Compositions and methods for treating creatine deficiencies

Abstract

This disclosure relates to nucleic acid constructs for expression of guanidinoacetate methyltransferase (GAMT) and / or expression of L-arginine:glycine amidinotransferase (AGAT), and viral vectors comprising said constructs useful for the treatment of creatine deficiency syndromes(CDDs). Also provided are methods and uses of the vectors disclosed herein for the treatment of CCDs.

Description

COMPOSITIONS AND METHODS FOR TREATING CREATINE DEFICIENCIESRELATED APPLICATION

[0001] This disclosure claims benefit of United States Provisional Patent Application serial no. 63 / 425,144 filed November 14, 2022, incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “29669. P69417PC00_SequenceListing. xml” (34,291 bytes), created on November 14, 2023, is herein incorporated by reference.FIELD

[0003] The present disclosure relates to the development of a nucleic acid construct comprising a transgene encoding the DNA sequence for a guanidinoacetate methyltransferase (GAMT) protein and / or a L-arginine:glycine amidinotransferase (AGAT) protein operably linked to a promoter, and a transcription termination site; vectors comprising said nucleic acid constructs; pharmaceutical compositions comprising said vector; and vectors or compositions for use in the treatment of creatine deficiency disorders (CDDs).INTRODUCTION

[0004] Creatine synthesis includes two enzymatic steps and a transporter known as SLC6A8 (FIG 1 ). L-arginine:glycine amidinotransferase (AGAT), encoded by the gene glycine amidinotransferase (GATM), converts arginine and glycine to ornithine and guanidinoacetate (GAA). GAA is methylated by GAMT in the presence of S-adenosyl-l- methionine to form creatine. Synthesis in the periphery takes place mostly in the kidneys and liver, where creatine will then enter the systemic circulation or uptake in several other tissues by a transporter called Solute Carrer Family 6 Member 8 (SLC6A8). The majority of cells that uptake creatine are located in the skeletal muscle, however the brain contains allcomponents required for creatine synthesis. AGAT and GAMT can be expressed in all brain cells, however they are not often co-expressed in the same cells7. Therefore, GAA must exit neuronal cells and be imported through SLC6A8 or astrocytic y-aminobutyric acid transporter to cells that express GAMT8-10SLC6A8 is not expressed in astrocytes resulting in low permeability of creatine through the blood brain barrier (BBB) since creatine may only enter through microcapillary endothelial cells7. This suggests that the brain relies heavily on endogenous synthesis for its creatine supply.

[0005] Cerebral creatine deficiency syndromes (CCDS) are inborn errors of creatine metabolism. The CCDS include AGAT deficiency, GAMT deficiency and SLC6A8 deficiency. AGAT and GAMT deficiency are autosomal recessive disorders which impair creatine biosynthesis, while SLC6A8 is an X-linked defect impairing creatine transport.41

[0006] Cerebral creatine deficiency 2 (CCD2, OMIM 612736) is an autosomal recessive genetic disorder resulting in loss of function of the guanidinoacetate methyltransferase (GAMT) enzyme, leading to low levels of creatine in the brain and accumulation of GAA in the brain and bodily fluids11-13. Symptoms include epilepsy, intellectual disability, developmental delay, and disordered extrapyramidal movement. Cerebral creatine deficiency 2 (CCDS2) is the most severe form of CCDS due to accumulation of guanidinoacetate (GAA) which has been found to be neurotoxic and increase epileptic symptoms11 14'15.

[0007] Cerebral creatine deficiency 3 (CCD3, OMIM 602360) is an autosomal recessive genetic disorder resulting in loss of function of the AGAT enzyme, leading to reduced production of GAA, which is the rate limiting step in creatine biosynthesis. Symptoms include intellectual disability, developmental delay and myopathy.42

[0008] AGAT and GAMT are essential enzymes in the biosynthesis of creatine, an important molecule in energy recycling. Creatine’s primary function is energy metabolism through recycling of ATP via the phosphocreatine-creatine system1-4The reversible reaction of creatine to its phosphorylated form functions to provide ADP for oxidative phosphorylation or glycolysis and ATP for energetic processes of the cell. Creatine has been widely known for its role in muscle development, however it has recently been elucidated that it plays an important role in brain function. Within the last 20 years, three inborn errorsof creatine metabolism have been described, all of which have severe neurological manifestations suggesting a critical role for creatine synthesis and transport within the central nervous system (CNS). Creatine is a known competitive antagonist of GABAA receptors and has been recently implicated in neuromodulation5’6. It has also been suggested that creatine has anti-apoptotic and antioxidant properties which may participate in neuroprotection1’2’4.

[0009] AGAT is present primarily in the kidneys, though extra-renal expression is variable among species.43In the kidneys, it is located in the mitochondria of cells found in the proximal tubules of nephrons.43’44The AGAT enzyme acts at a rate limiting and tightly regulated step during the production of creatine and homoarginine.43’46Previous studies have shown that AGAT mutations affecting AGAT levels have been associated with chronic kidney disease (CKD).50Similarly, there are indications of secondary creatine deficiency in in chronic liver disease (affecting the brain, especially thalamus), chronic fatigues syndrome and other neurodegenerative disorders.47’48’49’50 51

[0010] Viral vectors are often used to deliver nucleic acids due to their ability to efficiently enter cells and exploit the host machinery to produce their gene products. To avoid viral replication and toxic effects, recombinant technology is used to delete the viral genes with the exception of the components required for viral assembly16’17. Adeno- associated virus (AAV) vectors are known in the art and represent one of the most promising in vivo gene delivery tools due to their ability to transduce several cell types with little to no cytotoxicity and produce long-term expression in several tissue types. The rate-limiting step of AAV-mediated gene expression is second strand synthesis which can be overcome by the generation of self-complementary AAV (scAAV)19-21. scAAV9 has a reduced packaging capacity meaning only expression cassettes less than 2.5 kb can be delivered by scAAV19-21. The small size of the GAMT gene (711 bps) allows it to be packaged into a scAAV9 vector for delivery to the CNS.SUMMARY

[0011] The present disclosure relates to nucleic acid constructs encoding a functional GAMT protein and / or a functional AGAT protein operably linked to a promoter andtranscription termination site, as well as viral vectors comprising said nucleic acid constructs for therapeutic replacement of dysfunctional GAMT and / or AGAT protein. This disclosure also relates to the production of AAV vectors including nucleic acids encoding the GAMT protein and / or the AGAT protein.

[0012] The present inventors have tested the first CNS-directed, AAV9-based gene therapy for the treatment of GAMT-D also known as CCDS2 and the treatment of AGAT-D also known as CCDS3. It was found that delivery of GAMT plasmid DNA to cellular models of GAMT-D effectively restored protein and mRNA expression of GAMT while increasing intracellular creatine content and decreasing GAA accumulation. Further, delivery of AGAT plasmid DNA to cellular models of AGAT-D effectively restored intracellular creatine content, but also showed increase in GAA. It was also found that delivery of AGAT and GAMT together, bringing the whole machinery for creatine production at one place, effectively restored intracellular creatine content without toxic accumulation of GAA in models of GAMT-D and AGAT-D. In murine models of GAMT-D, treatment with scAAV9.hGAMT delivered intrathecally, resulted in increased creatine content as well as significant decreases in GAA accumulation throughout the entire body including the CNS. Overall, it was found that scAAV9.hGAMT represents a promising gene therapy approach to treating GAMT-D.

[0013] Accordingly, the present disclosure provides a nucleic acid construct comprising a promoter, a transcription termination site, and a nucleotide sequence encoding a guanidinoacetate N -methyltransferase (GAMT) protein and / or a L-arginine:glycine amidinotransferase (AGAT) protein. It further provides a viral vector comprising said nucleic acid construct and methods of treating and preventing CDDs in a subject.

[0014] One aspect of the disclosure includes a nucleic acid construct comprising a nucleotide sequence encoding a GAMT protein and / or a L-arginine:glycine amidinotransferase (AGAT) protein operably linked to a promoter and a transcription termination site.

[0015] In an embodiment, the nucleotide sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the nucleotidesequence encoded by any one of SEQ ID NO: 1 , and SEQ ID NO: 2, and which retains GAMT activity.

[0016] In an embodiment, the nucleotide sequence encoding a GAMT protein has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2 or functional variants thereof.

[0017] In another embodiment, the GAMT protein has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0018] In an embodiment the nucleic acid construct comprises a nucleotide sequence encoding a L-arginine:glycine amidinotransferase (AGAT) protein operably linked to a promoter and a transcription termination site.

[0019] In an embodiment, the nucleotide sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoded by SEQ ID NO: 15, and which retains AGAT activity.

[0020] In an embodiment, the nucleotide sequence encoding the AGAT protein is SEQ ID NO: 15, or a functional variant thereof.

[0021] In another embodiment, the AGAT protein has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 16.

[0022] In yet another embodiment, the nucleic acid construct comprises a nucleotide sequence encoding a L-arginine:glycine amidinotransferase (AGAT) protein and a guanidinoacetate N-methyltransferase (GAMT) operably linked to a promoter and a transcription termination site.

[0023] In an embodiment, the promoter is a constitutive promoter or a tissue- or cellspecific promoter.

[0024] In another embodiment, the promoter is selected from the group comprising: chicken beta actin (CBA), chicken [3-actin hybrid (CBh), CMV early enhancer (CAG), Elongation Factor 1a (eF-1 a), simian virus 40 early promoter (SV40), humanphosphoglycerate kinase 1 (PGK), cytomegalovirus immediate-early promoter (CMV), human (3-actin (hACTB), synapsin, myelin basic protein and JeT synthetic promoter.

[0025] In an embodiment, the nucleic acid construct comprises a sequence as set forth in SEQ ID NO: 5, or a functional variant thereof.

[0026] Another aspect of the disclosure is a viral vector comprising the nucleic acid construct disclosed herein.

[0027] In an embodiment, the viral vector is an Adeno-Associated Virus (AAV) vector or a derivative thereof.

[0028] In a further embodiment, the AAV vector is selected from the group consisting of: AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9, or a derivative thereof.

[0029] In another embodiment, the viral vector is a chimeric, shuffled or capsid modified derivative of AAV.

[0030] Another aspect of the disclosure is a pharmaceutical composition comprising the nucleic acid construct or the viral vector disclosed herein, and a pharmaceutically acceptable carrier or diluent, for example, but not limited to, liposomes and lipid / polymer nanoparticles.

[0031] In an embodiment, the pharmaceutical composition is formulated for intraparenchymal, intravenous or intrathecal or any other systemic method of administration.

[0032] Yet another aspect of the disclosure is a method of treating or preventing a creatine deficiency syndrome (CCD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the viral vector or the pharmaceutical composition disclosed herein.

[0033] In an embodiment, the CCD is selected from Cerebral Creatine Deficiency Syndrome 2 (CCDS2), Cerebral Creatine Deficiency Syndrome 3 (CCDS2) and secondary creatine deficiencies.

[0034] In an embodiment, the viral vector or pharmaceutical composition is formulated for systemic, peritoneal, intraparenchymal, intravenous and / or intrathecal injection.

[0035] Another aspect of the disclosure is a use of the viral vector or the pharmaceutical composition disclosed herein, for the treatment of a Creatine Deficiency Syndrome (CDD) in a subject in need thereof.

[0036] Another aspect of the disclosure is a use of the viral vector or the pharmaceutical composition disclosed herein, for the manufacture of a medicament for the treatment of a Creatine Deficiency Syndrome (CDD).

[0037] A further aspect of the disclosure is the viral vector or the pharmaceutical composition disclosed herein, for use in the treatment of a Creatine Deficiency Syndrome (CDD) in a subject in need thereof.

[0038] In an embodiment, the viral vector or pharmaceutical composition is formulated for systemic, peritoneal, intraparenchymal, intravenous and / or intrathecal injection.

[0039] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended reference section.DRAWINGS

[0040] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0041] FIG 1 is the creatine synthesis pathway. Arginine and glycine are enzymatically converted to ornithine and guanidinoacetate by L-arginine:glycine amidinotransferase. GAA is transformed into creatine by the addition of a methyl group by guanidinoacetate methyltransferase through the use of S-adenosyl-l-methionine converted to S-adenosyl-l-homocysteine. Creatine can then be imported into other cells through the creatine transporter, SLC6A8. Creatine will enter the phosphocreatine system to participate in energy recycling via creatine kinase (CK). Creatine and phosphocreatine are non- enzymatically converted to creatinine for excretion in the urine. Creatine exerts a negative feedback loop on GATM at the transcriptional level to suppress AGAT production.

[0042] FIG 2 is a timeline for an in vivo study. Mice started in the study at 5 weeks of age where baseline serum collection was taken, and the immunosuppression regimen of rapamycin and prednisone began. A loading dose of 300 ug of Rapamycin and 24ug of prednisone was used on day 1 (R3P0.24) and then a dose of 100 ug of rapamycin and 24 ug of prednisone (R1 P0.24) was given daily until endpoint. At 6 weeks of age the mice were injected via an intrathecal lumbar puncture with 10 uL of either scAAV9.hGAMT at a dose of 2.5e11 vg / mouse or a vehicle solution. Serum was collected again at 8 and 10 weeks of age until study endpoint at 13 weeks of age in which all gross organs, CNS tissues and cardiac serum were collected for analyses.

[0043] FIG 3 is a set up for intrathecal injections. The mouse is anesthetized in an induction chamber prior to being placed on the nose cone. A 15-mL conical tube is placed under the hips of the mouse with its nose secured in the nose cone.

[0044] FIG 4 is an immunosuppression regimen for the long-term dosage study. Mice received a loading dose of 300 ug of Rapamycin and 24ug of prednisone was used on day 1 and then a dose of 100 ug of rapamycin and 24 ug of prednisone was given daily until endpoint at 13 weeks or until 16 weeks of age for long-term study animals. At 16 weeks of age prednisone was tapered by 4ug / week for 5 weeks. The immunosuppression regimen ended at 21 weeks of age.

[0045] FIG 5 is the sectioning of CNS tissues during euthanizations of mice. The tissues of the CNS were divided into three brain sections and two spinal cord sections to analyze the distribution of vector. Sections were further divided for all analyses. Tissues forqPCR analysis of the brain were collected and mixed together and split into two sections one for RNA isolation and one for DNA isolation to perform gene expression. Other sections were collected for biochemical analysis by LC-MS / MS, western blotting and histology analysis.

