS). The AAV may target the brain. The AAV may target the liver. The AAV may target the CNS and brain. The AAV may target the brain and liver. The AAV may target the CNS and liver. The AAV may target the CNS, brain and liver. Pharmaceutical Composition The fourth aspect of the present invention provides a pharmaceutical composition comprising the expression vector according to the third aspect and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any suitable pharmaceutical carrier for the selected mode of administration, for example intra-venous, intra-cisternal, intra-thecal or intra- cerebroventricular. The pharmaceutically acceptable carrier may be saline, buffered saline, lactose, starch, sucrose, mannitol, protamine, dicalcium phosphate, magnesium stearate, sodium saccharin, talcum, cellulose, cellulose derivatives, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride, combinations thereof and the like. Expression Vector or Pharmaceutical Composition for Use in Therapy The fifth aspect of the present invention provides an expression vector according to the third aspect, or the pharmaceutical composition according to fourth aspect, for use in therapy. Advantages of the expression vector or pharmaceutical composition for use in therapy of the present invention include expression of GLDC mRNA and GLDC protein (resulting in restoration of the glycine cleavage system (GCS) in a subject, leading to decreased levels of glycine in plasma and tissue (e.g., liver and brain) in a subject and restoration of the folate one- carbon metabolism (FOCM) in a subject. Figures 3 and 4 demonstrate that GLDC mRNA expression is achieved in vivo, in the brain and liver of mice treated with AAV-GLDC expression vectors, providing proof of GLDC transgene transcription. Figures 5-7 and 14 demonstrate GLDC protein expression in the plasma and brain of mice treated with AAV- GLDC expression vectors, providing proof of GLDC transgene translation. Figures 8 to 10 and 15A demonstrate the glycine concentration in plasma and brain tissue of mice treated with AAV-GLDC expression vectors and demonstrate that expression vectors of the invention provide normalisation of the glycine concentration, providing proof that the encoded GLDC functions as intended, by restoring activity of the glycine cleavage system (GCS), such that glycine level is depleted. Figures 11 and 12 demonstrate the metabolite concentrations of species associated with the folate one-carbon metabolism (FOCM) and demonstrate the restoration of this system when mice are treated with AAV-GLDC expression vectors. While current therapies (e.g., sodium benzoate) enable lowering of systemic glycine in a subject, no current therapy is known to provide restoration of the FOCM, as demonstrated by the presented invention. Figures 15B and 15C demonstrate that the betaine and choline concentrations in brain tissue of mice treated with AAV-GLDC expression vectors and demonstrate that expression vectors of the invention provide normalisation of the betaine and choline concentration. Betaine is a biomarker of impaired glycine cleavage system activity. Low betaine levels in the brain are associated with neurological impairments and lower betaine is, therefore, also a therapeutic target. Normalisation of betaine in treated mice confirms restoration of GLDC function. The expression vector or pharmaceutical composition for use in therapy may be for use in treating diseases caused by impaired function of GLDC, for example, non-ketotic hyperglycinemia (NKH). The disease may be severe NKH. The disease may be attenuated NKH. The expression vector or pharmaceutical composition for use in therapy may be administered to the blood stream. The expression vector or pharmaceutical composition for use in therapy may be administered intra-venously. The expression vector or pharmaceutical composition for use in therapy may be administered to the central nervous system (CNS). The expression vector or pharmaceutical composition for use in therapy may be administered intra-cisternally, intra- thecally, or intra-cerebroventricularly. The expression vector or pharmaceutical composition for use in therapy may be administered to the blood stream and the CNS, for example, the expression vector or pharmaceutical composition for use in therapy may be administered intra-venously and intra-cisternally, intra-thecally, or intra-cerebroventricularly. The expression vector or pharmaceutical composition for use in therapy may be used alone or in conjunction with other treatments. The expression vector or pharmaceutical composition for use in therapy, wherein the therapy is treating diseases caused by impaired function of GLDC, for example, non-ketotic hyperglycinemia (NKH) may be used alone, or in conjunction with other treatments. The expression vector or pharmaceutical composition for use treating diseases caused by impaired function of GLDC, for example, non-ketotic hyperglycinemia (NKH) may be used in conjunction with known treatments such as sodium benzoate treatment and / or anti- epileptic medication. The expression vector or pharmaceutical composition for use in therapy may be administered to a subject in need thereof in a therapeutically effective amount. Expression Vector for Use in Method of Treatment The sixth aspect of the present invention provides an expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use in a method of treating a disease caused by impaired function of GLDC. Advantages of the expression vector or pharmaceutical composition for use in therapy of the present invention include expression of GLDC mRNA and GLDC protein (resulting in restoration of the glycine cleavage system (GCS) in a subject, leading to decreased levels of glycine in plasma and tissue (e.g., liver and brain) in a subject and restoration of the folate one- carbon metabolism (FOCM) in a subject. Figures 3, 4 and 14 together demonstrate that GLDC mRNA expression is achieved in vivo, in the brain and liver of mice treated with AAV-GLDC expression vectors, providing proof of GLDC transgene transcription. Figures 5-7 demonstrate GLDC protein expression in the plasma and brain of mice treated with AAV-GLDC expression vectors, providing proof of GLDC transgene translation. Figures 8 to 10 and 15A demonstrate the glycine concentration in plasma and brain tissue of mice treated with AAV-GLDC expression vectors and demonstrate that expression vectors of the invention provide normalisation of the glycine concentration, providing proof that the encoded GLDC functions as intended, by restoring activity of the glycine cleavage system (GCS), such that glycine level is depleted. Figures 11 and 12 demonstrate the metabolite concentrations of species associated with the folate one-carbon metabolism (FOCM) and demonstrate the restoration of this system when mice treated with AAV-GLDC expression vectors. While current therapies (e.g., sodium benzoate) enable lowering of systemic glycine in a subject, no current therapy is known to provide restoration of the FOCM, as demonstrated by the presented invention. . Figures 15B and 15C demonstrate that the betaine and choline concentrations in brain tissue of mice treated with AAV-GLDC expression vectors and demonstrate that expression vectors of the invention provide normalisation of the betaine and choline concentration. The disease caused by impaired function of GLDC may be non-ketotic hyperglycinemia (NKH). The disease caused by impaired function of GLDC may be severe NKH. The disease caused by impaired function of GLDC may be attenuated NKH. The expression vector may comprise the expression vector cassette according to the second aspect. The expression vector cassette may be packaged into an adeno-associated virus (AAV) capsid. The AAV may be an AAV9. The AAV expression vector may target the central nervous system (CNS). The AAV expression vector may target the brain. The AAV expression vector may target the liver. The AAV may target the CNS and brain. The AAV may target the brain and liver. The AAV may target the CNS and liver. The AAV may target the CNS, brain and liver. The expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use may be administered to the blood stream. The expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use may be administered intra-venously. The expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use may be administered to the central nervous system (CNS). The expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use may be administered intra- cisternally, intra-thecally, or intra-cerebroventricularly. The expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use may be administered to the blood stream and the CNS, for example, the expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use may be administered intra-venously and intra- cisternally, intra-thecally, or intra-cerebroventricularly. The expression vector for use in a method of treating diseases caused by impaired function of GLDC, for example, non-ketotic hyperglycinemia (NKH), may be used alone, or in conjunction with other treatments. The expression vector for use for use in treating diseases caused by impaired function of GLDC, for example, non-ketotic hyperglycinemia (NKH), may be used in conjunction with with known treatments such as sodium benzoate treatment and / or anti- epileptic medication. The expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use may be administered to a subject in need thereof in a therapeutically effective amount. Method of Treatment The seventh aspect of the present invention provides a method of treating a disease caused by impaired function of GLDC, wherein the method comprises administering an expression vector comprising a sequence encoding glycine decarboxylase (GLDC) to a subject in need thereof. Advantages of the expression vector or pharmaceutical composition for use in therapy of the present invention include expression of GLDC mRNA and GLDC protein (resulting in restoration of the glycine cleavage system (GCS) in a subject, leading to decreased levels of glycine in plasma and tissue (e.g., liver and brain) in a subject and restoration of the folate one- carbon metabolism (FOCM) in a subject. Figures 3 and 4 and 14 together demonstrate that GLDC mRNA expression is achieved in vivo, in the brain and liver of mice treated with AAV- GLDC expression vectors, providing proof of GLDC transgene transcription. Figures 5-7 demonstrate GLDC protein expression in the plasma and brain of mice treated with AAV- GLDC expression vectors, providing proof of GLDC transgene translation. Figures 8 to 10 and 15A together demonstrate the glycine concentration in plasma and brain tissue of mice treated with AAV-GLDC expression vectors and demonstrate that expression vectors of the invention provide normalisation of the glycine concentration, providing proof that the encoded GLDC functions as intended, by restoring activity of the glycine cleavage system (GCS), such that glycine level is depleted. Figures 11 and 12 demonstrate the metabolite concentrations of species associated with the folate one-carbon metabolism (FOCM) and demonstrate the restoration of this system when mice treated with AAV-GLDC expression vectors. While current therapies (e.g., sodium benzoate) enable lowering of systemic glycine in a subject, no current therapy is known to provide restoration of the FOCM, as demonstrated by the presented invention. . Figures 15B and 15C demonstrate that the betaine and choline concentrations in brain tissue of mice treated with AAV-GLDC expression vectors and demonstrate that expression vectors of the invention provide normalisation of the betaine and choline concentration. The disease caused by impaired function of GLDC may be non-ketotic hyperglycinemia (NKH). The disease caused by impaired function of GLDC may be severe NKH. The disease caused by impaired function of GLDC may be attenuated NKH. The expression vector may comprise the expression vector cassette according to the second aspect. The expression vector cassette may be packaged into an adeno-associated virus (AAV) capsid. The AAV may be an AAV9. The AAV expression vector may target the central nervous system (CNS). The AAV expression vector may target the brain. The AAV expression vector may target the liver. The AAV may target the CNS and brain. The AAV may target the brain and liver. The AAV may target the CNS and liver. The AAV may target the CNS, brain and liver. The method of treating a disease caused by impaired function of GLDC may comprise administering the expression vector to the blood stream. The method of treating a disease caused by impaired function of GLDC may comprise administering the expression vector intra-venously. The method of treating a disease caused by impaired function of GLDC may comprise administering the expression vector to the central nervous system (CNS). The method of treating a disease caused by impaired function of GLDC may comprise administering the expression vector intra-cisternally, intra-thecally, or intra-cerebroventricularly. The method of treating a disease caused by impaired function of GLDC may comprise administering the expression vector to the blood stream and the CNS, for example, the method of treating a disease caused by impaired function of GLDC may comprise administering the expression vector intra-venously and intra-cisternally, intra-thecally or intra-cerebroventricularly. The method of treating a disease caused by impaired function of GLDC may comprises administering the expression vector alone, or in conjunction with other treatments. The method of treating a disease caused by impaired function of GLDCmay comprises administering the expression vector in conjunction with known treatments such as sodium benzoate treatment and / or anti-epileptic medication. The method of treating a disease caused by impaired function of GLDC may comprise administering an expression vector comprising a sequence encoding glycine decarboxylase (GLDC) to a subject in need thereof in a therapeutically effective amount. Method of Obtaining Optimized-Codon Sequence The eighth aspect of the present invention provides a method for obtaining a codon-optimised GLDC sequence for gene therapy the method comprising: applying a codon-optimisation algorithm to a wild-type GLDC sequence, to obtain a codon-optimised GLDC sequence, optionally, translating, in silico, the codon-optimised GLDC sequence to confirm identity of the encoded protein with wild-type GLDC protein, administering an expression vector comprising an expression vector cassette comprising the codon-optimised GLDC sequence from step a) or b) to a Gldc-deficient disease model, and determining rescue of the disease phenotype in the Gldc-deficient disease model, wherein rescue of the disease phenotype indicates that the sequence is a codon-optimised GLDC sequence for gene therapy. The codon-optimisation algorithm may be a publicly available codon-optimisation algorithm. For example, the codon-optimisation algorithm may be GenSmart Codon Optimisation tool, GeneArt GeneOptimizer, or Genewiz algorithm. The wild-type GLDC sequence may be a wild-type consensus sequence. The wild-type GLDC sequence may be a cDNA sequence. The wild-type GLDC sequence may be a wild-type consensus cDNA sequence. The ninth aspect of the present invention provides a codon-optimised GLDC sequence for gene therapy obtainable by the method according to the eighth aspect. Examples Example 1 Design of AAV-GLDC vector The cDNA wild-type consensus sequence of the main GLDC transcript (ENST00000321612.8: GLDC-201, SEQ ID No:1) sequence was obtained from public databases. Three publicly available codon-optimisation algorithms were selected: 1. GenSmart Codon Optimisation tool (https: / / www.genscript.com / gensmart-free-gene- codon-optimization.html) 2. GeneArt GeneOptimizer (https: / / www.thermofisher.com / uk / en / home / life- science / cloning / gene-synthesis / geneoptimizer.html) 3. Genewiz algorithm (https: / / www.genewiz.com / en-GB / Public / Services / Gene-Synthesis / Codon- Optimization / ) The wild-type consensus cDNA GLDC sequence was entered into each of these algorithms and the parameters were modified to exclude enzyme restriction sites. The algorithms each generated a codon-optimised sequence (codon-optimised sequence 1, SEQ ID No:2; codon- optimised sequence 2, SEQ ID No:3; codon-optimised sequence 3, SEQ ID No:4). These codon-optimised sequences were denoted Optz1, Optz2 and Optz3. Each codon-optimised sequence was virtually translated (translated in silico) to confirm 100% identity of the encoded protein with wild-type GLDC protein. Each of these sequences was then generated by DNA synthesis, using a commercial service, with the addition of flanking restriction cloning sites (Nhe1 and Not1) and Kozak sequence, and cloned into a standard pUC57 plasmid. Each codon-optimised GLDC sequence was subcloned, using restriction digestion with Nhe1 and Not1 restriction enzymes, into an AAV expression vector cassette or plasmid with the corresponding restriction sites, downstream of a ubiquitous mammalian promoter (CBh; truncated chicken beta-actin hybrid promoter) and upstream of a termination sequence (BGH Poly A, bovine growth hormone polyadenylation sequence). The promoter and termination sequences were flanked by inverted terminal repeats (ITR) derived from AAV2. The resulting expression vector cassettes (AAV-GLDC plasmid DNA) were isolated and sequenced to confirm correct cloning and sequence of the insert, position of ATG start codon and integrity of the stop codon. In addition to codon-optimisation of the GLDC transgene, further design considerations for the expression vector were assessed, including choice of capsid, choice of promoter, and size considerations. AAV vectors, have been shown to be effective platforms for gene therapy applications, and AAV9 in particular has been found to be an optimal AAV serotype for liver and CNS transduction. However, the packaging capacity of AAV vectors has been typically considered to be up to 5 kb (Wu et al 2010), corresponding to the size of the parent AAV genome. Therefore, expression vector cassette design for this vector type was carefully considered. A small promoter (CBh) was selected, to allow efficient packaging, and the length of the vector cassette sequence was tailored by shortening the distance between the end of the CBh promoter and the start codon of GLDC, i.e., by removal of nucleotides from the 5’UTR of the GLDC sequence. These tailored cassettes were packaged into AAV9 capsids and administered by intravenous injection (iv) to Gldc-deficient mice, day 1 post-natal. Details of the Gldc-deficient mice (NKH moue model) can be found below, in the section entitled “NKH mouse model”. Details of the tested cassettes is provided in Table 1 below. The expression vector cassettes or AAV-GLDC plasmids of Table 1 were packaged into AAV9 capsid (packaged by Vector Biolabs) to provide expression vectors. For large scale production, AAV-GLDC vector was co-transfected with Ad Helper vector and AAV rep / cap vector into HEK293 cells. The vector was purified using two CsCl gradients to obtain titre of 1-2 x 1013GC / mL. Table 1 No increase in GLDC mRNA expression (measured as below described in the section entitled “In vivo GLDC mRNA expression by quantitative real time RT-PCR”) above the minimal expression in Gldc-deficient mice was observed for cassette 1 in the liver or brain at 4-5 weeks of age. Analysis of the packaged vector by sequencing showed that the Gldc transgene had been truncated. Cassette 2 however, provided robust mRNA and protein expression (protein expression measured as below described in the section entitled "In vivo GLDC protein expression by immunoblot”), suggesting that shortening of the distance between the end of the promoter and the start codon enhances packaging and expression of the transgene. Cassette 3 also provided robust expression of mRNA and GLDC protein. Cassettes 4 to 7 show that further shortening of the distance between the end of the promoter and the start codon, in these cases by essentially removing the 5’UTR, provided robust expression of GLDC mRNA and protein. In vitro GLDC protein expression and quantification by immunoblot In order to create a system for testing expression of GLDC, GLDC null versions of Huh7 hepatocarcinoma cells were generated, in which deletion of GLDC was confirmed by Sanger sequencing and a functional assay demonstrating lack of cleavage of isotopically labelled glycine. Equimolar quantities of expression vector cassette (AAV plasmid) were used for transfection of Huh7 (wild-type) and Huh7 DGLDC (lacking GLDC) cells, using ~4 µg vector cassette and lipofectamine, with equal cell numbers (0.5 x105cells / mL) used for each condition. Cells were harvested at 72 hours post-transfection, lysed by sonication in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors, and an aliquot was taken for protein assay. Equal quantities of total protein were used for immunoblotting to detect GLDC. Samples were run on 10% NuPAGE gels and transferred to PVDF membranes. Membranes were blocked with tris-buffered saline tween®20 (TBST) containing 5% milk (w / v) and probed with anti-GLDC antibody (1:1,000; HPA002318 Anti-GLDC (rabbit), Atlas Antibodies) and a horseradish peroxidase (HRP)-conjugated secondary antibody (polyclonal anti-rabbit (goat) immunoglobulins / HRP, DAKO) with chemiluminescent detection (ECL Western Blotting Detection reagent). Membranes were stripped and re-probed with anti- GAPDH (MAB374, Millipore) and anti-beta actin (sc-47778, Santa Cruz Biotechnology) antibodies as house-keeping proteins for normalisation. Blots were scanned using quantitative densitometer (GS800; Bio-Rad) and bands were quantified using Image-Studio software. The immunoblots are shown in Figure 1 and sample details are given in Table 2 below.