[0046] FIG 6 is a western blot analysis of GAMT KO cells post-transfection with phGAMT at doses ranging from 1.25 ug to 5.00 ug. Following transfections with phGAMT protein lysates were collected and ran on a western blot using approximately 30 ug of protein per well. In lane 1 a low dose of 1 .25 ug of plasmid DNA was used. In lane 2 a dose of 2.00 ug was used. In lane 3 a dose of 3.75 ug was used and in lane 4 a dose of 5.00 ug was used. Lanes 5-6 represent negative controls of KO cells transfected with a plasmid only expressing GFP. Lanes 7-8 consist of a positive control of untransfected wild type (WT) HAP1 cells. Expression was restored to that greater than WT cells and showed saturation at approximately 3.75 ug.

[0047] FIG 7 is a western blot analysis of GAMT KO cells post-transfection with phGAMT (human) and pmGamt (murine) at a low and high dose. FIG 7A Following transfections with phGAMT protein lysates were collected and ran on a western blot. In lane 1 a low dose of 1.25 ug of plasmid DNA was used. In lane 2 a high dose of 3.75 ug was used. Lanes 3-4 represent negative controls of KO cells transfected with a plasmid only expressing GFP. Lanes 5-6 consist of a positive control of untransfected WT HAP1 cells. Expression was restored to that greater than WT cells. FIG 7B Following transfections with pmGamt protein lysates were collected and ran on a western blot. In lane 1 a low dose of 1.25 ug of plasmid DNA was used. In lane 2 a high dose of 3.75 ug was used. Lanes 3-4 represent negative controls of KO cells transfected with a plasmid only expressing GFP. Lanes 5-6 consist of a positive control of untransfected WT HAP1 cells. Lane 6 consists of protein lysate collect from mouse liver tissue. The antibody used seems to have a higher affinity for human GAMT protein. It is evident that expression of the GAMT protein was restored following transfections.

[0048] FIG 8 is the gene expression of phGAMT quantified by RT-qPCR via normalization to 18SRNA expression. Knockout cells were transfected with either a high dose (3.75 ug) or low dose (1 .25 ug) of phGAMT or a GFP plasmid control (n=4). RNA wasisolated and then reverse transcribed into cDNA for use in qPCR. FIG 8A The ACq value was determined by subtracting the Cq value of the housekeeping gene 18SRNA from the Cq of the target DNA. A smaller ACq value therefore indicates that the expression is closer to that of the housekeeping gene. There is a significant decrease in the ACq values between the treated and the untreated samples with an approximate 2-fold difference observed between each group (** p < 0.0082, **** p < 0.0001 ). FIG 8B The low dose was used as a normalization point for all samples considering the untreated sample does not have a detectable level of plasmid. An approximate 2 fold difference is observed in gene expression between each treatment group (** p < 0.0082, **** p < 0.0001 ).

[0049] FIG 9 is preliminary data showing intracellular creatine and GAA posttransfection of knockout GAMT cells as determined by LC-MS / MS. FIG 9A Treatment of GAMT knockout cells with both the murine GAMT (mGAMT) and human GAMT (hGAMT) plasmid significantly reduced GAA accumulation comparable to that observed in WT HAP1 cells (n=4, p < 0.0001 ). FIG 9B Treatment of GAMT knockout cells with both the mGamt and hGAMT plasmid significantly increased intracellular creatine content (n=4, * p < 0.01 , *** p < 0.0004).

[0050] FIG 10 is the intracellular creatine and GAA content following low dose and high dose transfection of KO GAMT cells with pmGAMT and phGAMT. FIG 10A treatment of GAMT knockout cells with both the mGAMT and hGAMT plasmid as a low (1 .25 ug) and high (3.75 ug) dose significantly reduced GAA accumulation comparable to that observed in WT HAP1 cells (n=4, p < 0.0001 ). FIG 10B There was no significant difference observed in intracellular creatine concentration between the treated and untreated KO cells, with the exception of the significant increase observed following the high does (3.75 ug) of the hGAMT plasmid. (n=4, * p < 0.0192, *** p < 0.0002, **** p< 0.0001 ).

[0051] FIG 11 is a Western blot analysis of mid-section of the brain and liver tissues following treatment with scAAV9. hGAMT. Protein was loaded at approximately 40 ug of protein per well. Wells 1 -3 are vehicle treated heterozygote mice, wells 4-6 are vehicle treated knockout mice and wells 7-9 are knockout mice treated with 2.5e11 vg of scAAV9. hGAMT. FIG 11A Western blot carried out with protein isolated from the midsection of the brain. FIG 11 B Western blot carried out with protein isolated from the livers.

[0052] FIG 12 is the intracellular creatine content in tissue samples of murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls determined by LC-MS / MS. Mice were treated with either 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC-MS / MS for guanidino compounds. An observable trend of increased creatine content in treated mice was observed compared to vehicle treated knockouts. This increase was found to only be significant in the liver, caudal section of the brain and lumbar section of the spinal cord. (n=4-7, *p<0.05, **p<0.007, ***p<0.0007, ****p<0.0001 )

[0053] FIG 13 the intracellular GAA content in tissue samples of murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls determined by LC-MS / MS. Mice were treated with either 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC- MS / MS for guanidino compounds. A significant decrease in GAA accumulation was observed in treated mice compared to vehicle treated knockouts in all tissues except the rostral section of the brain. (n=4-7, *p<0.05, **p<0.007, ***p<0.0007, ****p<0.0001 ).

[0054] FIG 14 the creatine and GAA content in serum collected from murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls determined by LC-MS / MS. Mice were treated with either 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Serum was collected at four time points at 5 weeks of age, 8 weeks, 10 weeks and at endpoint at 13 weeks. FIG 14A An increasing amount of creatine in the serum is observed over time from the 5-week baseline prior to treatment up to endpoint at 13 weeks of age. FIG 14B A significant increase in creatine was observed in the serum of treated knockouts compared to vehicle treated knockout controls at endpoint (n=5-7, *P<0.02, **p<0.0014, ****p<0.0001 ) FIG 14C A decreasing amount of GAA in the serum is observed over time from the 5-week baseline prior to treatment up to endpoint at 13 weeks of age. FIG 14D A significant decrease in GAA was observed in the serum of treated knockouts compared to vehicle treated knockout controls at endpoint (n=5-7, ***p<0.0008, ****p<0.0001 ).

[0055] FIG 15 is the intracellular creatine content of HAP1 cells following high dose and low dose transfection of phGA77W, phGA / WT and p GATM-GAMT. FIG 15A Treatmentof GATM KO cells with high (H,3.75 g) and low (L,1 .25 g) of phGATM significantly increased the intracellular creatine levels compared to both wildtype and KO levels (**** p < 0.0001 ) and treatment of GATM KO cells with high dose of phGATM-GAMT restored intracellular creatine levels to that of untreated WT HAP1 cells. FIG 15B Treatment of GAMT KO cells with phGAMT significantly increased the intracellular creatine levels compared to untreated KO levels in the case of the high dose only, no significant increase was found after treatment with the low dose (*p =0.0109). In contrast, treatment of GAMT KO cells with high and low doses of ph GA TM-GAMT restored intracellular creatine levels to that of greater than untreated WT HAP1 cells (**** p < 0.0001 ). FIG 15C Treatment of GATM KO cells with a high dose of phGATM alone significantly (**** p < 0.0001 ) raises GAA levels to that of GAMT KO cells (D) leading to potentially toxic effects. Treatment of GATM KO cells with a high and low doses of phGATM-GAMT does not result in any GAA accumulation. FIG 15D Treatment of GAMT KO cells with both high and low doses of phGAMT and phGATM-GAMT significantly reduces GAA accumulation down to WT levels (**** p < 0.0001 ).

[0056] FIG 16 is a western blot analysis of GAMT KO cells post-transfection with bicistronic plasmid at a low, medium, medium-high and high dose. Following transfections with the bicistronic plasmid protein lysates were collected and ran on a western blot. In lane 1 a high dose of 5 ug was used. In lane 2 a medium-high dose of 3.5 ug was used. In lane 3 a medium dose of 2.5 ug was used. In lane 4 a low dose of 1 ug was used. Lanes 5 and 6 represent HAP1 and GFP-transfected GAMT KO cells representing WT and KO treatment groups, respectively. AGAT (FIG 16A) and GAMT (FIG 16B) are shown relative to [3-actin (42 kDa) and lamin B1 (66 kDa) were used as internal loading controls.

[0057] FIG 17 is the creatine and GAA content in serum collected from murine models of GAMT-D treated with scAAV9.hGAMT with three different doses in a different set of experiments to prove dose-responsiveness and vehicle controls over the 13-weeks determined by LC-MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1.25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Serum was collected at four time points at 5 weeks of age, 8 weeks, 12 weeks and at endpoint at 13 weeks. FIG 17A A significant increase in creatine was observed in the serum of treated knockouts compared to vehicle treated knockout controls at endpoint. FIG 17B Asignificant decrease in GAA was observed in the serum of treated knockouts compared to vehicle treated knockout controls at endpoint. FIG 17C An increasing amount of creatine in the serum is observed over time from the 5-week baseline prior to treatment up to endpoint at 13 weeks of age. FIG 17D Amount of GAA in the serum observed over time from the 5- week baseline prior to treatment up to endpoint at 13 weeks of age. (***p<0.001 ; **** p < 0.0001 ).

[0058] FIG 18 is the creatine and GAA content in serum collected from murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls over the 26-weeks (long-term follow-up) determined by LC-MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1.25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Serum was collected at 5 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks and at endpoint at 26 weeks. FIG 18A A significant increase in creatine was observed in the serum of treated knockouts compared to vehicle treated knockout controls at endpoint FIG 18B An increasing amount of creatine in the serum is observed over time from the 5- week baseline prior to treatment up to endpoint at 26 weeks of age. FIG 18C GAA levels in the serum of treated knockouts compared to vehicle treated knockout controls at endpoint. FIG 18D Amount of GAA in the serum observed over time from the 5-week baseline prior to treatment up to endpoint at 26 weeks of age. (*p<0.05; *** p<0.001 ; **** p < 0.0001 ).

[0059] FIG 19 is the intracellular creatine content in lumbar and cervical spinal cord tissue samples of murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls determined by LC-MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1.25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC- MS / MS for guanidino compounds. A significant increase in intracellular creatine was observed at all doses. (*p<0.05;**p<0.01 ;***p<0.001 ;**** p < 0.0001 ).

[0060] FIG 20 is the intracellular GAA content in lumbar and cervical spinal cord tissue samples of murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls determined by LC-MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1.25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC-MS / MS for guanidino compounds. A significant decrease in intracellular GAA was observed at all doses. (**p<0.01 ; ***p<0.001 ;**** p < 0.0001 ).

[0061] FIG 21 is the intracellular creatine content in peripheral tissue samples of murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls determined by LC -MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1 ,25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC-MS / MS for guanidino compounds. A significant increase in creatine was detected at all doses in the muscle. A significant increase in creatine was detected at the medium and high doses in the liver. A significant increase in creatine was detected at the high dose in the kidney. (**p<0.01 ;***p<0.001 ;**** p < 0.0001 ).

[0062] FIG 22 is the intracellular GAA content in peripheral tissue samples of murine models of GAMT-D treated with scAAV9.hGA / WT and vehicle controls determined by LC- MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1.25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC-MS / MS for guanidino compounds. A significant decreased in GAA was detected at the high dose in the muscle. (**p<0.01 ;***p<0.001 ;**** p < 0.0001 ).

[0063] FIG 23 is the intracellular creatine content in brain tissue samples of murine models of GAMT-D treated with scAAV9.hGA / WT and vehicle controls determined by LC- MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1.25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC-MS / MS for guanidino compounds. A significant increase in creatine was detected at the medium and high doses in all samples. (*p<0.05; **p<0.01 ;***p<0.001 ;**** p < 0.0001 ).

[0064] FIG 24 is the intracellular GAA content in brain tissue samples of murine models of GAMT-D treated with scAAV9.hGAMT and vehicle controls determined by LC- MS / MS. Mice were treated with either 6.25e10 vg / mouse, 1.25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. Tissues were collected at endpoint and proteins were isolated for analysis by LC-MS / MS for guanidino compounds.A significant decrease in GAA was detected at all doses in all samples. (*p<0.05; **p<0.01 ;**** p < 0.0001 ).

[0065] FIG 25 is the muscular strength at 8-weeks and 12-weeks of age in murine models of GAMT-D treated with scAAV9.hGAMT and vehicle control. Mice were treated with with either 6.25e10 vg / mouse, 1 ,25e11 vg / mouse, 2.5e11 vg / mouse or a vehicle (veh) solution by intrathecal lumbar puncture. A significant improvement in muscular strength was observed at 12-weeks of age in mice treated with the high dose. (*p<0.05; **p<0.01 ).DESCRIPTION OF VARIOUS EMBODIMENTS

[0066] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0067] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.I. General Definitions

[0068] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0069] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0070] All numerical values herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0071] The terms “about”, “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0072] As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0073] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified.

[0074] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e. , the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of” or, when used in the claims, “consisting of’ will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0075] As used herein, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to inclusive or be open-ended, i.e., to mean including but not limited to, and do not exclude additional, unrecited elements or process steps. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively

[0076] The term “consisting” and its derivatives as used herein are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0077] The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and / or steps.

[0078] As used herein, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list ofelements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.

[0079] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0080] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular any feature described herein may be combined with any other feature or features described herein.II. Nucleic Acid Constructs and Viral Vectors

[0081] Described herein is a nucleic acid construct comprising a nucleotide sequence encoding a GAMT protein, operably linked to a promoter and a transcription termination site. As shown herein, delivery of such nucleic acid constructs via adeno-associated viral vectors results in increased creatine content and decreased GAA accumulation in mouse models of GAMT-D. Further shown herein is that delivery of such nucleic acid constructs via adeno- associated viral vectors results in increased creatine content in models of AGAT-D. Accordingly, one aspect of the disclosure includes a nucleic acid construct comprising a nucleotide sequence encoding a GAMT protein and / or a AGAT protein operably linked to a promoter and a transcription termination site.