[0003] Table 2 With reference to Figure 1, the top images shows the immunoblot for GLDC protein for each sample (upper band at ~100kDa). The bottom images show GAPDH immunoblot for the corresponding samples. GLDC was detected in wild-type cells (lane 1, 4, 11), absent in DGLDC cells (lane 2, 12), and partially restored with the vector comprsing GLDC (lane 14), GLDC-Optz1 (lane 18) and GLDC-Optz2 (lane 16), and GLDC-Optz3 (lane 6).. Therefore, each of codon-optimised GLDC sequences 1, 2 and 3 are shown to at least partially restore GLDC protein expression. Protein abundance was also quantified as above mentioned and is shown in Figure 2. GLDC abundance was normalised to GAPDH for each sample. The relative abundance was determined by comparison to a wild-type mock transfected cell line (set as 1.0). Optimised sequences 1 and 2 (AAV9-GLDC Optz 1 vector and AAV9-GLDC Optz 2 vector) showed an increase in relative GLDC abundance when compared with mock transfected GLDC null (GLDC deficient, DGLDC) cells, while AAV-9-Optz3 vector showed a decrease in relative GLDC abundance when compared with mock transfected GLDC null (GLDC deficient, DGLDC) cells. The vector comprising codon optimised GLDC sequence 2 (AAV9-GLDC Optz 2 vector), in particular, generated significantly greater relative GLDC protein abundance than the other sequences tested, including wild-type consensus GLDC, both in wild-type and DGLDC cells (see Figure 2). Codon-optimised GLDC sequence 2 has 77% identity to the wild- type consensus GLDC sequence, differing at 690 of 3063 bases from the cDNA wild-type consensus GLDC sequence. NKH mouse model In order to generate mice carrying a gene-trap allele of Gldc (here denoted GldcGT1), the EUCOMM, EUCG0001_D02 embryonic stem cell line from the European Mouse Mutant Cell Repository was obtained. Chimeric mice carrying the Gldc gene-trap allele were generated by blastocyst injection of ES cells (Embryonic Stem Cell Facility, UCL Institute of Child Health), and mated to 129 / Sv mice to test for germline transmission and to establish a colony of heterozygous mice. Subsequently, the Gldc gene-trap allele was crossed onto the C57Bl / 6 background for five generations in order to generate mice for experimental matings. Generation and characterisation of this mouse line is described in Pai et al. Nat Commun 6:6388 (2015); Leung et al.. J. Inherit.. Metab. Dis. 43: 1186-1198 (2020). Experimental litters were generated by heterozygous matings and homozygous mice were identified by PCR genotyping of genomic DNA. Animal studies were carried out under regulations of the Animals (Scientific Procedures) Act 1986 of the UK Government, and in accordance with the guidance issued by the Medical Research Council, UK in Responsibility in the Use of Animals for Medical Research (July 1993). Heterozygous GldcGT1 / +mice were paired overnight for timed mating. On the day of delivery (P0) or post-natal day 1 (P1), pups were genotyped by PCR using genomic DNA extracted from a tail tip sample. The neonatal GldcGT1 / GT1mice (P1 or P2) were used as the NKH mouse model (Gldc-deficient mice). In vivo GLDC mRNA expression by quantitative real time RT-PCR The neonatal GldcGT1 / GT1mice (P1 or P2) were administered with either an AAV9-mGldc vector or an AAV9-hGLDC Optz 2 vector (prepared as above detailed), by intra-venous injection (iv) into the facial vein (10 µL) and / or intra-cerebroventricular (ic) route (5 µL). Litters were weaned at 21 days and then maintained on a standard diet with weight monitored on a weekly basis. Mice were sacrificed at 5-6 weeks of age and blood was collected by cardiac puncture under terminal anaesthetic, transferred to lithium-heparin tubes (BD Microtainer) and immediately centrifuged for the isolation of plasma. Tissues were rinsed in cold PBS and immediately frozen on dry ice prior to storage at -80oC. RNA was isolated from tissue (brain and liver) using TRIzol reagent (Invitrogen), followed by chloroform extraction and RNA precipitation. First strand cDNA synthesis was performed using random hexamers (Superscript VILO cDNA synthesis kit). The abundance of Gldc mRNA was analysed using qRT–PCR (iTaq, BioRad) on BioRad CFX system, with each sample analysed in triplicate. Primers were located in exons 2 and 4 (5’- AGCATTGATGAGCTCATCGAG-3’ and 5’-TCCAGCAGGGAAGCGTTGGC-3’) of mouse Gldc and (5’-GCCCCTAGAAGCAGAGACTC-3’ and 5’-GGCACGGTTTTCTCGATCAG- 3’) of human codon-optimised sequence 2 GLDC to amplify GLDC but not the mutant transcript. Results were normalized to abundance of Gapdh mRNA (as Pai et al. Nat. Commun. 6: 6388, 2015). Analysis confirmed that administration of AAV9-mGldc vector resulted in increased expression of Gldc. Expression was significantly higher in brain and liver for treated mice than in untreated GldcGT1 / GT1(Gldc-deficient) mice, in the brain this was typically above wild-type levels (see Figure 3). Analysis confirmed that administration of AAV9-hGLDC Optz2 vector results in expression of GLDC in the NKH mouse model (see Figure 4). Endogenous Gldc was expressed in wild-type mice (Gldc+ / +), but at very low levels in GldcGT1 / GT1(NKH model) mice. Human GLDC was detected in treated GldcGT1 / GT1mice, showing mRNA expression from the vector is present at 6 weeks of age in brain tissue. In vivo GLDC protein expression by immunoblot Tissue samples were collected from mice that had received ic + iv administration of AAV- hGLDC-Optz2 vector at P1 (as generated for analysis of mRNA expression). Tissue was homogenised by sonication and protein concentration in the lysate determined (Qubit). Equal total protein loading was used for immunoblot and GLDC protein abundance was determined using anti-GLDC antibody, which GLDC recognises mouse and human GLDC (Atlas antibodies), with GAPDH (detected using anti-GAPDH, Santa Cruz) as loading control. This analysis demonstrated production of vector expressed GLDC protein in the brain (Figure 5) of treated mice. Endogenous mouse GLDC was detected in wild-type (+ / +; lanes 1,2) brain but is absent or very low abundance in GldcGT1 / GT1(NKH model; lane 3) mice. At 6 weeks of age, GLDC is abundant in brain tissue of GldcGT1 / GT1mice that had received administration of AAV- hGLDC-Optz2 (lanes 4-15) by iv + ic route at P1. This analysis also demonstrated production of vector expressed GLDC protein in the liver (Figure 7) of treated mice. At 6 weeks of age, endogenous mouse GLDC is detected in wild- type liver (+ / +; lanes 1,2 of each blot) but is absent in GldcGT1 / GT1(NKH model; lane 3 of each blot) mice. Human GLDC is detected in liver after AAV-hGLDC-opt2 treatment by iv + ic route at P1 (lanes 4-15). For histological analysis, formalin-fixed mouse brains were dehydrated, embedded in paraffin wax and sectioned at 7 µm thickness. For immunohistochemistry, primary and secondary antibodies were anti-GLDC (1:300, rabbit polyclonal, Atlas Antibodies) and Alexafluor secondary antibody (donkey anti-rabbit, 1:250, Invitrogen). For nuclear staining, cells were incubated with 4,6-diamidino-2-phenylindole (DAPI, 1:10,000 in PBS). Fluorescent images were collected on an Axiophot microscope (Zeiss) with a DC500 camera (Leica), using FireCam software (Leica). Immunohistochemistry confirmed that expression of vector mediated GLDC expression recapitulates endogenous sites of expression in the brain (see Figure 6). Endogenous expression in wild-type (+ / +) brain was detected throughout the brain (A). Higher magnification images (E-J) show expression in hippocampus (E-G) and cerebellum (H-J). GLDC is not detected in untreated Gldc-deficient (GldcGT1 / GT1) brain (B, I). In mice treated with AAV9- hGLDC-Optz2 by ic route, vector expressed GLDC is detected with widespread expression (C, F – male, D, G – female), including in hippocampus (F-G) and cerebellum (J). Glycine analysis by mass spectrometry Tissue extracts were prepared by sonication in phosphate buffered saline (PBS) containing 1x protease inhibitor cocktail (Roche). An aliquot was removed for protein determination by the Qubit assay. Tissue debris were removed by centrifugation (12,000 g at 4oC). Plasma and tissue lysate glycine were measured following the Kairos amino acid sample preparation and method (Waters corporation, UK). Protein was removed by the addition of 5-sulphosalicylic acid containing a specified quantity of amino acid internal standard in a ratio of 1:1 (sample:internal standard solution). Samples were derivatised using AccQ.Tag reagent. Glycine was resolved and detected using CORTECS UPLC C18, 1.6 μm, 2.1 x 150 mm column on an UPLC coupled to XEVO-TQS mass spectrometer (Waters Corporation, UK). Plasma and tissue glycine reflects liver glycine cleavage activity. Plasma and tissue glycine is therefore elevated in untreated GldcGT1 / GT1(NKH model) mice (Pai et al. Nat. Commun.6: 6388, 2015; Leung et al. J. Inherit. Metab. Dis. 43:1186-1198, 2020). Glycine concentration in plasma and tissue of wild-type, GldcGT1 / GT1and treated GldcGt1 / GT1mice was determined by mass spectrometry as above. Following administration of AAV- mGldc vector at P1 by iv injection we observed lowering of plasma glycine in male and female GldcGT1 / GT1mice compared with vehicle-treated controls (Figure 8). Plasma glycine is elevated in vehicle treated GldcGT1 / GT1mice compared with wild-type (+ / +) littermates (**significantly different, p<0.001). At 6 weeks after treatment at P1 plasma glycine was lower in GldcGT1 / GT1mice treated by iv+ic route, than in vehicle treated GldcGt1 / GT1mice (* significant difference (*p<0.01). The effect of iv +ic treatment among GldcGtT1 / GT1mice was observed among both male and female mice (significant difference between mice of same sex, p<0.05). (n = 19 + / +, 21 GldcGT1 / GT1, 19 GldcGT1 / GT1iv+iv treated, 9 GldcGT1 / GT1ic treated). This confirmed that AAV-expressed mouse GLDC exhibits enzymatic activity. Expression in brain (ic only group) showed similar plasma glycine concentration to the untreated GldcGT1 / GT1samples, however it had not been expected that expression in the brain would alter plasma glycine. Note that female (F) GldcGT1 / GT1mutant mice have a trend toward higher baseline glycine. Following administration of GldcGT1 / GT1with AAV9-mGldc, AAV9-hGLDC, AAV9-hGLDC- Optz1 and AAV9-hGLDC-Optz2 vectors at P1 by iv, as above described, lowering of plasma glycine in male and female GldcGT1 / GT1mice compared with vehicle-treated controls was observed (Figure 9). Compared with untreated GldcGT1GT1there is a significant reduction in plasma glycine in mice treated with AAV-hGLDC and AAV-hGLDC-Otpz2. Plasma glycine in GldcGT1 / GT1mice is significantly higher than in (+ / +) wild-type (##p<0.001). Compared with untreated (vehicle control) GLDC-deficient mice there is a significant reduction in mean plasma glycine in GLDC-deficient mice treated with AAV9-mGLDC (**p<0.001), AAV9-hGLDC (*p<0.02), and AAV9-hGDLDC-Optz2 (**p<0.001). For human GLDC vectors n = 11-12 mice per vector (4-7 mice / sex / vector). This confirmed that AAV-expressed human GLDC exhibits enzymatic activity. Brain tissue glycine was elevated in GldcGT1 / GT1(NKH model) mice. Brain tissue glycine was determined in wild-type and GldcGT1 / GT1(NKH model) mice after vehicle or AAV-GLDC treatment (AAV9-mGldc and AAV9-hGLDC-Optz2) at P1. The data shows that glycine abundance is normalised (lowered in comparison to the Gldc-deficient control) by treatment with AAV9-GLDC using either mouse or human-optimised vector (Figure 10). Brain tissue glycine did not differ with sex in control mice (+ / + or GldcGT1 / GT1) so samples were grouped by genotype / treatment. Glycine is significantly more abundant in the brain of GldcGT1 / GT1than in wild-type (** p<0.01). Each treatment led to significantly lower glycine abundance in brain of GldcGT1 / GT1mice than in vehicle treated controls (* significant difference to control GldcGT1 / GT1, p<0.05). Analysis of metabolites in folate one-carbon metabolism and related-metabolites by mass spectrometry Tissue extracts were prepared by homogenization of samples by sonication in buffer containing 20 mM ammonia acetate, 0.1% ascorbic acid, 0.1% citric acid and 100 mM dithiothreitol at pH 7. An aliquot was removed for protein determination by the Qubit assay. Protein was removed by precipitation by addition of two volumes of acetonitrile and centrifugation (12,000 g at 4oC). Supernatants were transferred, lyophilized, stored at -80°C and re-suspended in dH2O before analysis. Folate analysis was carried out by UPLC-MS / MS. Lyophilized samples were resuspended in 30 mL water (milli-Q) and centrifuged for 5 min at 12,000 g at 4oC. Metabolites were resolved by reversed-phase UPLC (Acquity UPLC BEH C18 column, Waters Corporation, UK). Solvents for UPLC were as follows: Buffer A, 5% methanol, 95% Milli-Q water and 5 mM dimethylhexylamine at pH 8.0; Buffer B, 100% methanol, 5 mM dimethylhexylamine. The column was equilibrated with 95% Buffer A: 5% Buffer B. The sample injection volume was 25 mL. The UPLC protocol consisted of 95% Buffer A: 5% Buffer B for 1 min, followed by a gradient of 5–60% Buffer B over 9 min and then 100% Buffer B for 6 min before re- equilibration for 4 min. The metabolites were eluted at a flow rate of 0.5mL / min and the wash step with 100% Buffer B was at flow rate of 0.6mL / min. The UPLC was coupled to a XEVO- TQS mass spectrometer (Waters Corporation) operating in negative-ion mode using the following settings: capillary 2.5 kV, source temperature 150oC, desolvation temperature 600oC, cone gas flow rate 150 L / h , and desolvation gas flow rate 1,200 L / h. Folates were measured by multiple reaction monitoring with optimized cone voltage and collision energy for precursor and product ions (as described Pai et al. Nat Commun. 