[0082] The term “nucleic acid construct of the disclosure” as used herein refers to a nucleic acid molecule comprising an expression cassette, the expression cassette comprising a DNA sequence encoding a GAMT protein and / or a AGAT protein operably linked to a promoter and a transcription termination site. In an embodiment, the DNA sequence encoding a GAMT protein comprises a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, or a functional variant of any thereof. In an embodiment, the nucleicacid encodes a GAMT protein having an amino acid sequence as set forth in SEQ ID NO: 4 or a functional variant of any thereof. In an embodiment, the DNA sequence encoding a AGAT protein comprises a nucleotide sequence set forth in SEQ ID NO: 15, or a functional variant of any thereof. In an embodiment, the nucleic acid encodes a AGAT protein having an amino acid sequence as set forth in SEQ ID NO: 16 or a functional variant thereof. In a further embodiment, the DNA sequence encoding both a AGAT and a GAMT protein comprises a nucleotide sequence set forth in SEQ ID NO: 11 , or a functional variant thereof. In an embodiment, the nucleic acid encodes a AGAT and a GAMT protein having an amino acid sequence as set forth in SEQ ID NO: 12, or a functional variant thereof.

[0083] The term, “L-arginine:glycine amidinotransferase” or “AGAT” as used herein refers to a protein that participates in the two-step production of creatine by enzymatically converting arginine and glycine to ornithine and guanidinoacetate. The AGAT protein is encoded by the glycine amidinotransferase (GATM) gene. Defects in this gene result in arginine:glycine amidinotransferase deficiency, an inborn error of creatine synthesis, which causes cognitive disability, language impairment and behavioural disorders.

[0084] The term “AGAT activity” as used herein refers to a protein that is known to catalyze the transfer of the amidino group of L-arginine to glycine to generate guanidinoacetate.

[0085] The term “guanidinoacetate N-methyltransferase” or “GAMT” as used herein refers to a protein that participates in the two-step production of creatine by donating a methyl group from S-adenosylmethionine to guanidinoacetate (GAA). Defects in this gene have been implicated in neurologic syndromes and muscular hypotonia due to creatine deficiency and accumulation of guanidinoacetate in the brain of affected individuals. Two transcript variants encoding different isoforms have been described for this gene. Pseudogenes of this gene are found on chromosomes 2 and 13.

[0086] The term “GAMT activity” as used herein refers to a protein that is known to convert GAA into creatine by donating a methyl group from adenosylmethionine to GAA.

[0087] The term “nucleic acid molecule” and its derivatives, as used herein, are intended to include unmodified DNA or RNA or modified DNA or RNA. For example, thenucleic acid molecules or polynucleotides of the disclosure can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecules can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule” embraces chemically, enzymatically, or metabolically modified forms. The term “polynucleotide” shall have a corresponding meaning.

[0088] The term “operably linked” as used herein refers to a relationship between two components that allows them to function in an intended manner. For example, where a coding sequence is operably linked to a promoter, the promoter actuates expression of the coding sequence.

[0089] The term “promoter” or “promoter sequence” generally refers to a regulatory DNA sequence capable of being bound by an RNA polymerase to initiate transcription of a downstream (i.e. 3’) sequence to generate an RNA. Suitable promoters may be derived from any organism and may be bound or recognized by any RNA polymerase. Suitable promoters for the expression cassette will be known to the skilled person. In some embodiments, the promoter is an inducible promoter. Examples of inducible promoters include, without limitation, a tetracycline response element (TRE) (e.g. Tet-ON or Tet-OFF systems), ponA- inducible expression systems (Agilent Technologies), or cumate-inducible promoters such as CuO (System Biosciences). In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters include human Ubiquitin C (UBC), human Elongation Factor 1 a (EF1A), human phosphoglycerate kinase 1 (PGK), simian virus 40 early promoter (SV40) (GeneBank accession number J02400.1 ), cytomegalovirus immediate-early promoter (CMV), chicken b-Actin promoter coupled with CMV earlyenhancer (CAG) and EF1 -HTLV. In some embodiments, the promoter is a tissue- or cellspecific promoter. In an embodiment, the promoter is a synthetic promoter such as JeT.

[0090] The term “transcription termination site” as used herein refers generally to a polyadenylation signal (pA) that terminates transcription of messenger RNA (mRNA). As used herein, the phrase “polyadenylation signal” refers to sequences from various genes that can be added to mammalian vectors to ensure proper mRNA processing and stability. For example, a 100-200 nucleotide polyadenylate tail can be added to the 3’ end of a coding sequence to protect mRNA from degradatory action of phosphatases and nucleases. Suitable pAs may be derived from any organism and are known to the skilled person. Examples of pA signals include, without limitation, rabbit beta-globin pA (GeneBank accession number K03256), SV40 late polyA, and hGH polyA and strong bovine growth hormone pA (BGHpA).

[0091] The term “functional variant” as used herein includes modifications of the nucleic acid or polypeptide sequences disclosed herein that perform substantially the same function as the nucleic acid molecules or polypeptides disclosed herein in substantially the same way. For example, the functional variant may comprise sequences having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to the sequences disclosed herein. In the case of nucleic acids, functional variants include nucleotide sequences that hybridize to the nucleic acid sequences set out above, under at least moderately stringent hybridization conditions, optionally stringent hybridization conditions, or the functional variant nucleic acid sequences may comprise degenerate codon substitutions or codon-optimized nucleic acid sequences. In the case of polypeptides, the functional variant may also comprise conservatively substituted amino acid sequences of the sequences disclosed herein.

[0092] In an embodiment, the functional variant sequences comprise sequences having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to the sequences disclosed herein.

[0093] The term “sequence identity” as used herein refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determ ine the percent identity of two am ino acid sequences or of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = [number of identical overlapping positions] I [total number of positions] X 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI- BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 -17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can bedetermined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0094] In one embodiment, the functional variants include nucleotide sequences that hybridize to the nucleic acid sequences described herein, under at least moderately stringent hybridization conditions, optionally stringent hybridization conditions.

[0095] With reference to nucleic acids, the terms “anneal” and “hybridize” as used herein refer to the ability of a nucleic acid to non-covalently interact with another nucleic acid through base-pairing. The terms “complementary” or “complementary nucleic acid” refer to a nucleic acid or a portion of a nucleic acid that is able to anneal with a nucleic acid of a given sequence. In some cases, this is referred to as the “reverse complement” of a given sequence.

[0096] By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. The term “at least moderately stringent hybridization conditions” encompasses stringent hybridization conditions and moderately stringent hybridization conditions. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5°C - 16.6 (Log10 [Na+]) + 0.41 (%(G+C) - 600 / I), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1 % mismatch may be assumed to result in about a 1 °C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In some embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt’s solution / 1 .0% SDS at Tm - 5°C based on the above equation,followed by a wash of 0.2x SSC / 0.1 % SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001 .

[0097] In another embodiment, the functional variant nucleic acid sequences comprise degenerate codon substitutions or codon-optimized nucleic acid sequences. The term “degenerate codon substitution” as used herein refers to variant nucleic acid sequences in which the second and / or third base of a codon is substituted with a different base that does not result in a change in the amino acid sequence encoded therein. The term “codon- optimized” as used herein refers to a variant nucleic acid molecule comprising one or more degenerate codon substitutions that reflect the codon usage bias of a particular organism. Accordingly, in an embodiment, the nucleic acid construct disclosed herein comprises a codon-optimized or degenerate nucleotide sequence of SEQ ID NO:1 and / or SEQ ID NO:2. In an embodiment, the nucleic acid construct disclosed herein comprises a codon-optimized or degenerate nucleotide sequence of SEQ ID NO: 15. In another embodiment, the nucleic acid construct disclosed herein comprises a codon-optimized or degenerate nucleotide sequence of SEQ ID NO: 11. In an embodiment, the nucleic acid construct disclosed herein comprises a codon-optimized or degenerate nucleotide sequence of SEQ ID NO: 5.

[0098] In an embodiment, the nucleic acid construct disclosed herein comprises a nucleic acid molecule that encodes a polypeptide having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the protein encoded by any one of SEQ ID NO:1 or SEQ ID NO:2, and which retains GAMT activity.

[0099] In an embodiment, the nucleic acid construct disclosed herein comprises a nucleic acid molecule that encodes a polypeptide having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the protein encoded by SEQ ID NO: 16 and which retains AGAT activity.

[0100] In an embodiment, the nucleic acid construct disclosed herein comprises a nucleic acid molecule that encodes a polypeptide having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the protein encoded by SEQ ID NO: 12 and which retains GAMT and AGAT activity.

[0101] The term “monocistronic” as used herein, refers to a nucleic acid molecule which contains a single gene or protein.

[0102] In an embodiment, the nucleic acid molecule is a monocistronic nucleic acid molecule.

[0103] In another embodiment, the monocistronic nucleic acid molecule encodes a GAMT protein.

[0104] In a further embodiment, the monocistronic nucleic acid molecule encodes a AGAT protein.

[0105] The term “bicistronic” as used herein, refers to a nucleic acid molecule which contains two genes or proteins.

[0106] In an embodiment, the nucleic acid molecule is a bicistronic nucleic acid molecule.

[0107] In a further embodiment, the bicistronic nucleic acid molecule encodes a GAMT protein and a AGAT protein.

[0108] In an embodiment, the bicistronic nucleic acid molecule contains two promoters.

[0109] In an embodiment, the bicistronic nucleic acid molecule contains one promoter.

[0110] In an embodiment, two or more polypeptides encoded by a polynucleotide described herein can be separated by an intervening sequence encoding a linker polypeptide. In some embodiments the linker is a cleavage-susceptible linker. In some embodiments, the polypeptides of interest are expressed as fusion proteins linked by acleavage-susceptible linker polypeptide. In some embodiments, cleavage susceptible linker polypeptide(s).

[0111] In an embodiment, the bicistronic nucleic acid molecule contains a linker.

[0112] In a further embodiment, the linker is P2A.

[0113] In an embodiment, the linker has a nucleotide sequence encoded by SEQ ID NO: 13. In an embodiment, the linker has an amino acid sequence encoded by SEQ ID NO: 14.

[0114] In an embodiment, the nucleic acid construct further comprises, an enhancer, a post-transcription regulatory sequence, one or more sequences that facilitate incorporation of the nucleic acid into a viral particle and / or integration into the host genome, or any combination thereof, operably linked to the nucleic acid encoding the GAMT protein. In another embodiment, the nucleic acid construct further comprises, an enhancer, a posttranscription regulatory sequence, one or more sequences that facilitate incorporation of the nucleic acid into a viral particle and / or integration into the host genome, or any combination thereof, operably linked to the nucleic acid encoding the AGAT protein. In yet another embodiment, the nucleic acid construct further comprises, an enhancer, a post-transcription regulatory sequence, one or more sequences that facilitate incorporation of the nucleic acid into a viral particle and / or integration into the host genome, or any combination thereof, operably linked to the nucleic acid encoding the AGAT and the GAMT protein. Post transcriptional regulatory sequences include, for example, without limitation, sequences of nucleotides that when placed in an AAV transfer plasmid results in the increased or decreased expression of the transgene. As used herein, the phrase “enhancer” refers to a sequence of nucleotides that argument the activity of a promoter in an orientation, position, and distance-dependent manner. Enhancers play a significant role in the regulation of tissue-specific gene expression in high eukaryotes but have been repurposed for use in recombinant DNA technologies to impact the transcriptional activity of an associated promoter. Typically, a frans-acting gene regulatory protein binds the enhancer in order to affect transcriptional activity of the associated promoter.

[0115] Also described herein is a viral construct comprising a nucleic acid construct described herein. Viral constructs are made of DNA or RNA and they contain some of the genetic material of the viruses they are derived from (such as lentivirus, retrovirus, AAV and adenoviruses). For example, viral constructs may include sequences that facilitate incorporation of the nucleic acid into a viral particle and / or integration into the host genome. In some embodiments, the viral construct may include inverted terminal repeats (ITRs) for example from an AAV such as AAV9, or other viral sequences. Viral constructs have been modified to carry and to deliver a gene of interest that will produce a protein or an RNA of interest and can be used for example for the treatment of diseases by gene therapy. Suitable viral constructs are known in the art and depend on the type of viral vectors and viruses being used.Viral Vectors

[0116] One aspect of the disclosure is a viral vector comprising a nucleic acid construct disclosed herein. Replication incompetent viral vectors are particularly useful in gene therapy applications as they allow for efficient transduction of delivery of a transgene to target tissues. Differences between viral vectors include availability of tropisms, packaging capacity, safety, and transduction efficiencies in different tissues.

[0117] The term “viral vector” as used herein is intended to include viral particles or virus-like particles capable of transduction of a target cell. Common viral vectors include, but are not limited to, HIV-derived lentiviral vectors, retroviral vectors, adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. Other viral vectors may be derived from rhabdovirus (such as vesicular stomatitis virus (VSV)), or herpes virus (such CMV and HSV-1 ). Typical components of the viral vector are the structural components of the viral particle, such as the proteins making the capsid and the envelope of the vector. Other components are the enzymes involved in the replication of the vector RNA or DNA. Such enzymes can be also involved in the synthesis, maturation or transport of the virus RNA. These enzymes can also be involved in the processing and maturation of viral components, as well as in the integration of the genome of the virus into the cell chromosomes. Enzymes that are components of the viral vectors can also be involved in the reverse transcription of the virus genomic RNA into DNA. Other components of the vector can be protein or peptidethat regulate the replication, transcription, transport or translation of the genes or gene products of the viral vector. Such factors can also activate or decrease the expression of cellular genes and they can modulate the defense mechanism of the cells against viruses.

[0118] Several viral vectors are well known in the art including adenovirus, adenoviral associated virus (AAV), lentivirus, retrovirus, and herpes simplex virus 1. Accordingly, in an embodiment, the viral vector is a lentivirus, adenovirus, adenoviral associated virus (AAV), retrovirus, or herpes simplex virus 1 vector. Optionally, the viral vector is an AAV vector.

[0119] AAV is particularly useful for gene therapy applications as it elicits a limited immune response, exhibits a wide variety of serotypes, and has a stable expression profile. Accordingly, in an embodiment, the viral vector is an AAV vector or a derivative thereof. Optionally, the AAV vector is selected from the group consisting of AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, or a derivative thereof.