2015; Leung et al. Cell Reports 2017). In order to minimize variation in sample or running conditions between genotypes, samples of each genotype were analysed randomly. For betaine and choline analysis, frozen tissue samples were sent to Metabolon Ltd (USA) for metabolite profiling by LC-MS / MS. Loss of GLDC activity leads to suppression of folate one-carbon metabolism (FOCM), owing to reduced supply of glycine-derived one carbon units (Pai et al. Nat. Commun.6: 6388, 2015; Leung et al. Cell Reports, 21: 1795-1808, 2017). The abundance of tetrahydrofolate (THF) is increased and the abundance of 5-methyl tetrahydrofolate (5-methylTHF) is decreased in GldcGT1 / GT1mice compared with wild-type (+ / +) in brain at 6 weeks of age. Mice treated with AAV9-hGLDC-Optz2 and AAV9-mGldc by iv+ic or ic only route at P1 show normalisation of folate levels in brain tissue at 6 weeks of age (Figure 11). This data confirms that glycine cleavage activity is restored by AAV-expressed protein in brain tissue. Mice treated with AAV- mGLDC by iv+ic route and mice treated AAV9-hGLDC-Optz2 show normalisation of folate levels (*significant difference to untreated, p<0.05). The FOCM intermediates betaine and choline show diminished abundance in brain of GldcGT1GT1(NKH model) mice at 6 weeks of age. These levels are restored in the brain following ic administration of AAV9-CBh-mGldc (with or without concomitant iv administration), showing correction of brain FOCM (Figure 12). Betaine and choline are significantly less abundant in brain of untreated Gldc-deficient mice (*p<0.05) but are normalised by treatment with AAV-mGldc by iv+ic or ic route at P1 (followed by sample collection at 6 weeks of age). Example 2 AAV-mediated expression of GLDC under control of the GFAP promoter In a further study the CBh promoter was replaced by a GFAP promoter in order to drive expression of GLDC in astrocytes which are a key target cell type in which GLDC is expressed. We generated AAV-GFAP-GLDC-optz2 plasmid vector (containing the optimised cDNA encoding human GLDC with a 0.7 kb GFAP promoter). The vector was packaged in AAV9 capsid. The vector AAV-CBh-GLDC-optz2 plasmid vector was used as the starting material. The CBh promoter was removed and replaced with a GFAP (0.7) promoter which contains key elements of the 2.2Kb human GFAP promoter and recapitulates the expression pattern of the parental promoter (cloning performed using commercial service). The 0.7kb GFAP promoter used is the gfaABC1D promoter as described Lee et al Glia 56: 481-493, 2008. Methods AAV9-GFAP-hGLDCoptz2 was administered to neonatal GLDC-deficient (GldcGT1 / GT1) by intra-cerebroventricular route (5 µl injection volume). Mice were maintained for 12 weeks. Blood was collected for quantification of glycine and expression of GLDC was investigated by immunohistochemistry using an anti-GLDC antibody (Atlas Antibodies). Glycine, betaine and choline abundance were determined by liquid chromatography tandem mass spectrometry (Metabolon). Results Administration of AAV9-GFAP-hGLDCoptz2 achieves GLDC protein expression in the brain In adult mice which received neonatal administration of AAV9-GFAP-GLDC via intra- cerebroventricular route, immnohistochemical analysis showed that GLDC expression was widespread in the brain of GLDC-deficient mice (GldcGT1 / GT1) which lack endogenous expression (Figure 14). Expression sites included cortex, hippocampus, cerebellum and olfactory bulbs. Expression of GLDC via AAV9-GFAP-hGLDCoptz2 treatment leads to normalisation of metabolic abnormalities in brain of GLDC-deficient mice Mass spectrometry based analysis was used to evaluate biomarkers and therapeutic targets which are downstream of impaired GLDC expression. Glycine accumulates in the brain of GLDC-deficient compared with wild-type mice and is partially corrected by AAV9-GFAP- hGLDCoptz2 treatment (p=0.06, n = 6 per group)(Figure 15A). A power calculation shows that sample size of 8 per group would be required to show statistical significance with p<0.05. Betaine and choline are found at lower abundance in the brain of GLDC-deficient mice (Figure 15B-C), as a consequence of impaired folate one-carbon metabolism (FOCM). In GLDC- deficient mice treated with AAV9-GFAP-GLDC the abundance of betaine is significantly increased compared with vehicle-treated GLDC-deficient controls (Figure 15B) and there is a trend towards increased choline (Figure 15C). The abundance of betaine and choline in brain of treated GLDC-deficient mice does not significantly differ from wild-type controls. Sequences used in the present invention SEQ ID No:1 – Consensus, wild-type hGLDC cDNA sequence ATGCAGTCCTGTGCCAGGGCGTGGGGGCTGCGCCTGGGCCGCGGGGTCGGGGGCGGCCGC CGCCTGGCTGGGGGATCGGGGCCGTGCTGGGCGCCGCGGAGCCGGGACAGCAGCAGTGGC GGCGGGGACAGCGCCGCGGCTGGGGCCTCGCGCCTCCTGGAGCGCCTTCTGCCCAGACAC GACGACTTCGCTCGGAGGCACATCGGCCCTGGGGACAAAGACCAGAGAGAGATGCTGCAG ACCTTGGGGCTGGCGAGCATTGATGAATTGATCGAGAAGACGGTCCCTGCCAACATCCGT TTGAAAAGACCCTTGAAAATGGAAGACCCTGTTTGTGAAAATGAAATCCTTGCAACTCTG CATGCCATTTCAAGCAAAAACCAGATCTGGAGATCGTATATTGGCATGGGCTATTATAAC TGCTCAGTGCCACAGACGATTTTGCGGAACTTACTGGAGAACTCAGGATGGATCACCCAG TATACTCCATACCAGCCTGAGGTGTCTCAGGGGAGGCTGGAGAGTTTACTCAACTACCAG ACCATGGTGTGTGACATCACAGGCCTGGACATGGCCAATGCATCCCTGCTGGATGAGGGG ACTGCAGCCGCAGAGGCACTGCAGCTGTGCTACAGACACAACAAGAGGAGGAAATTTCTC GTTGATCCCCGTTGCCACCCACAGACAATAGCTGTTGTCCAGACTCGAGCCAAATATACT GGAGTCCTCACTGAGCTGAAGTTACCCTGTGAAATGGACTTCAGTGGAAAAGATGTCAGT GGAGTGTTGTTCCAGTACCCAGACACGGAGGGGAAGGTGGAAGACTTTACGGAACTCGTG