[0120] The term “AAV derivative” as used herein describes a recombinant AAV produced by combining AAV helper plasmids from different AAV serotypes to produce AAV capsids with the combined advantages of more than one serotype. An AAV derivative may further refer to a shuffled AAV derivative which used herein describes an AAV virus containing mutations produced through directed evolutionary or related recombination techniques including but not limited to DNA shuffling. The term “AAV derivative” may also refer to a capsid-modified AAV that can be produced by pseudo typing the sequences of two or more AAV serotypes producing an AAV vector combining characteristics of the two or more serotypes. In an embodiment, the AAV vector is a chimeric, shuffled or capsid modified derivative of AAV.and Kits

[0121] In one embodiment there is provided a pharmaceutical composition comprising a nucleic acid construct or viral vector described herein, and a pharmaceutically acceptable carrier or diluent. The composition may be formulated for use or prepared for administration to a subject using pharmaceutically acceptable formulations known in the art including liposomes or lipid nanoparticles. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington’sPharmaceutical Sciences (2003 - 20thedition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. The term “pharmaceutically acceptable” means compatible with the treatment of animals, in particular, humans.

[0122] On this basis, the pharmaceutical compositions could include an active compound or substance, such as a nucleic acid construct or viral vector described herein, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isosmotic with the physiological fluids. The methods of combining viral vectors the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the delivery or transport of the active compound to specified sites within the body, organ, tissue, or cell.

[0123] As used herein, the term “diluent” refers to a pharmaceutically acceptable carrier which does not inhibit a physiological activity or property of an active compound, such as lipoxin or a lipoxin analogue, to be administered and does not irritate the subject and does not abrogate the biological activity and properties of the administered compound. Diluents include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservative salts, preservatives, binders, excipients, disintegration agents, lubricants, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0124] The pharmaceutical compositions, formulations, dosages, etc. described herein can be administered for example, by parenteral, intravenous, intrathecal, subcutaneous, or intramuscular administration in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

[0125] The nucleic acid constructs or viral vectors described herein are suitably formulated in a conventional manner into compositions using one or more carriers or diluents. Accordingly, the present description also includes a composition comprising one or more nucleic acid constructs or viral vectors described herein and a carrier or diluent. The nucleic acid constructs or viral vectors described herein are suitably formulated into pharmaceuticalcompositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Accordingly, the present description further includes a pharmaceutical composition comprising the nucleic acid constructs or viral vectors described herein, and a pharmaceutically acceptable carrier. In some embodiments the pharmaceutical compositions are used in the treatment of any of the diseases, disorders or conditions described herein. In an embodiment, the disease, disorder, or condition is a creatine deficiency disorder. In an embodiment, the disease, disorder, or condition is a primary creatine deficiency. In another embodiment, the disease, disorder, or condition is secondary creatine deficiency. In another embodiment the disease, disorder, or condition is a cerebral creatine deficiency disorder. In a further embodiment, the disease, disorder, or condition is GAMT-D. In yet another embodiment, the disease, disorder or condition is AGAT-D.

[0126] In an embodiment, the secondary creatine deficiencies include chronic kidney disease, chronic liver disease, cancer, muscle disorders, myalgic encephalomyelitis / chronic fatigue syndrome, and neurodegenerative conditions.

[0127] Muscle disorders include, but are not limited to, for example fibromyalgia.

[0128] Neurodegenerative disorders include, but are not limited to, Parkinson’s disease and Alzheimer’s disease.

[0129] In some embodiments, the nucleic acid constructs or viral vectors described herein are formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion. Formulations for injection are, for example, presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. In some embodiments, the compositions take such forms as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulating agents such as suspending, stabilizing and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Alternatively, nucleic acid constructs or viral vectors described herein are suitably in a sterile powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0130] Also provided are kits comprising a nucleic acid construct, viral vector, or pharmaceutical composition as described herein, along with suitable container or packaging and / or instructions for the use thereof, such as for the treatment of CCDS2 in a subject.III. Methods and Uses

[0131] As described in the Examples, scAAV9.hGAMT can effectively restore the protein and mRNA expression of GAMT while increasing intracellular creatine content and reducing GAA accumulation in treated cellular and murine models of guanidinoacetate methyltransferase deficiency (GAMT-D). Therefore, GAMT gene therapy is useful in treating GAMT-D or Cerebral Creatine Deficiency Syndrome 2 (CCDS2). Accordingly, one aspect of the disclosure is a method of treating or preventing CCDS2 in a subject in need thereof, comprising administering a therapeutically effective amount of a nucleic acid construct or viral vector disclosed herein to the subject. Another aspect of the disclosure includes use of a nucleic acid construct or viral vector described herein to treat CCDS2. An aspect also includes use of a nucleic acid construct or viral vector described herein in the manufacture of a medicament for treating CCDS2. An aspect also includes a nucleic acid construct or viral vector described herein for use in treating CCDS2.

[0132] In an embodiment, the use or method of treating or preventing CCDS2 comprises administering the therapeutically effective amount of a vector disclosed herein by intravenous, intrathecal, and / or systemic injection.

[0133] The term “Cerebral Creatine Deficiency Syndrome 2” (CCDS2) also known as guanidinoacetate methyltransferase deficiency (GAMT-D) describes a hereditary condition inherited in an autosomal recessive manner. CCDS2 is caused by mutations in the GAMT gene and is characterized by developmental delay / regression, severe disturbances in expressive and cognitive speech, intractable seizures and movement disturbances, and severe depletion of creatine and / or phosphocreatine in the brain, and accumulation of guanidinoacetic acid (GAA) in the brain and bodily fluids.

[0134] As described in the Examples, hAGAT can effectively increase intracellular creatine content in treated cellular models of AGAT-D. Therefore, AGAT gene therapy can be useful in treating AGAT-D or Cerebral Creatine Deficiency Syndrome 3 (CCDS3).Accordingly, one aspect of the disclosure is a method of treating or preventing CCDS3 in a subject in need thereof, comprising administering a therapeutically effective amount of a nucleic acid construct or viral vector disclosed herein to the subject. Another aspect of the disclosure includes use of a nucleic acid construct or viral vector described herein to treat CCDS3. An aspect also includes use of a nucleic acid construct or viral vector described herein in the manufacture of a medicament for treating CCDS3. An aspect also includes a nucleic acid construct or viral vector described herein for use in treating CCDS3.

[0135] In an embodiment, the use or method of treating or preventing CCDS3 comprises administering the therapeutically effective amount of a vector disclosed herein by intravenous, intrathecal and / or systemic injection.

[0136] The term “Cerebral Creatine Deficiency Syndrome 3” (CCDS3) also known as L-arginine:glycine amidinotransferase deficiency (AGAT-D) describes a hereditary condition inherited in an autosomal recessive manner. CCDS3 is caused by mutations in the GATM gene and is characterized by developmental delay / regression, myopathy, delayed speech and motor skills, autistic behaviours, general failure to thrive, and low levels of guanidinoacetic acid (GAA) in the brain and bodily fluids.

[0137] As further described in the Examples, hGATM-P2A-GAMT can effectively restore the protein and mRNA expression of AGAT and / or GAMT while increasing intracellular creatine content without toxic accumulation of GAA in treated cellular models of AGAT-D and GAMT-D. Therefore, combined AGAT and GAMT gene therapy is useful in treating AGAT-D or Cerebral Creatine Deficiency Syndrome 3 (CCDS3) and / or GAMT-D or Cerebral Creatine Deficiency Syndrome 2 (CCDS2). Accordingly, one aspect of the disclosure is a method of treating or preventing CCDS2 and / or CCDS3 in a subject in need thereof, comprising administering a therapeutically effective amount of a nucleic acid construct or viral vector disclosed herein to the subject. Another aspect of the disclosure includes use of a nucleic acid construct or viral vector described herein to treat CCDS2 and / or CCDS3. An aspect also includes use of a nucleic acid construct or viral vector described herein in the manufacture of a medicament for treating CCDS2 and / or CCDS3. An aspect also includes a nucleic acid construct or viral vector described herein for use in treating CCDS2 and / or CCDS3.

[0138] In an embodiment, the use or method of treating or preventing CCDS2 and / or CCDS3 comprises administering the therapeutically effective amount of a vector disclosed herein by intravenous, intrathecal, and / or systemic injection.

[0139] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease (e.g. maintaining a patient in remission), preventing disease or preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment methods comprise administering to a subject a therapeutically effective amount of a nucleic acid construct or viral vector described herein and optionally consists of a single administration, or alternatively comprises a series of administrations.

[0140] “Palliating” a disease, disorder or condition means that the extent and / or undesirable clinical manifestations of a disease, disorder or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to not treating the disorder.

[0141] The term “prevention” or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a disease, disorder or condition or manifesting a symptom associated with a disease, disorder or condition.

[0142] The term “subject” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Optionally, the term “subject” includes mammals that have been diagnosed with a CDDs, optionally the CCD is CCDS2 or CCDS3. In an embodiment, the subject is a mammal. In another embodiment, the subject is human. In one embodiment, the term “subject” refers to a human having, or suspected of having a CDDs, optionally the CCD is CCDS2 or CCDS3.

[0143] The term “subject in need thereof” refers to a subject that could benefit from the method(s) or treatment(s) described herein, and optionally refers to a subject with, or optionally a subject with increased risk of CCD, such as a subject with a strong genetic predisposition.

[0144] The term “administered” or “administering” as used herein means administration of a therapeutically effective amount of a compound or composition of the disclosure to a cell either in cell culture or in a subject. The nucleic acid constructs or viral vectors described herein may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. For example, the nucleic acid constructs or viral vectors described herein may be administered by parenteral administration and the pharmaceutical compositions formulated accordingly. In some embodiments, administration is by means of a pump for periodic or continuous delivery.

[0145] The nucleic acid constructs or viral vectors described herein may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. For example, the nucleic acid constructs or viral vectors described herein may be administered by parenteral administration and the pharmaceutical compositions formulated accordingly. In some embodiments, administration is by means of a pump for periodic or continuous delivery. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington’s Pharmaceutical Sciences (2000 - 20thedition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0146] Parenteral administration includes systemic delivery routes other than the gastrointestinal (Gl) tract, and includes, for example intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, intrapulmonary (for example, by use of an aerosol), and intrathecal modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0147] As used herein, the phrase “intrathecal” means existing or taking place within, or administered into the fluid-filled space between the thin layers of tissue that cover the brain and spinal cord.

[0148] As used herein, the phrase “intravenous” means existing or taking place within, or administered into, a vein or veins. Intravenous delivery of gene therapy vectors allows for widespread delivery and transduction to organs and tissues in a subject.

[0149] As used herein, the phrase “systemic” means existing or taking place within, or administered into, the circulatory system. Systemic delivery of gene therapy vectors allows for widespread delivery and transduction to organs and tissues in a subject.

[0150] As used herein, the phrase “effective amount” or “therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, in the context of treating CCDS2, an effective amount is an amount that for example increases creatine and / or phosphocreatine levels in the brain, and / or decreases the accumulation of GAA in the brain and bodily fluids compared to the response obtained without administration of the compound. For a further example, in the context of treating CCDS3, an effective amount is an amount that for example increases creatine and / or phosphocreatine levels in the brain, and / or increases the production of GAA compared to the response obtained without administration of the compound. Effective amounts may vary according to factors such as the disease state, age, sex, and weight of the animal. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0151] Suitable administration schedules may include, without limitation, at least once a week, from about one time per two weeks, three weeks or one month, about one time per week to about once daily. The length of the treatment period may depend on a variety of factors, such as the severity of the disease, disorder or condition, the age of the subject, the concentration and / or the activity of the nucleic acid constructs or viral vectors described herein. It will also be appreciated that the effective dosage of the nucleic acid constructs or viral vectors described herein used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration isrequired. For example, the nucleic acid construct or viral vector described herein are administered to the subject in an amount and for duration sufficient to treat the subject.Examples

[0152] The following non-limiting examples are illustrative of the present disclosure:Example 1: Materials and MethodsCell Culture

[0153] The GAMT knockout HAP1 cell line was provided and produced by the Schulze lab. The GAMT gene was targeted by introducing a frameshift mutation in the first exon eliminating GAMT expression. Cells were maintained in Iscove Modified Dulbecco Medium (IMDM) substituted with 1 % penicillin-streptomycin and 10% dialyzed fetal bovine serum (FBS). Dialyzed FBS was created by putting FBS in a dialysis membrane and placing it in a 0.15M NaCI solution that is changed every 4 hours and left over night on a stir plate at 4°C. This process helps to eliminate small molecules such as creatine. Cells were grown in the media for at least two weeks prior to transfection. Cells were maintained in an incubator at 37°C and 5% CO2.

[0154] The GATM knockout HAP1 cell line was previously created by the Walia lab using guide RNA (gRNA) (unpublished). The GATM gene was targeted by a gene deletion indicated by GATM forward and reverse primers. Single cells with the interrupted AGAT expression were then selected and created a colony from the single clone. Cells were maintained in Iscove Modified Dulbecco Medium (IMDM) supplemented with 1 % penicillinstreptomycin and 10% dialyzed fetal bovine serum (FBS). The dialyzed FBS solution was created by aliquoting FBS into a dialysis membrane, then placed in a 0.15M NaCI solution on a stir plate at 4°C. The NaCI solution was changed every hour 6 times before being left overnight. This process was required to eliminate small molecules such as creatine. Cells were maintained in an incubator at 37°C with 5% CO2 and grown in the specialized media for a minimum of two weeks prior to transfections.Plasmids and VectorsGAMT vectors

[0155] The GAMT vectors included the GAMT cDNA from either human or mouse sequences. The nucleic acid construct was under the control of the synthetic JeT promoter and followed by a poly-adenylation signal. The entire sequence was flanked by inverted terminal repeats (ITR) to allow for packaging into the self-complementary AAV9 vector, with the 3’ ITR have a mutated terminal resolution site to allow for self-complementary folding32. The designed vectors were synthesized with the codon optimized transgenes sequences for optimal expression (Biobasics, Markham, ON). The plasmid was transformed by addition to competent E. coli bacterial cells and isolated by miniprep in accordance with the kit protocol (QIAprep Spin Miniprep Kit, Qiagen) for use in transfection. DNA concentration and purity were determined using a Nanodrop 2000 (Thermo Fisher). Plasmids were sent to Aldevron, LCC for larger plasmid prep and to UNC Vector Core for viral vector preparation (UNC Vector Core, UNC School of Medicine). To test the GAMT vectors, GAMT knockout cells were transfected with both human GAMT and murine GAMT vectors. Protein isolate from transfected cells were also analyzed for GAMT protein expression and LC MS / MS was conducted to analyze GAA and creatine levels in cell lysates.GATM vector and bicistronic GATM-GAMT vector

[0156] The monocistronic GATM vector contained the GATM cDNA, which was coupled with the GAMT cDNA in the bicistronic GATM-GAMT vector, all from codon optimized human sequences (hGATM, hGATM-GAMT). These constructs both used a synthetic JeT promotor, followed by a poly-adenylation signal, and the entire sequence was flanked by inverted terminal repeats (ITRs). The 3’ ITR was mutated in the terminal resolution site to allow for self-complementary folding32. Additionally, the GATM-GAMT vector contained a peptide 2A sequence (P2A), derived from a picornavirus, between the GATM and GAMT sequences. The P2A linker is only 19 amino acids long and causes ribosomal skipping during translation, which results in a missing peptide bond effectively separating the AGAT and GAMT proteins. The designed vectors were synthesized with the codon optimized transgene sequences for optimal expression (Biobasics, Markham, ON). The plasmid was transformed with competent E. coli bacterial cells and isolated by maxiprep in accordance with the kit protocol (QIAprep Spin Maxiprep Kit, Qiagen) for use intransfection. DNA concentration and purity were determined via a Nanodrop 2000 (Thermo Fisher). Plasmids were sent to UNC Vector Core for viral vector preparation (UNC Vector Core, UNC School of Medicine).TransfectionsGAMT-D in vitro study

[0157] For transfection, cells were be seeded in a 6-well plate at approximately 500,000 cells per well determined by manual cell counting using Trypan Blue (Gibco, 15250061 ). The GAMT plasmids were introduced to the cells by Lipofectamine 3000 (Invitrogen, L3000001 ) mediated transfection according to the manufacturer’s protocol at a low dose of 1 .25 pg and a high dose of 3.75 pg. Cells were treated with plasmids containing the human or murine GAMT construct or a GFP plasmid to provide a visual of transfection efficiency (Table 1 ). GAMT knockouts treated with GFP plasmids served as negative controls while wild-type HAP1 cells served as positive controls. After 48 hours, cells were subject to protein or RNA isolation for their respective analyses.Table 1. In vitro study design for transfection of cellular models of GAMT-D.Cohort Cell Type Treatment Dose (pg of DNA) Rationale1 WT HAP 1 None 0 Positive control2 GAMT - / - Vehicle - GFP 1.25 Negative Control3 GAMT - / - Vehicle - GFP 3.75 Negative Control4 GAMT - / - hGAMT 1.25 Low dose, human5 GAMT - / - hGAMT 3.75 High dose, human6 GAMT - / - mGcimt 1.25 Low dose, murine7 GAMT - / - mGamt 3.75 High dose, murineAGAT-D in vitro study

[0158] Cells were manually counted using Trypan Blue (Gibco, 15250061 ) and seeded into 6-well plates at 400 000 cells per well. The plasmids (hGATM, hGATM-GAMT, and GFP) underwent Lipofectamine 3000 (Invitrogen, L3000001 ) mediated transfection in the desired HAP1 cells (Table 2) according to the manufacturer’s protocol at a low dose of 1.25pg and high dose of 3.75pg. GA TM knockout (KO) cells transfected with GFP plasmids served as a visual for transfection efficiency and as a negative control and wild-type (WT)HAP1 cells as positive controls. The cells were harvested for either protein or RNA isolation 48-hours post transfection for the respective analyses.Table 2 In vitro study design for transfection of cellular models of AGAT-DCohort Cell type Treatment Dose (pg of RationalDNA) 1 Vehicle- GFP 3.75pg Positive control 1 Vehicle- GFP 1.25 pg Positive control Vehicle- GFP 1.25 pg Negative control Vehicle- GFP 3.75 pg Negative control hGATM 1.25pg Low dose, AGAT only hGATM 3.75pg High dose, AGAT only hGATM-GAMT 1.25pg Low dose, AGAT+GAMT hGATM-GAMT 3.75pg High dose, AGAT+GAMT 1 hGATM 1.25 pg Overexpression1 hGATM-GAMT 1.25 pg OverexpressionAnimal Models and In Vivo Study Design

[0159] The GAMT-D mice were obtained from the Schulze Laboratory and maintained on a 12-hr light cycle from 7 a.m. to 7 p.m. The GAMT-D mouse model has a neomycin cassette introduced into the first exon of the murine Gamt gene creating a frameshift mutation that results in an early stop codon. Gamt knockout mice tend to be smaller in size and mimic the biochemical phenotype of the human disease with increased intracellular GAA and decreased creatine content. Little to no behavioral differences have been observed so far, however one group has observed differences in a grip strength test and the Barnes maze. All experimental protocols and procedures will be performed in accordance with the Canadian Council on Animal Care and will be approved by the Queen’s University Animal Care Committee.

[0160] For the in vivo study, animals were injected intrathecally with 2.5 x 1011vector genomes (vg) per mouse of the scAAV9.hGAMT vector or a vehicle control (Table 3). Vectors were made up in 1X PBS with 5% sorbitol for the appropriate dosage while vehicleinjections were performed with 1X PBS with 5% sorbitol only (Nationwide). The study timeline is shown in FIG 2.Table 3. Study design for in vivo study.Cohort Genotype Mice (n) Dose Injection Type Endpoint(vg / mouse) (weeks)1 Gamt+ / - 5 0 IT 132 Gamt '- 5 0 IT 133 Gamt '- 5 2.5E11 IT 13For dosage studies mice were injected intrathecally via lumbar puncture with varying doses of the scAAV9.hGAMT vector ranging from 6.25e10 vg / mouse to 2.5e11 vg / mouse and were sacrificed at a short-term endpoint of 13 weeks or a long term endpoint of 26 weeks of age. Serum was collected monthly beginning at 5 weeks of age and mice also underwent a muscular strength test every month. Tissue and serum samples were processed by LC- MS / MS to quantify creatine and GAA content.Genotyping

[0161] Genotyping was performed by standard PCR on DNA extracted from ear notches using the Extracta DNA prep for PCR kit (Quantabio). Primers were as follows: Forward primer 1 : 5’-GGTCTCCCAACGCTCCATCACT-3’, reverse primer: 5’- CCTCAGGCTCCCACCCACTTG-3’ and forward primer 2: 5’-AGGCCTACCCGCTTCCATTG-3’. Samples were prepared using the Advanced 2X HS-Red Taq PCR kit (Wisent).Intrathecal (IT) Injections

[0162] The protocol for performing intrathecal injections was adapted from previous literature35 36At 6 weeks of age, mice were anesthetized by inhalation of isoflurane at 4- 5% for induction and maintained at 1 -3% for injection. The mice were placed with their head in a nose cone while the hips are elevated by a 15-mL conical tube (FIG 3). The back of the mouse was shaved and sterilized and the location between L5 and L6 was palpated to mark the injection spot. A Hamilton syringe with a 30-gauge needle was loaded with the vector at a volume of 10 pL for a dose of 2.5x1011vector genomes (vg) per mouse. The syringe wasinserted at a 90° angle from the spine with the needle bevel facing up. Once it contacts the spinal column the syringe was bent to a 50-30° angle such that it can enter the subarachnoid space between L5 and L6. Proper penetration was indicated by a tail flick - a movement of the tail in the shape of an S. When in the appropriate spot, the vector was injected slowly, and the needle kept in place from a few seconds before turning the bevel down and being removed. The mice were recovered in a clean cage and monitored for several minutes after injections to ensure there is no paralysis caused by the injection.Immunosuppression and Blood Collection

[0163] To reduce the immune response against the vector, mice received an immunosuppression regimen from 5 weeks of age to 13 weeks of age. Rapamycin (LC- Laboratories, R-5000) at 1 mg / kg / day and Prednisone (Sigma Aldrich, P6254) at 0.24 mg / kg / day were given daily by oral gavage. For long term studies the immunosuppression regimen was as described in FIG 4.

[0164] Blood collections were performed bi-weekly by collecting approximately 100pL of blood from the saphenous vein. The serum was collected by separation from the blood sample through centrifugation. For long term studies blood and urine collections occurred monthly.Euthanizations

[0165] Tissue samples were collected at the designated short-term endpoint of 13 weeks. The mice were euthanized by CO2 asphyxiation after which a cardiac puncture was performed. Mice were then perfused with 10 mL of 1X PBS. Visceral organs collected include the liver, heart, gonad, lung, spleen, kidney, and muscle and were sectioned for their respective analyses. The brain was sectioned into rostral, mid-section and caudal regions while the spinal cord was sectioned into lumbar and cervical sections (FIG 5). Organ designated for RNA isolation were stored in RNALater Solution (Invitrogen) at -80°C. All organs were frozen at -20°C until processing for their respective analyses.Quantitative Polymerase Chain Reaction (qPCR)

[0166] Copy numbers of the scAAV9hGAMT vectors and mouse genomic DNA was determined by qPCR. Total DNA was extracted from tissues using the gSYNC DNA Extraction Kit (GS100, Geneaid) and total DNA concentration were determined using a Nanodrop 2000. Quantitative PCR (qPCR) reactions were caried out using the SYBR Green Master mix (BioRad) on a Biorad CFX96 Touch Real-Time PCR Detection System. Plasmid DNA (hGAMT) was used as the standard for quantitation of the vector. Mouse genomic DNA was purified as a standard for mouse genomic DNA quantitation. Primers for the scAA V9. hGAMT vector and mouse LaminB2 primers for quantitation of mouse genomic DNA are found in Table 4. To determine the relative copy number variation of the viral vectors in each organ the values were reported as double-stranded copies of the GAMT vector per two double-stranded copies of the mouse LaminB2 locus. This gave an approximate measure of the vector genome copies per diploid mouse genome found in the assessed tissues.

[0167] Gene expression was analyzed by isolation of RNA for use in RT-qPCR. RNA was isolated from cells using GeneJET RNA Purification kit (Thermo Scientific, K0732) and from animal tissues using the EZ-10 DNAaway RNA Miniprep Kit. cDNA was synthesized using the Quantitect Reverse Transcription kit (Qiagen). The forward and reverse primers were used as described above. RNA18S was used as the reference gene for the in vitro analysis to quantify gene expression. Ubiquitin- C (UBC) and 0-Actin (ACTB) were used for in vivo analysis. All MIQE guidelines were followed to ensure best qPCR practices37.Table 4. Primers used for qPCR analysis.Target Forward Primer Reverse Primer hGAMT GGATTCTTGGGAAGCCCGTG GGGGTGATCATCTGGGGGAALaminB2 (Lrnnb2) GGACCCAAGGACTACCTCAAGGG AGGGCACCTCCATCTCGGAAAC18S rRNA GTGGAGCGATTTGTCTGGTT AACGCCACTTGTCCCTCTAA(RNA 18S)B-Actin (Actb) TGGCTCCTAGCACCATGAAG AAACGCAGCTCAGTAACAGTCUbiquitin-C (Ubc) GCCCAGTGTTACCACCAAGA CCCATCACACCCAAGAACALiquid Chromatography Tandem Mass Spectrometry (LC-MS / MS)

[0168] Preparation of samples was performed as previously described38 39For cell lysates, cells were washed with 1X PBS and then scraped into tubes and centrifuged. Cellpellets were resuspended in water and then sonicated. The samples were then centrifuged again, and the supernatant taken for analysis. Protein concentration was determined by taking an OD280 reading using a Nanodrop 2000 (Thermo ScientificTM, ND-2000) where 1 Abs = 1 mg / mL of protein. For animal samples the brain tissue, liver and serum were analyzed for guanidino compound levels. Extraction of protein from the tissue will be similar to the above-described methods and serum will be analyzed directly. The LC-MS / MS system located in the Schulze Lab consists of an API4000 QTRAP mass spectrometer (Applied Biosystems Inc.) and an Agilent 1200 series HPLC (Agilent Technologies). The conditions for creatine metabolite separation and sample preparation were done as following: a small 10 uL amount of sample was mixed with 10pL of an internal standard solution containing 5 stable isotopes of CT metabolites at 100 pmol / l concentration for ornithine-de, arginine-d?, creatine-ds, and creatinine-ds. Proteins were precipitated in methanol and evaporated under nitrogen gas. Residues were dissolved in a 3M butanol- HCI solution and incubated at 60°C. Following cooling the samples were evaporated again under nitrogen gas and residues suspended in methanol. The samples were loaded for analysis by LC-MS / MS of creatine metabolites including ornithine, arginine, creatine, GAA, and creatinine. The raw data reads in umol / L were normalized to the protein concentration in umol / ug of protein where applicable.Western Blotting

[0169] To extract protein lysate from the cellular models, 1X radioimmunoprecipitation assay (RIPA) buffer (Cell Signalling Technology, 9806) was added to each well and the cells were briefly incubated on ice. The cells were scraped into tubes and sonicated (20% power, 10 seconds / sample, twice). Cell debris was removed by centrifugation. Tissue samples were weighed out and added to a bead tube for homogenization with RIPA buffer. The homogenate was incubated in the RIPA buffer for 10 minutes on ice. The tissue was sonicated (20% power, 10 seconds / sample, twice) and debris was removed by centrifugation and collection of the supernatant. Protein concentration was determined using the Pierce BCA Protein Assay Kit following the manufacturer protocol (Thermo Fisher, 23225).