GAGAGAGCTCATCAGAGTGGGAGCCTGGCCTGCTGTGCTACTGACCTTTTAGCTTTGTGC ATCTTGAGGCCACCTGGAGAATTTGGGGTAGACATCGCCCTGGGCAGCTCCCAGAGATTT GGAGTGCCACTGGGCTATGGGGGACCCCATGCAGCATTTTTTGCTGTCCGAGAAAGCTTG GTGAGAATGATGCCTGGAAGAATGGTGGGGGTAACAAGAGATGCCACTGGGAAAGAAGTG TATCGTCTTGCTCTTCAAACCAGGGAGCAACACATTCGGAGAGACAAGGCTACCAGCAAC ATCTGTACAGCTCAGGCCCTCTTGGCGAATATGGCTGCCATGTTTGCAATCTACCATGGT TCCCATGGGCTGGAGCATATTGCTAGGAGGGTACATAATGCCACTTTGATTTTGTCAGAA GGTCTCAAGCGAGCAGGGCATCAACTCCAGCATGACCTGTTCTTTGATACCTTGAAGATT CAGTGTGGCTGCTCAGTGAAGGAGGTCTTGGGCAGGGCCGCTCAGCGGCAGATCAATTTT CGGCTTTTTGAGGATGGCACACTTGGTATTTCTCTTGATGAAACAGTCAATGAAAAAGAT CTGGACGATTTGTTGTGGATCTTTGGTTGTGAGTCATCTGCAGAACTGGTTGCTGAAAGC ATGGGAGAGGAGTGCAGAGGTATTCCAGGGTCTGTGTTCAAGAGGACCAGCCCGTTCCTC ACCCATCAAGTGTTCAACAGCTACCACTCTGAAACAAACATTGTCCGGTACATGAAGAAA CTGGAAAATAAAGACATTTCCCTTGTTCACAGCATGATTCCACTGGGATCCTGCACCATG AAACTGAACAGTTCGTCTGAACTCGCACCTATCACATGGAAAGAATTTGCAAACATCCAC CCCTTTGTGCCTCTGGATCAAGCTCAAGGATATCAGCAGCTTTTCCGAGAGCTTGAGAAG GATTTGTGTGAACTCACAGGTTATGACCAGGTCTGTTTCCAGCCAAACAGCGGAGCCCAG GGAGAATATGCTGGACTGGCCACTATCCGAGCCTACTTAAACCAGAAAGGAGAGGGGCAC AGAACGGTTTGCCTCATTCCGAAATCAGCACATGGGACCAACCCAGCAAGTGCCCACATG GCAGGCATGAAGATTCAGCCTGTGGAGGTGGATAAATATGGGAATATCGATGCAGTTCAC CTCAAGGCCATGGTGGATAAGCACAAGGAGAACCTAGCAGCTATCATGATTACATACCCA TCCACCAATGGGGTGTTTGAAGAGAACATCAGTGACGTGTGTGACCTCATCCATCAACAT GGAGGACAGGTCTACCTAGACGGGGCAAATATGAATGCTCAGGTGGGAATCTGTCGCCCT GGAGACTTCGGGTCTGATGTCTCGCACCTAAATCTTCACAAGACCTTCTGCATTCCCCAC GGAGGAGGTGGTCCTGGCATGGGGCCCATCGGAGTGAAGAAACATCTCGCCCCGTTTTTG CCCAATCATCCCGTCATTTCACTAAAGCGGAATGAGGATGCCTGTCCTGTGGGAACCGTC AGTGCGGCCCCATGGGGCTCCAGTTCCATCTTGCCCATTTCCTGGGCTTATATCAAGATG ATGGGAGGCAAGGGTCTTAAACAAGCCACGGAAACTGCGATATTAAATGCCAACTACATG GCCAAGCGATTAGAAACACACTACAGAATTCTTTTCAGGGGTGCAAGAGGTTATGTGGGT CATGAATTTATTTTGGACACGAGACCCTTCAAAAAGTCTGCAAATATTGAGGCTGTGGAT GTGGCCAAGAGACTCCAGGATTATGGATTTCACGCCCCTACCATGTCCTGGCCTGTGGCA GGGACCCTCATGGTGGAGCCCACTGAGTCGGAGGACAAGGCAGAGCTGGACAGATTCTGT GATGCCATGATCAGCATTCGGCAGGAAATTGCTGACATTGAGGAGGGCCGCATCGACCCC AGGGTCAATCCGCTGAAGATGTCTCCACACTCCCTGACCTGCGTTACATCTTCCCACTGG GACCGGCCTTATTCCAGAGAGGTGGCAGCATTCCCACTCCCCTTCGTGAAACCAGAGAAC AAATTCTGGCCAACGATTGCCCGGATTGATGACATATATGGAGATCAGCACCTGGTTTGT ACCTGCCCACCCATGGAAGTTTATGAGTCTCCATTTTCTGAACAAAAGAGGGCGTCTTCT TAG SEQ ID No:2 – Codon-optimised hGLDC cDNA sequence 1 (Optz 1) ATGCAGAGTTGTGCTCGGGCTTGGGGGCTGAGGCTGGGCAGGGGAGTCGGGGGAGGG AGGAGGCTGGCTGGAGGAAGCGGACCTTGTTGGGCCCCCAGGTCCAGAGACAGCTCC TCTGGAGGAGGCGATAGCGCCGCCGCCGGAGCCAGCCGGCTGCTGGAGCGCCTGCTG CCAAGGCACGACGATTTCGCCAGGAGACACATCGGCCCCGGCGACAAGGATCAGAGG GAGATGCTGCAGACCCTGGGCCTGGCCTCTATCGACGAGCTGATCGAGAAGACAGTG CCTGCCAATATCCGGCTGAAGCGCCCTCTGAAGATGGAGGATCCAGTGTGCGAGAAC GAGATCCTGGCCACCCTGCACGCCATCAGCTCCAAGAATCAGATCTGGCGCTCCTAT ATCGGCATGGGCTACTATAACTGTTCTGTGCCACAGACAATCCTGAGGAACCTGCTG GAGAATTCCGGCTGGATCACCCAGTACACACCTTATCAGCCAGAGGTGTCCCAGGGC AGGCTGGAGTCTCTGCTGAATTATCAGACCATGGTGTGCGACATCACAGGCCTGGAT ATGGCCAACGCCTCTCTGCTGGACGAGGGAACCGCTGCCGCCGAGGCCCTGCAGCTG TGCTACAGGCACAACAAGCGGCGCAAGTTTCTGGTGGATCCAAGATGTCACCCCCAG ACCATCGCCGTGGTGCAGACACGGGCCAAGTATACCGGCGTGCTGACAGAGCTGAAG CTGCCATGCGAGATGGACTTTAGCGGCAAGGACGTGAGCGGCGTGCTGTTCCAGTAC CCCGACACCGAGGGCAAGGTGGAGGATTTCACAGAGCTGGTGGAGAGGGCACACCAG TCTGGCAGCCTGGCCTGCTGTGCAACCGACCTGCTGGCCCTGTGCATCCTGCGCCCC CCTGGAGAGTTTGGAGTGGATATCGCCCTGGGCTCTAGCCAGAGGTTCGGCGTGCCA CTGGGATATGGAGGACCTCACGCAGCCTTCTTTGCCGTGCGCGAGAGCCTGGTGAGG ATGATGCCCGGCAGAATGGTGGGCGTGACCAGAGACGCCACAGGCAAGGAGGTGTAC CGGCTGGCCCTGCAGACCAGGGAGCAGCACATCAGGAGAGATAAGGCCACCTCCAAC ATCTGCACAGCACAGGCCCTGCTGGCCAATATGGCAGCAATGTTCGCCATCTACCAC GGCAGCCACGGCCTGGAGCACATCGCAAGGAGGGTGCACAATGCCACCCTGATCCTG TCCGAGGGCCTGAAGAGAGCAGGACACCAGCTGCAGCACGACCTGTTCTTTGATACA CTGAAGATCCAGTGCGGCTGTTCTGTGAAGGAGGTGCTGGGAAGAGCAGCACAGAGG CAGATCAATTTCCGGCTGTTTGAGGACGGCACCCTGGGCATCAGCCTGGACGAGACA GTGAACGAGAAGGATCTGGACGATCTGCTGTGGATCTTTGGATGCGAGTCCTCTGCC GAGCTGGTGGCAGAGTCCATGGGAGAGGAGTGTAGGGGAATCCCAGGAAGCGTGTTC AAGCGCACCTCCCCTTTTCTGACACACCAGGTGTTCAACTCCTACCACTCTGAGACA AATATCGTGAGATATATGAAGAAGCTGGAGAACAAGGACATCTCCCTGGTGCACTCT ATGATCCCCCTGGGCAGCTGTACCATGAAGCTGAATAGCTCCTCTGAGCTGGCCCCT ATCACATGGAAGGAGTTTGCCAACATCCACCCCTTCGTGCCTCTGGATCAGGCCCAG GGCTACCAGCAGCTGTTTCGGGAGCTGGAGAAGGACCTGTGCGAGCTGACCGGCTAT GATCAGGTGTGCTTCCAGCCTAATAGCGGAGCACAGGGAGAGTACGCAGGCCTGGCC ACAATCCGGGCCTATCTGAACCAGAAGGGAGAGGGACACAGGACCGTGTGCCTGATC CCTAAGTCTGCCCACGGCACAAATCCAGCCAGCGCCCACATGGCCGGCATGAAGATC CAGCCCGTGGAGGTGGACAAGTACGGCAATATCGATGCCGTGCACCTGAAGGCCATG GTGGACAAGCACAAGGAGAACCTGGCCGCCATCATGATCACCTATCCTAGCACAAAC GGCGTGTTTGAGGAGAATATCTCCGACGTGTGCGATCTGATCCACCAGCACGGAGGA CAGGTGTACCTGGATGGAGCCAACATGAATGCACAAGTGGGAATCTGCAGGCCTGGC GACTTTGGCAGCGACGTGAGCCACCTGAACCTGCACAAGACCTTCTGTATCCCACAC GGAGGAGGAGGACCTGGAATGGGCCCAATCGGCGTGAAGAAGCACCTGGCCCCATTC CTGCCCAACCACCCTGTGATCTCCCTGAAGAGGAATGAGGACGCATGCCCAGTGGGA ACAGTGTCTGCCGCACCTTGGGGAAGCTCCTCTATCCTGCCAATCAGCTGGGCCTAC ATCAAGATGATGGGAGGCAAGGGCCTGAAGCAGGCAACCGAGACAGCCATCCTGAAC GCCAATTACATGGCCAAGAGGCTGGAGACACACTATAGAATCCTGTTTAGGGGCGCC AGAGGCTATGTGGGCCACGAGTTTATCCTGGACACAAGACCATTCAAGAAGAGCGCC AACATCGAGGCAGTGGACGTGGCAAAGCGGCTGCAGGATTACGGCTTTCACGCACCA ACCATGTCCTGGCCAGTGGCAGGCACCCTGATGGTGGAGCCCACAGAGTCTGAGGAC AAGGCCGAGCTGGACAGGTTCTGCGATGCCATGATCTCTATCAGACAGGAGATCGC CGACATCGAGGAGGGCCGCATCGATCCCAGGGTGAATCCTCTGAAGATGTCTCCT CACAGCCTGACCTGCGTGACCAGCTCCCACTGGGACAGACCATATAGCAGGGAGGTG GCAGCATTCCCACTGCCCTTTGTGAAGCCAGAGAACAAGTTCTGGCCCACCATCGCC AGAATCGACGATATCTACGGCGATCAGCACCTGGTGTGCACATGCCCTCCTATGGA AGTCTACGAATCACCTTTTAGCGAGCAGAAGAGAGCCTCATCCTGA SEQ ID No:3 - Codon-optimised hGLDC cDNA sequence 2 (Optz 2) ATGCAGTCTTGTGCTAGAGCCTGGGGACTGAGACTCGGCAGAGGTGTTGGCGGAG GAAGAAGGCTTGCTGGCGGATCTGGACCTTGTTGGGCCCCTAGAAGCAGAGACTC TTCTAGCGGCGGAGGCGATTCTGCTGCTGCTGGTGCTTCTCGGCTGCTGGAAAGA CTGCTGCCCAGACACGACGACTTCGCCAGAAGGCATATCGGCCCTGGCGACAAGG ACCAGAGAGAGATGCTGCAGACACTGGGCCTCGCCAGCATCGACGAGCTGATCGA GAAAACCGTGCCTGCCAACATCCGGCTGAAGCGGCCTCTGAAGATGGAAGATCCC GTGTGCGAGAACGAGATCCTGGCCACACTGCACGCCATCAGCAGCAAGAACCAGA TTTGGCGGAGCTACATCGGCATGGGCTACTACAATTGCAGCGTGCCCCAGACCAT CCTGCGGAACCTGCTCGAGAATAGCGGCTGGATCACCCAGTACACCCCTTACCAG CCTGAGGTGTCACAGGGCAGACTGGAATCCCTGCTGAACTACCAGACAATGGTCT GCGACATCACCGGCCTGGACATGGCTAATGCCAGCCTGCTGGATGAGGGAACAGC CGCTGCTGAAGCTCTGCAGCTGTGCTACAGACACAACAAGCGGCGGAAGTTCCTG GTGGACCCCAGATGTCACCCTCAGACAATTGCCGTGGTGCAGACCAGAGCCAAGT ACACCGGCGTGCTGACAGAGCTGAAGCTGCCCTGCGAGATGGACTTCTCTGGCAA GGATGTGTCTGGCGTGCTGTTTCAGTACCCCGACACCGAGGGCAAAGTGGAAGAT TTCACCGAGCTGGTGGAACGGGCCCACCAGTCTGGATCTCTGGCCTGTTGTGCCA CTGATCTGCTGGCCCTGTGTATCCTCAGACCTCCTGGCGAGTTCGGCGTGGACAT