[0170] Western blots were performed in accordance with most standard protocols. Briefly, 30 pg of protein was loaded into each well along with a ladder (Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards, 1610375, BioRad) and proteins were separated via SDS-PAGE on a 12.5% polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane. The membrane was blocked with a 5% skim milk solution and then incubated overnight with the primary anti-GAMT rabbit polyclonal antibody (A304-182A, Bethyl Laboratories). Following several washes, the secondary antibody (Goat anti-rabbit IgG, HRP, A27036) was added followed by another set of wash steps. Proteins were visualized by the chemiluminescent detection method using Immobilon Western chemiluminescent HRP substrate reagents (Millipore Sigma, WBKLS0500). The western blot was imaged using the Azure Biosystems C600 imaging system. The 0-Actin protein was as an internal control to show equal protein loading between wells. The membrane was washed following imaging for the GAMT target protein and then incubated with the primary anti-actin antibody produced in rabbit (Sigma Aldrich, A2066-100UL) overnight and imaged following the same steps as above for secondary antibody staining. phGAMT, phGATM, phGATM-GAMT study (related to FIG 15)

[0171] Protein was extracted from cells using 1X radioimmunoprecipitation assay (RIPA) buffer (cell Signaling Technology, 9806) mixed with 1 % phenylmethylsulphonyl fluoride (PMSF) (Thermo Scientific, 36978). The mixture was added to each well and then incubated on ice for 5 minutes. The cells were then scraped into tubes and sonicated (20% power, 10 seconds / samples) twice. Cell debris was removed by centrifugation and the final protein concentration was measured using the Pierce BCA Protein Assay Kit following the manufacturer protocol (Thermo Fisher, 23225). Western blots were performed in accordance with most standard protocols. Precision Plus Protein™ (Kaleidoscope™ Prestained Protein Standards, 1610375, BioRad) ladder was used to determine molecular size and 20pg of protein was loaded into each well. The proteins were separated via SDS- PAGE on a 12.5% polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was blocked with a 5% skim milk, 1 X tris-buffered saline with 0.1 % Tween® 20 detergent (TBST) solution and incubated overnight with the primary anti-GATM rabbit polyclonal antibody (NBP1 -89211 , Novus Biologicals) on a shaker at 4°C. Following severalwashes with 1X TBST, the membrane was incubated with the secondary antibody (Goat anti-rabbit IgG, HRP, A27036) for 1 hour on a shaker at room temperature. After, the membrane underwent more wash steps, the proteins were then visualized by the chemiluminescent detection method using Immobilon Western chemiluminescent HRP substrate reagents (Millipore Sigma, WBKLS0500). The blot was imaged using the Azure Biosystems c600 imaging system. The membrane was washed following the imaging for the AGAT target proteins and then underwent the same primary antibody incubation with anti- GAPDH (5174S, BioLabs) overnight as the internal control, followed by the same goat antirabbit secondary antibody as described above. The internal control is used to show equal protein loading between wells.Statistical Analysis

[0172] All statistical analysis was performed in Graph Pad Prism 9. In most cases, a one-way ANOVA with a Tukey’s multiple comparison test was used to compare treatment groups.Example 2: An In vitro study showed restored GAMT expression and intracellular creatine content in a treated cellular model of GAMT-D.

[0173] To confirm that the construct could successfully restore GAMT expression when delivered to cells, in vitro experiments were performed with plasmid DNA. A cellular model of GAMT-D was created and provided by the Shulze Lab in a HAP1 cell line. For all below experiments, wildtype (WT) HAP1 cells were used as positive controls while knockout (KO) cells treated with a plasmid containing GFP only were used as negative controls. For the purpose of these experiments, cells were grown in media supplemented with dialyzed FBS to limit the presence of small molecules such as creatine that could impact the results, particularly analysis of creatine and GAA content. Cells were transfected using Lipofectamine 3000 according to the kit protocol.GAMT protein expression is restored following transfections of cellular models of GAMT-D with plasmids carrying the designed GAMT construct.

[0174] To confirm protein expression could be restored following transfection of GAMT KO cells a western blot was performed. Cells were transfected in a 6-well plate. Severaldoses of plasmid ranging from 1 .25 ug to 5.0 ug of plasmid DNA were used for the human plasmid showing saturation past 3.75 ug, thus going forward this was chosen as the high dose for the other experiments (FIG 6). The 0-Actin (ACTB) protein was used as an internal control to ensure approximately equivalent protein loading across wells. It was evident that transfection of GAMT KO cells with the designed GAMT plasmids successfully restored protein expression showing a dose response with greater expression at the higher dose (FIG 7).GAMT gene expression was restored following transfections of cellular models of GAMT-D with plasmids carrying the designed GAMT construct confirmed by RT-qPCR.

[0175] Following transfections with the high and low dose of the hGAMT plasmid DNA cells were lysed and subject to RNA isolation. RNA was then reverse transcribed into cDNA for analysis of gene expression by qPCR. Reference genes RNA18S and 0-Actin were used to normalize and quantify gene expression. Since the untreated knockouts are assumed to have zero expression of the plasmid and any signal would be considered noise or equivalent to that of a non-template control, the low dose of 1 .25 ug was used to normalize and obtain the AACq value. As is standard for qPCR data, to determine significance the log of the 2’AACqvalues were taken to perform an ANOVA to compare all groups. The gene expression of hGAMT was significantly increased following treatment of knockout cells with phGAMT in a dose response manner with an approximate two-fold increase in expression between the low dose (1 .75 ug) and the high dose (3.75 ug) (FIG 8). This data indicated that the hGAMT plasmid can successfully restore gene expression in a cellular model of GAMT-D. Data collected using the ACTB reference gene showed a similar trend, however there was more variability observed between replicates and the fold-changes were not as consistent (not shown).Intracellular creatine content was increased while GAA content was decreased in treated cellular models of GAMT-D.

[0176] To obtain preliminary data, transfections were carried out using a single dose ranging from 1 .25 ug to 5.00 ug of pDNA in a 6-well plate. Only one well was transfected for each dose to first determine if there was any effect of pDNA on creatine and GAA levels andto determine a baseline for data analysis by LC-MS / MS for the cell lysates. Treatment of knockout cells with pmGamt or phGAMT significantly reduced GAA accumulation decreasing it to almost undetectable levels (p< 0.001 ) with no significant differences observed between the wildtype cells and treated knockouts (FIG 9A). Treatment was also able to significantly increase intracellular creatine content, however to a lesser extent that was expected given the dramatic reduction in the GAA substrate (FIG 9B).

[0177] Following this preliminary experiment, the low dose of 1 .25 ug and a high dose of 3.75 ug of pDNA was used to transfect knockout GAMT cells (n=4). Once again, a significant reduction in GAA was observed at all doses (FIG 10A). However, intracellular creatine was only increased significantly by the 3.75 ug phGAMT treatment (FIG 10B).Example 3: A small short-term in vivo study in murine models of GAMT-D showed treatment with scAAV9.hGAMT effectively restored GAMT expression and intracellular creatine content in comparison to untreated controls.GAMT protein expression was restored in the livers and brain tissues of treated animals of GAMT-D.

[0178] Protein was extracted from liver and brain tissue (mid-section) following euthanization of all mice in the study. GAMT is expressed at a very low level in the brain and it was previously found that GAMT protein expression in the brain was not detectable in even wildtype mice. GAMT is predominantly expressed in the liver and protein expression is most detectable on a western blot in the liver tissue. For the purpose of this study a western blot was performed for both the liver and the brain to explore whether the therapy may significantly restore GAMT expression within the CNS such that it may be detectable on a western blot. There was no detectable expression of GAMT in the brain of treated mice or heterozygotes (FIG 1 1A). Detectable protein expression is not necessary to restore GAMT function in the brain given that it is naturally expressed at a low level in the brain in healthy mice40. Vector distribution in brain tissues and analysis of intracellular creatine content should be sufficient to determine whether the therapy was effective within the CNS. It was found that treated mice however, had detectable GAMT expression in the liver even greater than that of the heterozygote mice (FIG 11 B). This was indicative of successful genedelivery of GAMT by the scAAV9.hGAMT vector leading to increased protein expression in the liver.Intracellular creatine content was increased while GAA was significantly decreased in several tissues including the CNS in treated mice compared to untreated controls.Several tissues were collected including all sections of the CNS (FIG 5) as well as muscle and liver samples. Serum was collected at endpoints as well as biweekly starting at a baseline of 5 weeks prior to injections. There was a trend observed showing increased intracellular creatine in all tissue types, however this was only significant in some tissues given the small scale of the study (FIG 12). GAA accumulation was significantly decreased in treated animals in all tissues except for the rostral brain section (FIG 13). There was a significant increase in creatine content in the serum collected at endpoint and this increase was observable over time from 5 week until endpoint at 13 weeks (FIG 14A,B). Similarly, a significant decrease in GAA accumulation was observed in the serum at end point which could be seen over time (FIG 14C-D).Example 4: An in vitro study showed increased intracellular creatine content in a treated cellular model of AG AT-D and GAMT-D.

[0179] AGAT-D cells treated with a monocistronic AGAT coding plasmid demonstrated elevated creatine levels (FIG 15A), but very high levels of GAA that mimic untreated GAMT-D cells (FIG 15C). However, a bicistronic plasmid demonstrated creatine production (FIG 15A) without GAA accumulation (FIG 15C). In GAMT-D cells, both the monocistronic and bicistronic plasmids, increased creatine (FIG 15B) and completely reduced GAA levels (FIG 15D).

[0180] Western blot confirmed that the bicistronic AGAT-GAMT vector successfully restored AGAT expression (FIG 16A) and GAMT expression (FIG 16B).

[0181] Example 5: An in vivo dose study showed increased creatine levels in GAMT knockout mice in long-term

[0182] GAMT-D mice treated with low (6.25e10vg / mouse), medium (1.25e11vg / mouse) and high (2.5e11vg / mouse) doses of scAAV9.GAMT showed increased creatine levels in serum collected at both the 13 week (FIG 17A) and 26-week endpoint (FIG 18A).Increased creatine levels were also detected in the lumbar and cervical spinal cord in the low, medium and high dose as compared to untreated knockout mice (FIG 19). Increased creatine levels were detected in the muscle at all doses tested as compared to untreated knockout mice (FIG 21A). Creatine levels were increased in the liver at medium and high doses (FIG 21 B) and in the kidney at the high dose only as compared to untreated knockout mice (FIG 21 C). The levels of creatine at in the muscle and liver at the high dosage was not significantly different than vehicle treated heterozygous nice (FIG 21A,B).

[0183] In the brain, creatine was increased at the medium and high doses in the midsection, caudal-section and rostral section (FIG 23).

[0184] Example 6: An in vivo dose study shows reduced GAA accumulation in GAMT knockout mice in a dose dependent manner

[0185] GAMT-D mice treated with low (6.25e10vg / mouse), medium (1.25e11vg / mouse) and high (2.5e11vg / mouse) doses of sc.AAV9.GAMT showed reduced GAA accumulation in serum collected at the 13-week endpoint (FIG 17B). However, no significant differences were observed in GAA accumulation at the 26-week endpoint (FIG 18C).

[0186] GAA accumulation was also reduced in the lumbar and cervical spinal cord for all doses as compared to knockout controls (FIG 20). In the muscle, GAA accumulation was significant reduced at only the high dose as compared to untreated knockout controls (FIG 22A). In the kidney and liver, no significant differences in GAA accumulation were observed (FIG 22B, C). However, it was noted that in the liver the GAA accumulation at the high dose was significantly lower than the medium dose (FIG 22B).

[0187] In the brain, GAA accumulation was reduced at all doses in the mid-section, caudal-section and rostral-section as compared to untreated knockout controls (FIG 24).Example 7: An in vivo study shows improved muscular strength in mice treated with SCAAV9.GAMT

[0188] GAMT-D mice treated with the high dose (2.5e11vg / mouse) demonstrated improved muscular strength on the mesh test as compared to untreated knockout controls at the 13-week endpoint. Notably, the mice treated with the high dose were not significantlydifferent than control heterozygous control mice (FIG 25A). Th same effect was seen in both male and female mice (FIGs 25B,C).

[0189] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.

[0190] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present description is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.Sequences:SEQ ID NO: 1: Optimized Human GAMT Coding SequenceATGAGCGCCCCCAGCGCCACACCCATCTTCGCCCCTGGGGAGAACTGCAGCCCCGC TTGGGGAGCAGCCCCTGCCGCCTACGATGCCGCCGACACTCACTTGCGGATTCTTG GGAAGCCCGTGATGGAGCGGTGGGAGACCCCCTACATGCACGCCCTCGCCGCCGC CGCCAGCAGCAAGGGGGGGAGGGTGCTGGAGGTGGGGTTTGGCATGGCCATCGCC GCCAGCAAGGTGCAGGAGGCCCCCATCGATGAACATTGGATCATCGAGTGCAACGA TGGGGTGTTCCAGCGGCTGCGCGACTGGGCCCCCAGGCAGACCCACAAAGTCATCC CCCTGAAGGGCCTGTGGGAGGATGTGGCCCCCACCCTGCCCGATGGGCACTTCGAC GGCATCCTGTACGACACATACCCCCTGAGCGAGGAGACCTGGCACACACACCAGTT CAACTTCATCAAAAACCACGCCTTCCGGCTGCTGAAGCCCGGCGGGGTGCTGACATA CTGCAACCTGACCTCCTGGGGCGAGCTGATGAAGTCCAAGTACAGCGACATCACCAT CATGTTCGAGGAGACACAGGTGCCCGCCCTGCTGGAGGCCGGGTTCAGGAGGGAG AACATCAGGACAGAGGTCATGGCCCTGGTGCCCCCCGCCGACTGCCGGTACTACGC CTTCCCCCAGATGATCACCCCCCTGGTGACAAAGGGCTGASEQ ID NO: 2: Human GAMT Coding SequenceATGAGCGCCCCCAGCGCGACCCCCATCTTCGCGCCCGGCGAGAACTGCAGCCCCGCGTGGGGGGCGGCGCCCGCGGCCTACGACGCAGCGGACACGCACCTGCGCATCCTGGGCAAGCCGGTGATGGAGCGCTGGGAGACCCCCTATATGCACGCGCTGGCCGCCGCCGCCTCCTCCAAAGGGGGCCGGGTCCTGGAGGTGGGCTTTGGCATGGCCATCGCAGCGTCAAAGGTGCAGGAGGCGCCCATTGATGAGCATTGGATCATCGAGTGCAATGACGGCGTCTTCCAGCGGCTCCGGGACTGGGCCCCACGGCAGACACACAAGGTCATCCCCTTGAAAGGCCTGTGGGAGGATGTGGCACCCACCCTGCCTGACGGTCACTTTGATGGGATCCTGTACGACACGTACCCACTCTCGGAGGAGACCTGGCACACACACCAGTTCAACTTCATCAAGAACCACGCCTTTCGCCTGCTGAAGCCGGGGGGCGTCCTCACCTACTGCAACCTCACCTCCTGGGGGGAGCTGATGAAGTCCAAGTACTCAGACATCACCATCATGTTTGAGGAGACGCAGGTGCCCGCGCTGCTGGAGGCCGGCTTCCGGAGGGAGAACATCCGTACGGAGGTGATGGCGCTGGTCCCACCGGCCGACTGCCGCTACTACGCCTTCCCACAGATGATCACGCCCCTGGTGACCAAAGGCTGASEQ ID NO: 3 Human GAMT AAV VectorGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGAACAGAATTCTGAGGGCGGAGTTAGGGCGGAGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTGGGCGGAGAATGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTTCCGGAAAGCCACCATGAGCGCCCCCAGCGCCACACCCATCTTCGCCCCTGGGGAGAACTGCAGCCCCGCTTGGGGAGCAGCCCCTGCCGCCTACGATGCCGCCGACACTCACTTGCGGATTCTTGGGAAGCCCGTGATGGAGCGGTGGGAGACCCCCTACATGCACGCCCTCGCCGCCGCCGCCAGCAGCAAGGGGGGGAGGGTGCTGGAGGTGGGGTTTGGCATGGCCATCGCCGCCAGCAAGGTGCAGGAGGCCCCCATCGATGAACATTGGATCATCGAGTGCAACGATGGGGTGTTCCAGCGGCTGCGCGACTGGGCCCCCAGGCAGACCCACAAAGTCATCCCCCTGAAGGGCCTGTGGGAGGATGTGGCCCCCACCCTGCCCGATGGGCACTTCGACGGCATCCTGTACGACACATACCCCCTGAGCGAGGAGACCTGGCACACACACCAGTTCAACTTCATCAAAAACCACGCCTTCCGGCTGCTGAAGCCCGGCGGGGTGCTGACATACTGCAACCTGACCTCCTGGGGCGAGCTGATGAAGTCCAAGTACAGCGACATCACCATCATGTTCGAGGAGACACAGGTGCCCGCCCTGCTGGAGGCCGGGTTCAGGAGGGAGAACATCAGGACAGAGGTCATGGCCCTGGTGCCCCCCGCCGACTGCCGGTACTACGCCTTCCCCCAGATGATCACCCCCCTGGTGACAAAGGGCTGAAATAAAGAGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTTGTGTGACGTCGACTTACACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGASEQ ID NO: 4: GAMT Protein Sequence (translation of SEQ ID NOs: 1 and 2)MSAPSATPIFAPGENCSPAWGAAPAAYDAADTHLRILGKPVMERWETPYMHALAAAASSKGGRVLEVGFGMAIAASKVQEAPIDEHWIIECNDGVFQRLRDWAPRQTHKVIPLKGLWEDVAPTLPDGHFDGILYDTYPLSEETWHTHQFNFIKNHAFRLLKPGGVLTYCNLTSWGELMKSKYSDITIMFEETQVPALLEAGFRRENIRTEVMALVPPADCRYYAFPQMITPLVTKGSEQ ID NO: 5: Murine GAMT Coding SequenceATGAGCTCTAGCGCTGCTAGTCCTCTGTTCGCTCCTGGCGAGGACTGTGGACCTGCCTGGAGAGCCGCCCCTGCTGCCTATGATGCTTCTGATACACATCTGCAGATCCTGGGAAAACCAGTGATGGAAAGATGGGAGACCCCTTATATGCACGCTCTGGCCGCTGCTGCTGCTTCTAGAGGAGGAAGAGTGCTGGAGGTGGGATTCGGCATGGCTATTGCTGCCTCTAGAGTGCAGCAGGCCCCAATCGAGGAGCACTGGATTATTGAATGTAATGACGGAGTGTTCCAGAGATTACAGGATTGGGCTCTGAGACAGCCTCATAAAGTGGTGCCACTGAAGGGACTGTGGGAGGAGGTGGCTCCTACACTGCCTGATGGCCACTTTGACGGAATCCTGTATGACACATATCCTCTTTCTGAAGAAGCTTGGCACACACACCAGTTTAACTTTATCAAGAATCATGCTTTTAGACTGCTGAAAACAGGAGGAGTGCTGACATATTGTAATCTGACATCTTGGGGCGAGCTGATGAAATCTAAATATACAGATATCACAACAATGTTCGAGGAAACACAGGTGCCTGCTCTGCAGGAGGCCGGATTTCTGAAGGAAAATATCTGCACAGAAGTGATGGCTCTGGTGCCTCCTGCTGATTGTAGATATTATGCTTTTCCTCAGATGATCACACCTCTGGTGACCAAGCACTGASEQ ID NO: 6: Murine G AMT AAV VectorGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGAGCAGCCGGCCGGGCGGAGTTAGGGCGGAGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTGGGCGGAGAATGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTTCCGGAAAGCCACCATGAGCTCTAGCGCTGCTAGTCCTCTGTTCGCTCCTGGCGAGGACTGTGGACCTGCCTGGAGAGCCGCCCCTGCTGCCTATGATGCTTCTGATACACATCTGCAGATCCTGGGAAAACCAGTGATGGAAAGATGGGAGACCCCTTATATGCACGCTCTGGCCGCTGCTGCTGCTTCTAGAGGAGGAAGAGTGCTGGAGGTGGGATTCGGCATGGCTATTGCTGCCTCTAGAGTGCAGCAGGCCCCAATCGAGGAGCACTGGATTATTGAATGTAATGACGGAGTGTTCCAGAGATTACAGGATTGGGCTCTGAGACAGCCTCATAAAGTGGTGCCACTGAAGGGACTGTGGGAGGAGGTGGCTCCTACACTGCCTGATGGCCACTTTGACGGAATCCTGTATGACACATATCCTCTTTCTGAAGAAGCTTGGCACACACACCAGTTTAACTTTATCAAGAATCATGCTTTTAGACTGCTGAAAACAGGAGGAGTGCTGACATATTGTAATCTGACATCTTGGGGCGAGCTGATGAAATCTAAATATACAGATATCACAACAATGTTCGAGGAAACACAGGTGCCTGCTCTGCAGGAGGCCGGATTTCTGAAGGAAAATATCTGCACAGAAGTGATGGCTCTGGTGCCTCCTGCTGATTGTAGATATTATGCTTTTCCTCAGATGATCACACCTCTGGTGACCAAGCACTGAAATAAAGAGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTTGTGTGGCAGTCGACGCCCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGASEQ ID NO: 7: 5’ ITRGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGASEQ ID NO: 8: 3’ ITRCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGASEQ ID NO: 9: JeT promoter + Kozak sequenceGGGCGGAGTTAGGGCGGAGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTGGGCGGAGAATGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTTCCGGA AAGCCACCSEQ ID NO: 10: Poly A RegionAATAAAGAGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTTGTGTGSEQ ID NO: 11 : Optimized Human AGAT-P2A-GAMT Coding SequenceATGTTAAGGGTGAGGTGCCTGAGGGGCGGCAGCAGGGGCGCCGAGGCCGTGCACTACATCGGGTCCAGGCTGGGCCGGACACTGACTGGCTGGGTGCAGCGCACATTCCAGAGCACCCAGGCGGCCACAGCCTCCAGCAGAAACTCTTGTGCGGCTGATGATAAGGCTACAGAGCCCCTGCCTAAGGACTGCCCAGTGAGCTCATACAACGAGTGGGATCCTCTCGAAGAGGTCATCGTGGGGCGGGCCGAGAACGCCTGCGTGCCCCCATTCACTATTGAGGTGAAGGCAAACACTTACGAGAAATACTGGCCATTCTACCAGAAGCAGGGGGGCCACTACTTTCCCAAGGACCACTTGAAGAAGGCCGTCGCCGAGATCGAGGAGATGTGCAATATTCTTAAAACTGAAGGCGTGACCGTGCGGAGGCCCGATCCAATTGACTGGTCCCTGAAGTATAAAACCCCCGATTTCGAAAGTACCGGCCTGTATAGCGCCATGCCACGGGACATTCTGATCGTCGTCGGGAACGAAATCATTGAGGCCCCCATGGCCTGGCGGTCCCGGTTCTTCGAGTACCGGGCCTACCGGAGCATCATCAAGGATTACTTCCACAGGGGCGCCAAGTGGACCACAGCCCCTAAGCCCACAATGGCCGATGAGCTGTACAACCAGGATTACCCCATTCACAGCGTGGAGGACCGGCACAAGCTGGCCGCCCAGGGGAAGTTTGTGACAACAGAGTTCGAGCCCTGCTTCGACGCCGCCGACTTTATCAGGGCCGGGAGGGACATCTTTGCCCAGCGGAGCCAGGTGACAAACTACCTGGGGATCGAGTGGATGCGGAGGCACCTGGCCCCTGATTACAGGGTGCACATCATCTCCTTCAAGGACCCCAACCCCATGCACATTGATGCCACTTTCAACATCATCGGGCCTGGGATCGTGCTCAGCAACCCCGACCGGCCCTGCCACCAGATCGACCTGTTCAAGAAGGCCGGCTGGACCATCATCACCCCCCCCACACCCATCATCCCTGACGACCACCCCCTCTGGATGTCCAGCAAGTGGCTGAGCATGAACGTGCTGATGCTGGATGAGAAGAGAGTGATGGTCGATGCCAACGAGGTGCCCATCCAGAAGATGTTCGAGAAGCTCGGGATCACCACCATTAAGGTCAACATCAGGAACGCCAACAGCCTGGGGGGGGGCTTTCACTGCTGGACCTGCGACGTGAGGCGGCGGGGGACCCTGCAGTCCTACCTGGACGCCACAAACTTCAGCCTGCTGAAGCAGGCCGGGGACGTGGAGGAGAACCCCGGTCCAATGAGCGCCCCCTCCGCCACCCCCATTTTCGCCCCTGGCGAGAACTGCAGCCCCGCATGGGGAGCCGCCCCCGCCGCCTACGATGCCGCCGATACACATCTGAGGATTCTGGGAAAGCCTGTGATGGAAAGATGGGAGACACCCTACATGCATGCCCTGGCCGCCGCAGCCAGCAGCAAGGGCGGCCGGGTGCTAGAAGTGGGCTTTGGGATGGCCATCGCTGCCAGCAAGGTGCAGGAGGCCCCCATTGATGAGCACTGGATCATCGAGTGCAACGATGGGGTCTTTCAGAGGCTGAGGGACTGGGCCCCCAGGCAGACACACAAAGTGATCCCCCTGAAGGGGCTGTGGGAGGACGTGGCCCCCACCCTGCCCGATGGCCACTTCGACGGCATCCTGTACGATACCTACCCCCTGAGCGAGGAGACATGGCACACCCACCAGTTCAACTTCATCAAGAACCACGCCTTTAGGCTGCTGAAGCCTGGCGGGGTGCTGACATACTGTAACCTGACTAGTTGGGGAGAGCTGATGAAGTCCAAGTACTCCGATATCACCATCATGTTTGAGGAGACCCAGGTGCCCGCCCTGCTGGAGGCCGGCTTCCGGCGGGAGAACATTAGGACCGAGGTCATGGCCCTGGTGCCCCCCGCCGACTGCCGGTACTACGCCTTCCCCCAGATGATCACCCCCTTAGTGACTAAGGGGTGASEQ ID NO: 12: Optimized Human AGAT-P2A-GAMT protein sequenceMLRVRCLRGGSRGAEAVHYIGSRLGRTLTGWVQRTFQSTQAATASSRNSCAADDKATEPLPKDCPVSSYNEWDPLEEVIVGRAENACVPPFTIEVKANTYEKYWPFYQKQGGHYFPKDHLKKAVAEIEEMCNILKTEGVTVRRPDPIDWSLKYKTPDFESTGLYSAMPRDILIVVGNEIIEAPMAWRSRFFEYRAYRSIIKDYFHRGAKWTTAPKPTMADELYNQDYPIHSVEDRHKLAAQGKFVTTEFEPCFDAADFIRAGRDIFAQRSQVTNYLGIEWMRRHLAPDYRVHIISFKDPNPMHIDATFNIIGPGIVLSNPDRPCHQIDLFKKAGWTIITPPTPIIPDDHPLWMSSKWLSMNVLMLDEKRVMVDANEVPIQKMFEKLGITTIKVNIRNANSLGGGFHCWTCDVRRRGTLQSYLDATNFSLLKQAGDVEENPGPMSAPSATPIFAPGENCSPAWGAAPAAYDAADTHLRILGKPVMERWETPYMHALAAAASSKGGRVLEVGFGMAIAASKVQEAPIDEHWIIECNDGVFQRLRDWAPRQTHKVIPLKGLWEDVAPTLPDGHFDGILYDTYPLSEETWHTHQFNFIKNHAFRLLKPGGVLTYCNLTSWGELMKSKYSDITIMFEETQVPALLEAGFRRENIRTEVMALVPPADCRYYAFP QMITPLVTKGSEQ ID NO: 13: P2A Coding SequenceGCCACAAACTTCAGCCTGCTGAAGCAGGCCGGGGACGTGGAGGAGAACCCCGGTCC AASEQ ID NO: 14: P2A Protein SequenceATNFSLLKQAGDVEENPGPSEQ ID NO: 15: Optimized Human AGAT (G T ) Coding SequenceATGTTAAGGGTGAGGTGCCTGAGGGGCGGCAGCAGGGGCGCCGAGGCCGTGCACTACATCGGGTCCAGGCTGGGCCGGACACTGACTGGCTGGGTGCAGCGCACATTCCAGAGCACCCAGGCGGCCACAGCCTCCAGCAGAAACTCTTGTGCGGCTGATGATAAGGCTACAGAGCCCCTGCCTAAGGACTGCCCAGTGAGCTCATACAACGAGTGGGATCCTCTCGAAGAGGTCATCGTGGGGCGGGCCGAGAACGCCTGCGTGCCCCCATTCACTATTGAGGTGAAGGCAAACACTTACGAGAAATACTGGCCATTCTACCAGAAGCAGGGGGGCCACTACTTTCCCAAGGACCACTTGAAGAAGGCCGTCGCCGAGATCGAGGAGATGTGCAATATTCTTAAAACTGAAGGCGTGACCGTGCGGAGGCCCGATCCTATTGACTGGTCCCTGAAGTATAAAACCCCCGATTTCGAAAGTACCGGCCTGTATAGCGCCATGCCACGGGACATTCTGATCGTCGTCGGGAACGAAATCATTGAGGCCCCCATGGCCTGGCGGTCCCGGTTCTTCGAGTACCGGGCCTACCGGAGCATCATCAAGGATTACTTCCACAGGGGCGCCAAGTGGACCACAGCCCCTAAGCCCACAATGGCCGATGAGCTGTACAACCAGGATTACCCCATTCACAGCGTGGAGGACCGGCACAAGCTGGCCGCCCAGGGGAAGTTTGTGACAACAGAGTTCGAGCCCTGCTTCGACGCCGCCGACTTTATCAGGGCCGGGAGGGACATCTTTGCCCAGCGGAGCCAGGTGACAAACTACCTGGGGATCGAGTGGATGCGGAGGCACCTGGCCCCTGATTACAGGGTGCACATCATCTCCTTCAAGGACCCCAACCCCATGCACATTGATGCCACTTTCAACATCATCGGGCCTGGGATCGTGCTCAGCAACCCCGACCGGCCCTGCCACCAGATCGACCTGTTCAAGAAGGCCGGCTGGACCATCATCACCCCCCCCACACCCATCATCCCTGACGACCACCCCCTCTGGATGTCCAGCAAGTGGCTGAGCATGAACGTGCTGATGCTGGATGAGAAGAGAGTGATGGTCGATGCCAACGAGGTGCCCATCCAGAAGATGTTCGAGAAGCTCGGGATCACCACCATTAAGGTCAACATCAGGAACGCCAACAGCCTGGGGGGGGGCTTTCACTGCTGGACCTGCGACGTGAGGCGGCGGGGGACCCTGCAGTCCTACCTGGACTAASEQ ID NO: 16: Optimized Human AGAT Protein SequenceMLRVRCLRGGSRGAEAVHYIGSRLGRTLTGWVQRTFQSTQAATASSRNSCAADDKATEPLPKDCPVSSYNEWDPLEEVIVGRAENACVPPFTIEVKANTYEKYWPFYQKQGGHYFPKDHLKKAVAEIEEMCNILKTEGVTVRRPDPIDWSLKYKTPDFESTGLYSAMPRDILIVVGNEIIEAPMAWRSRFFEYRAYRSIIKDYFHRGAKWTTAPKPTMADELYNQDYPIHSVEDRHKLAAQGKFVTTEFEPCFDAADFIRAGRDIFAQRSQVTNYLGIEWMRRHLAPDYRVHIISFKDPNPMHIDATFNIIGPGIVLSNPDRPCHQIDLFKKAGWTIITPPTPIIPDDHPLWMSSKWLSMNVLMLDEKRVMVDANEVPIQKMFEKLGITTIKVNIRNANSLGGGFHCWTCDVRRRGTLQSYLD*SEQ ID NO: 17: Optimized Human AGAT -P2A- GAMT full viral vector sequenceGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGAGCAGAATTCTGGGGGCGGAGTTAGGGCGGAGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTGGGCGGAGAATGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTTCCGGAAAGCCACCATGTTAAGGGTGAGGTGCCTGAGGGGCGGCAGCAGGGGCGCCGAGGCCGTGCACTACATCGGGTCCAGGCTGGGCCGGACACTGACTGGCTGGGTGCAGCGCACATTCCAGAGCACCCAGGCGGCCACAGCCTCCAGCAGAAACTCTTGTGCGGCTGATGATAAGGCTACAGAGCCCCTGCCTAAGGACTGCCCAGTGAGCTCATACAACGAGTGGGATCCTCTCGAAGAGGTCATCGTGGGGCGGGCCGAGAACGCCTGCGTGCCCCCATTCACTATTGAGGTGAAGGCAAACACTTACGAGAAATACTGGCCATTCTACCAGAAGCAGGGGGGCCACTACTTTCCCAAGGACCACTTGAAGAAGGCCGTCGCCGAGATCGAGGAGATGTGCAATATTCTTAAAACTGAAGGCGTGACCGTGCGGAGGCCCGATCCAATTGACTGGTCCCTGAAGTATAAAACCCCCGATTTCGAAAGTACCGGCCTGTATAGCGCCATGCCACGGGACATTCTGATCGTCGTCGGGAACGAAATCATTGAGGCCCCCATGGCCTGGCGGTCCCGGTTCTTCGAGTACCGGGCCTACCGGAGCATCATCAAGGATTACTTCCACAGGGGCGCCAAGTGGACCACAGCCCCTAAGCCCACAATGGCCGATGAGCTGTACAACCAGGATTACCCCATTCACAGCGTGGAGGACCGGCACAAGCTGGCCGCCCAGGGGAAGTTTGTGACAACAGAGTTCGAGCCCTGCTTCGACGCCGCCGACTTTATCAGGGCCGGGAGGGACATCTTTGCCCAGCGGAGCCAGGTGACAAACTACCTGGGGATCGAGTGGATGCGGAGGCACCTGGCCCCTGATTACAGGGTGCACATCATCTCCTTCAAGGACCCCAACCCCATGCACATTGATGCCACTTTCAACATCATCGGGCCTGGGATCGTGCTCAGCAACCCCGACCGGCCCTGCCACCAGATCGACCTGTTCAAGAAGGCCGGCTGGACCATCATCACCCCCCCCACACCCATCATCCCTGACGACCACCCCCTCTGGATGTCCAGCAAGTGGCTGAGCATGAACGTGCTGATGCTGGATGAGAAGAGAGTGATGGTCGATGCCAACGAGGTGCCCATCCAGAAGATGTTCGAGAAGCTCGGGATCACCACCATTAAGGTCAACATCAGGAACGCCAACAGCCTGGGGGGGGGCTTTCACTGCTGGACCTGCGACGTGAGGCGGCGGGGGACCCTGCAGTCCTACCTGGACGCCACAAACTTCAGCCTGCTGAAGCAGGCCGGGGACGTGGAGGAGAACCCCGGTCCAATGAGCGCCCCCTCCGCCACCCCCATTTTCGCCCCTGGCGAGAACTGCAGCCCCGCATGGGGAGCCGCCCCCGCCGCCTACGATGCCGCCGATACACATCTGAGGATTCTGGGAAAGCCTGTGATGGAAAGATGGGAGACACCCTACATGCATGCCCTGGCCGCCGCAGCCAGCAGCAAGGGCGGCCGGGTGCTAGAAGTGGGCTTTGGGATGGCCATCGCTGCCAGCAAGGTGCAGGAGGCCCCCATTGATGAGCACTGGATCATCGAGTGCAACGATGGGGTCTTTCAGAGGCTGAGGGACTGGGCCCCCAGGCAGACACACAAAGTGATCCCCCTGAAGGGGCTGTGGGAGGACGTGGCCCCCACCCTGCCCGATGGCCACTTCGACGGCATCCTGTACGATACCTACCCCCTGAGCGAGGAGACATGGCACACCCACCAGTTCAACTTCATCAAGAACCACGCCTTTAGGCTGCTGAAGCCTGGCGGGGTGCTGACATACTGTAACCTGACTAGTTGGGGAGAGCTGATGAAGTCCAAGTACTCCGATATCACCATCATGTTTGAGGAGACCCAGGTGCCCGCCCTGCTGGAGGCCGGCTTCCGGCGGGAGAACATTAGGACCGAGGTCATGGCCCTGGTGCCCCCCGCCGACTGCCGGTACTACGCCTTCCCCCAGATGATCACCCCCTTAGTGACTAAGGGGTGAAATAAAGAGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTTGTGTGGCAGTCGACGTGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGASEQ ID NO: 18: Optimized Human AGAT (GATM) Full Viral Vector SequenceGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGAGCCTAGGGGGCGGAGTTAGGGCGGAGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTGGGCGGAGAATGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTTCCGGAAAGCCACCATGTTAAGGGTGAGGTGCCTGAGGGGCGGCAGCAGGGGCGCCGAGGCCGTGCACTACATCGGGTCCAGGCTGGGCCGGACACTGACTGGCTGGGTGCAGCGCACATTCCAGAGCACCCAGGCGGCCACAGCCTCCAGCAGAAACTCTTGTGCGGCTGATGATAAGGCTACAGAGCCCCTGCCTAAGGACTGCCCAGTGAGCTCATACAACGAGTGGGATCCTCTCGAAGAGGTCATCGTGGGGCGGGCCGAGAACGCCTGCGTGCCCCCATTCACTATTGAGGTGAAGGCAAACACTTACGAGAAATACTGGCCATTCTACCAGAAGCAGGGGGGCCACTACTTTCCCAAGGACCACTTGAAGAAGGCCGTCGCCGAGATCGAGGAGATGTGCAATATTCTTAAAACTGAAGGCGTGACCGTGCGGAGGCCCGATCCTATTGACTGGTCCCTGAAGTATAAAACCCCCGATTTCGAAAGTACCGGCCTGTATAGCGCCATGCCACGGGACATTCTGATCGTCGTCGGGAACGAAATCATTGAGGCCCCCATGGCCTGGCGGTCCCGGTTCTTCGAGTACCGGGCCTACCGGAGCATCATCAAGGATTACTTCCACAGGGGCGCCAAGTGGACCACAGCCCCTAAGCCCACAATGGCCGATGAGCTGTACAACCAGGATTACCCCATTCACAGCGTGGAGGACCGGCACAAGCTGGCCGCCCAGGGGAAGTTTGTGACAACAGAGTTCGAGCCCTGCTTCGACGCCGCCGACTTTATCAGGGCCGGGAGGGACATCTTTGCCCAGCGGAGCCAGGTGACAAACTACCTGGGGATCGAGTGGATGCGGAGGCACCTGGCCCCTGATTACAGGGTGCACATCATCTCCTTCAAGGACCCCAACCCCATGCACATTGATGCCACTTTCAACATCATCGGGCCTGGGATCGTGCTCAGCAACCCCGACCGGCCCTGCCACCAGATCGACCTGTTCAAGAAGGCCGGCTGGACCATCATCACCCCCCCCACACCCATCATCCCTGACGACCACCCCCTCTGGATGTCCAGCAAGTGGCTGAGCATGAACGTGCTGATGCTGGATGAGAAGAGAGTGATGGTCGATGCCAACGAGGTGCCCATCCAGAAGATGTTCGAGAAGCTCGGGATCACCACCATTAAGGTCAACATCAGGAACGCCAACAGCCTGGGGGGGGGCTTTCACTGCTGGACCTGCGACGTGAGGCGG CGGGGGACCCTGCAGTCCTACCTGGACTAAAGATCTAATAAAGAGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTTGTGTGCAATTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAReferences:1. 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Claims