TGCCCTTGGAAGCAGCCAGAGATTCGGAGTGCCTCTCGGATATGGCGGACCTCAC GCCGCCTTTTTTGCCGTGCGAGAGTCTCTCGTGCGGATGATGCCTGGCAGAATGG TCGGAGTGACCAGAGATGCCACCGGCAAAGAGGTGTACAGACTGGCCCTGCAGAC AAGAGAGCAGCACATCAGAAGAGACAAGGCCACCAGCAACATCTGCACCGCTCAA GCCCTGCTGGCCAATATGGCCGCCATGTTCGCCATCTACCACGGCTCTCACGGCC TGGAACACATTGCCAGAAGAGTGCACAACGCCACACTGATCCTGAGCGAGGGACT GAAGAGAGCCGGTCATCAGCTGCAGCACGACCTGTTCTTCGACACCCTGAAGATC CAGTGCGGCTGCAGCGTGAAAGAGGTGCTGGGTAGAGCTGCCCAGCGGCAGATCA ACTTCAGACTGTTCGAGGATGGCACCCTGGGCATCAGCCTGGACGAGACAGTGAA CGAGAAGGACCTGGACGACCTGCTGTGGATCTTTGGCTGTGAAAGCAGCGCCGAA CTGGTGGCCGAGTCTATGGGCGAAGAGTGTAGAGGCATCCCCGGCAGCGTGTTCA AGCGGACAAGCCCATTTCTGACCCACCAGGTGTTCAACAGCTACCACAGCGAGAC AAACATCGTGCGGTACATGAAGAAGCTCGAGAACAAGGACATCTCCCTGGTGCAC AGCATGATCCCTCTGGGCTCTTGCACCATGAAGCTGAACAGCAGCTCCGAGCTGG CCCCTATCACCTGGAAAGAGTTCGCCAACATTCACCCCTTCGTGCCCCTGGATCA GGCCCAGGGATATCAGCAGCTGTTCAGAGAGCTGGAAAAGGACCTGTGCGAGCTG ACCGGCTACGACCAAGTGTGCTTCCAGCCTAATTCTGGCGCCCAGGGCGAATATG CCGGACTGGCCACAATCAGAGCCTACCTGAACCAGAAAGGCGAGGGCCACAGAAC CGTGTGTCTGATCCCTAAAAGCGCCCACGGCACAAACCCTGCCTCTGCTCATATG GCCGGAATGAAGATTCAGCCCGTGGAAGTGGATAAGTACGGCAACATCGACGCCG TGCACCTGAAGGCCATGGTGGACAAGCACAAAGAGAACCTGGCCGCTATCATGAT CACATACCCCAGCACCAACGGCGTGTTCGAAGAGAACATCAGCGACGTGTGCGAC CTGATCCACCAGCATGGCGGACAGGTTTACCTGGACGGCGCCAACATGAATGCCC AAGTGGGCATCTGCAGACCCGGCGATTTTGGCTCCGATGTGTCCCACCTGAACCT GCACAAGACATTCTGCATCCCTCATGGCGGTGGCGGCCCTGGAATGGGACCTATT GGCGTGAAGAAGCACCTGGCTCCATTTCTGCCTAATCACCCCGTGATCAGCCTGA AGCGGAACGAGGATGCTTGTCCTGTGGGCACCGTTTCTGCTGCCCCTTGGGGCTC TAGCTCCATCCTGCCTATCAGCTGGGCCTACATCAAGATGATGGGCGGCAAGGGC CTGAAGCAGGCCACAGAGACAGCCATCCTGAACGCCAACTACATGGCCAAGAGAC TGGAAACCCACTACCGGATCCTGTTTAGAGGCGCCAGAGGCTATGTGGGCCACGA GTTCATCCTGGACACCCGGCCTTTTAAGAAGTCCGCCAATATCGAGGCCGTGGAC GTGGCCAAAAGGCTGCAGGATTACGGCTTTCACGCCCCTACCATGAGCTGGCCTG TGGCCGGAACACTGATGGTGGAACCTACCGAGAGCGAGGACAAGGCCGAGCTGGA TAGATTCTGCGACGCCATGATCAGCATCCGGCAAGAGATCGCCGACATCGAGGAA GGCAGAATCGACCCCAGAGTGAACCCACTGAAGATGTCCCCACACAGCCTGACCT GTGTGACCAGCAGCCACTGGGACAGACCCTACTCTAGAGAGGTGGCCGCCTTTCC TCTGCCTTTCGTGAAGCCTGAGAACAAGTTCTGGCCCACAATCGCCCGGATCGAC GACATCTATGGCGATCAGCACCTCGTGTGCACCTGTCCTCCAATGGAAGTGTACG AGAGCCCCTTCAGCGAGCAGAAGAGGGCCAGTTCTTGA SEQ ID No:4 - Codon-optimised hGLDC cDNA sequence 3 (Optz 3) ATGCAGAGCTGCGCTAGAGCTTGGGGACTGAGGCTCGGAAGAGGCGTCGGAGGAG GCAGAAGGCTCGCCGGAGGATCCGGACCTTGTTGGGCTCCCAGATCTAGAGACAG CAGCTCCGGAGGAGGAGATAGCGCTGCCGCTGGAGCCTCCAGACTGCTGGAAAGA CTGCTCCCTAGGCACGATGACTTCGCTAGAAGACACATCGGCCCCGGCGACAAGG ATCAGAGGGAGATGCTCCAGACCCTCGGACTGGCCAGCATCGATGAACTGATCGA AAAAACAGTGCCCGCCAACATTAGACTCAAGAGACCTCTGAAGATGGAAGACCCC GTGTGCGAAAACGAAATTCTGGCCACACTCCATGCCATCAGCTCCAAGAACCAGA TTTGGAGAAGCTACATCGGCATGGGCTACTATAACTGCTCCGTGCCTCAAACCAT TCTGAGGAATCTGCTGGAGAACTCCGGATGGATCACCCAGTACACCCCTTATCAG CCCGAGGTGTCCCAAGGAAGACTGGAGTCCCTCCTCAATTACCAGACCATGGTGT GCGACATTACCGGCCTCGACATGGCCAACGCCTCTCTGCTGGATGAGGGAACAGC TGCCGCCGAAGCCCTCCAGCTCTGCTACAGACACAATAAGAGAAGAAAGTTCCTC GTCGATCCCAGATGCCACCCCCAAACAATCGCCGTGGTGCAAACAAGAGCCAAAT ATACCGGCGTGCTCACCGAGCTCAAGCTCCCTTGCGAGATGGATTTTTCCGGCAA GGACGTGAGCGGAGTGCTCTTCCAGTACCCCGACACCGAGGGAAAGGTCGAGGAC TTCACCGAGCTCGTCGAGAGAGCTCACCAGAGCGGATCTCTGGCTTGCTGCGCTA CAGATCTGCTGGCTCTCTGCATTCTGAGACCCCCCGGCGAGTTCGGCGTGGACAT TGCTCTGGGATCCTCCCAAAGATTCGGCGTCCCCCTCGGATATGGAGGCCCCCAC GCTGCTTTCTTTGCTGTGAGGGAGTCTCTGGTGAGAATGATGCCCGGCAGAATGG TGGGCGTCACCAGAGACGCCACCGGCAAGGAGGTCTATAGGCTGGCTCTGCAGAC AAGGGAGCAGCATATTAGAAGGGACAAGGCCACATCCAACATCTGTACAGCTCAA GCTCTGCTGGCCAACATGGCTGCCATGTTCGCTATCTATCACGGCAGCCACGGAC TGGAGCACATTGCTAGAAGGGTGCATAACGCCACACTGATTCTGTCCGAAGGACT CAAAAGAGCCGGACACCAGCTGCAGCACGATCTGTTTTTCGATACACTGAAGATC CAATGCGGCTGTAGCGTCAAGGAAGTGCTGGGAAGAGCTGCCCAGAGGCAGATCA ATTTTAGACTGTTTGAGGATGGCACACTGGGAATTTCTCTGGACGAGACCGTGAA CGAAAAGGATCTGGACGATCTGCTGTGGATCTTTGGCTGTGAGAGCTCCGCTGAA CTGGTGGCCGAGAGCATGGGAGAGGAGTGTAGAGGCATCCCCGGCTCCGTCTTTA AGAGAACCTCCCCCTTTCTGACCCACCAAGTGTTTAACTCCTATCACTCCGAAAC AAATATTGTGAGATACATGAAAAAGCTCGAGAACAAGGACATCAGCCTCGTGCAT TCCATGATTCCTCTCGGCAGCTGCACAATGAAACTGAACTCCAGCTCCGAGCTGG CCCCCATCACATGGAAGGAATTTGCCAACATCCACCCTTTCGTGCCTCTGGACCA AGCCCAAGGCTATCAGCAACTCTTTAGAGAGCTGGAGAAGGACCTCTGTGAGCTC ACCGGCTATGACCAAGTGTGTTTCCAGCCTAATTCCGGAGCCCAAGGCGAATACG CCGGACTCGCCACCATCAGAGCCTATCTGAACCAGAAGGGAGAGGGCCATAGGAC AGTGTGTCTGATCCCCAAGAGCGCCCATGGCACAAACCCCGCCAGCGCCCACATG GCCGGAATGAAGATTCAGCCCGTGGAGGTGGACAAGTATGGCAATATCGACGCCG TCCATCTCAAAGCCATGGTCGACAAGCACAAGGAGAACCTCGCCGCTATCATGAT CACCTATCCCTCCACCAACGGCGTCTTCGAGGAGAACATTAGCGATGTCTGCGAT CTGATCCACCAGCATGGAGGCCAAGTGTATCTGGACGGAGCCAACATGAACGCTC AAGTGGGCATTTGCAGACCCGGCGATTTCGGAAGCGATGTGAGCCATCTGAATCT GCATAAGACCTTTTGCATTCCCCACGGAGGAGGCGGACCCGGCATGGGCCCTATC GGAGTCAAAAAGCATCTGGCTCCTTTTCTGCCCAACCATCCCGTGATCTCCCTCA AGAGAAACGAGGACGCTTGTCCCGTGGGCACAGTGAGCGCCGCCCCTTGGGGATC CAGCAGCATTCTCCCTATCAGCTGGGCTTATATCAAAATGATGGGAGGAAAGGGA CTCAAGCAAGCCACAGAGACCGCTATTCTGAACGCTAATTACATGGCCAAAAGGC TCGAGACCCACTATAGAATTCTGTTTAGAGGCGCTAGAGGATATGTCGGCCATGA GTTTATTCTGGACACAAGACCCTTCAAAAAGAGCGCCAACATCGAAGCCGTGGAT GTCGCCAAGAGACTGCAAGACTACGGATTCCATGCCCCCACAATGAGCTGGCCCG TGGCTGGCACACTGATGGTGGAACCTACCGAATCCGAGGACAAGGCTGAGCTGGA CAGATTCTGCGACGCCATGATTTCCATTAGACAAGAGATCGCTGACATTGAGGAA GGAAGGATCGACCCCAGAGTCAACCCCCTCAAGATGTCCCCCCACTCTCTGACAT GCGTCACCAGCAGCCACTGGGATAGACCCTATAGCAGAGAAGTCGCTGCTTTCCC TCTGCCCTTTGTCAAACCCGAGAACAAGTTCTGGCCCACCATCGCTAGGATCGAC GACATCTACGGAGACCAGCACCTCGTGTGTACATGCCCTCCTATGGAGGTGTACG AGAGCCCTTTCAGCGAGCAGAAGAGAGCCTCCAGCTGA SEQ ID No:5 – hGLDC protein sequence MQSCARAWGLRLGRGVGGGRRLAGGSGPCWAPRSRDSSSGGGDSAAAGASRLLERLLPRH DDFARRHIGPGDKDQREMLQTLGLASIDELIEKTVPANIRLKRPLKMEDPVCENEILATL HAISSKNQIWRSYIGMGYYNCSVPQTILRNLLENSGWITQYTPYQPEVSQGRLESLLNYQ TMVCDITGLDMANASLLDEGTAAAEALQLCYRHNKRRKFLVDPRCHPQTIAVVQTRAKYT GVLTELKLPCEMDFSGKDVSGVLFQYPDTEGKVEDFTELVERAHQSGSLACCATDLLALC ILRPPGEFGVDIALGSSQRFGVPLGYGGPHAAFFAVRESLVRMMPGRMVGVTRDATGKEV YRLALQTREQHIRRDKATSNICTAQALLANMAAMFAIYHGSHGLEHIARRVHNATLILSE GLKRAGHQLQHDLFFDTLKIQCGCSVKEVLGRAAQRQINFRLFEDGTLGISLDETVNEKD LDDLLWIFGCESSAELVAESMGEECRGIPGSVFKRTSPFLTHQVFNSYHSETNIVRYMKK LENKDISLVHSMIPLGSCTMKLNSSSELAPITWKEFANIHPFVPLDQAQGYQQLFRELEK DLCELTGYDQVCFQPNSGAQGEYAGLATIRAYLNQKGEGHRTVCLIPKSAHGTNPASAHM AGMKIQPVEVDKYGNIDAVHLKAMVDKHKENLAAIMITYPSTNGVFEENISDVCDLIHQH GGQVYLDGANMNAQVGICRPGDFGSDVSHLNLHKTFCIPHGGGGPGMGPIGVKKHLAPFL PNHPVISLKRNEDACPVGTVSAAPWGSSSILPISWAYIKMMGGKGLKQATETAILNANYM AKRLETHYRILFRGARGYVGHEFILDTRPFKKSANIEAVDVAKRLQDYGFHAPTMSWPVA GTLMVEPTESEDKAELDRFCDAMISIRQEIADIEEGRIDPRVNPLKMSPHSLTCVTSSHW DRPYSREVAAFPLPFVKPENKFWPTIARIDDIYGDQHLVCTCPPMEVYESPFSEQKRASS

[0004] Ĵ5

Claims

Claims 1. A nucleotide sequence comprising a codon-optimised nucleotide sequence encoding glycine decarboxylase (GLDC).

2. The nucleotide sequence according to claim 1, wherein the codon-optimised nucleotide sequence comprises a sequence at least 90% identical to SEQ ID No:2 (Optz 1), SEQ ID No:3 (Optz 2), or SEQ ID No:4 (Optz 3), optionally wherein the codon-optimised nucleotide sequence is SEQ ID No:3 (Optz 2).

3. An expression vector cassette encoding GLDC, wherein the expression vector cassette comprises: a. a 5’ inverted terminal repeat (ITR); b. a promoter; c. a sequence selected from a wild-type GLDC sequence, a human GLDC sequence, a consensus GLDC sequence at least 70% identical to SEQ ID No:1, or a codon-optimised GLDC sequence; d. a termination sequence; and e. a 3’ inverted terminal repeat (ITR), wherein the distance between the end of the promoter and the start codon of the GLDC sequence is 201 bp or less.

4. The expression vector cassette according to claim 3, wherein the expression vector cassette comprises the nucleotide sequence of claim 1 or 2.

5. The expression vector cassette according to claim 3 or 4, wherein: i) the promoter is a promoter comprising from about 0.6 kb to about 1.0 kb, optionally wherein the promoter is a truncated chicken beta-actin hybrid promoter (CBh)or a truncated human glial fibrillary acidic protein (GFAP) promoter, and / or ii) the termination sequence is bovine growth hormone polyadenylation sequence (BGH-PolyA), and / or iii) the 5’ ITR and 3’ ITR are derived from adeno-associated virus 2 (AAV2).

6. The expression vector cassette of claims 3 to 5, wherein the distance between the end of the promoter and the start codon of the GLDC sequence is 15 bp or less.

7. The expression vector cassette of claims 3 or 4, wherein the GLDC sequence comprises a truncated 5’ untranslated region (UTR) of 60 bp or less.

8. The expression vector cassette of claim 7, wherein the 5’ UTR of the GLDC sequence is absent.

9. An expression vector capable of expressing GLDC comprising the expression vector cassette according to any of claims 3 to 8.

10. The expression vector according to claim 9, wherein the expression vector cassette is packaged into an adeno-associated virus (AAV) capsid, optionally wherein the AAV targets the central nervous system (CNS), further optionally wherein the AAV targets the brain, still further optionally wherein the AAV is an AAV9.

11. A pharmaceutical composition comprising the expression vector according to claim 9 or 10 and a pharmaceutically acceptable carrier.

12. The expression vector according to claim 9 or 10, or the pharmaceutical composition according to claim 11, for use in therapy.

13. An expression vector comprising a sequence encoding glycine decarboxylase (GLDC) for use in a method of treating a disease caused by impaired function of GLDC.

14. The expression vector for the use of claim 13, wherein the disease is non-ketotic hyperglycinemia (NKH), optionally wherein the NKH is: a) severe NKH, or b) attenuated NKH.

15. The expression vector for the use of claim 13 or 14, wherein the expression vector comprises the expression vector cassette according to any of claims 3 to 8.

16. The expression vector for the use of claim 13 to 15, wherein the expression vector cassette is packaged into an adeno-associated virus (AAV) capsid, optionally wherein the AAV targets the central nervous system (CNS), further optionally wherein the AAV targets the brain, further optionally wherein the AAV targets the liver, still further optionally wherein the AAV is an AAV9.

17. The expression vector for the use according to any of claims 13 to 16, wherein the method comprises administering the expression vector to: a) the blood stream, optionally intra-venously, and / or b) the central nervous system, optionally intra-cisternally, intra-thecally, or intra- cerebroventricularly.

18. A method of treating a disease caused by impaired function of GLDC, wherein the method comprises administering an expression vector comprising a sequence encoding glycine decarboxylase (GLDC) to a subject in need thereof.

19. The method according to claim 18, the wherein the disease caused by impaired function of GLDC is non-ketotic hyperglycinemia (NKH), optionally wherein the NKH is: a. severe NKH, or b. attenuated NKH.

20. The method according to claim 18 or 19, wherein the expression vector comprises the expression vector cassette according to any of claims 3 to 8.

21. The method according to claim 18 to 20, wherein the expression vector cassette is packaged into an adeno-associated virus (AAV) capsid, optionally wherein the AAV targets the central nervous system (CNS), further optionally wherein the AAV targets the brain, further optionally wherein the AAV targets the liver, still further optionally wherein the AAV is an AAV9.

22. The method of treating NKH according to any of claims 18 to 21, wherein the method comprises administering the expression vector to:a) the blood stream, optionally intra-venously, and / or b) the central nervous system, optionally intra-cisternally, intra-thecally, or intra- cerebroventricularly.

23. A method for obtaining a codon-optimised GLDC sequence for gene therapy the method comprising: a. applying a codon optimisation algorithm to a wild-type GLDC sequence, to obtain a codon-optimised GLDC sequence, b. optionally, translating, in silico, the codon-optimised GLDC sequence to confirm identity of the encoded protein with wild-type GLDC protein, and c. administering an expression vector comprising an expression vector cassette comprising the codon-optimised GLDC sequence from step a) or b) to a Gldc- deficient disease model, and d. determining rescue of the disease phenotype in the Gldc-deficient disease model, wherein rescue of the disease phenotype indicates that the sequence is a codon- optimised GLDC sequence for gene therapy.

24. A codon-optimised GLDC sequence for gene therapy obtainable by the method according to claim 23.

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