CLAIMS1. A nucleic acid construct comprising a nucleotide sequence encoding a guanidinoacetate N-methyltransferase (GAMT) protein and / or a L-arginine:glycine amidinotransferase (AGAT) protein operably linked to a promoter and a transcription termination site.

2. The nucleic acid construct of claim 1 , wherein the nucleotide sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoded by any one of SEQ ID NO: 1 , and SEQ ID NO: 2, and which retains GAMT activity.

3. The nucleic acid construct of claim 1 or 2, wherein the nucleotide sequence encoding the GAMT protein is selected from SEQ ID NO:1 , and SEQ ID NO:2, or a functional variant thereof.

4. The nucleic acid construct according to any one of claims 1 -3, wherein the GAMT protein has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

5. The nucleic acid construct of claim 1 , comprising a nucleotide sequence encoding a L- arginine:glycine amidinotransferase (AGAT) protein operably linked to a promoter and a transcription termination site.

6. The nucleic acid construct of claim 5, wherein the nucleotide sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoded by SEQ ID NO: 15, and which retains AGAT activity.

7. The nucleic acid construct of claim 5 or 6, wherein the nucleotide sequence encoding the AGAT protein is SEQ ID NO: 15, or a functional variant thereof.

8. The nucleic acid construct according to any one of claims 5-7, wherein the AGAT protein has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 16.

9. The nucleic acid construct of claim 1 -8, comprising a nucleotide sequence encoding a L- arginine:glycine amidinotransferase (AGAT) protein and a guanidinoacetate N- m ethyltransferase (GAMT) operably linked to a promoter and a transcription termination site.

10. The nucleic acid construct according to any one of claims 1 to 9, wherein the promoter is a constitutive promoter or a tissue- or cell-specific promoter.11 . The nucleic acid construct according to claim 10, wherein the promoter is selected from the group comprising: chicken beta actin (CBA), chicken (3-actin hybrid (CBh), CMV early enhancer (CAG), Elongation Factor 1 a (eF-1 a), simian virus 40 early promoter (SV40), human phosphoglycerate kinase 1 (PGK), cytomegalovirus immediate-early promoter (CMV), human (3-actin (hACTB), synapsin, myelin basic protein and JeT synthetic promoter.

12. The nucleic acid construct of any one of claims 1 to 11 , comprising a sequence as set forth in SEQ ID NO:5, or a functional variant thereof.

13. A viral vector comprising the nucleic acid construct according to any one of claims 1 to 12.

14. The viral vector according to claim 13, wherein the viral vector is an Adeno-Associated Virus (AAV) vector or a derivative thereof.

15. The viral vector according to claim 14, wherein the AAV vector is selected from the group consisting of: AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9, or a derivative thereof.

16. The viral vector according to claim 14 or claim 15 wherein the viral vector is a chimeric, shuffled or capsid modified derivative of AAV.

17. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 1 to 12 or the viral vector of any one of claims 13 to 16, and a pharmaceutically acceptable carrier or diluent.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is formulated for intraparenchymal, intravenous, intrathecal or any other systemic method of administration.

19. Use of the viral vector according to any one of claims 13 to 16 or the pharmaceutical composition of claim 17 or claim 18, for the treatment of a Creatine Deficiency Syndrome (CDD) in a subject in need thereof.

20. Use of the viral vector according to any one of claims 13 to 16 or the pharmaceutical composition of claim 17 or claim 18, for the manufacture of a medicament for the treatment of a Creatine Deficiency Syndrome (CDD).21 . The use of claim 19 or claim 20, wherein the viral vector or pharmaceutical composition is formulated for systemic, peritoneal, intraparenchymal, intravenous and / or intrathecal injection.

22. The viral vector according to any one of claims 13 to 16 or the pharmaceutical composition of claim 17 or claim 18 for use in the treatment of a Creatine Deficiency Syndrome (CDD) in a subject in need thereof.

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