WO2024103166A1 - Compositions et méthodes de traitement de déficits en créatine - Google Patents

Compositions et méthodes de traitement de déficits en créatine Download PDF

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WO2024103166A1
WO2024103166A1 PCT/CA2023/051522 CA2023051522W WO2024103166A1 WO 2024103166 A1 WO2024103166 A1 WO 2024103166A1 CA 2023051522 W CA2023051522 W CA 2023051522W WO 2024103166 A1 WO2024103166 A1 WO 2024103166A1
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gamt
nucleic acid
protein
creatine
acid construct
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Jagdeep WALIA
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01002—Guanidinoacetate N-methyltransferase (2.1.1.2)
    • C—CHEMISTRY; METALLURGY
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    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/04—Amidinotransferases (2.1.4)
    • C12Y201/04001—Glycine amidinotransferase (2.1.4.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • 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).
  • GAT guanidinoacetate methyltransferase
  • AAT L-arginine:glycine amidinotransferase
  • Creatine synthesis includes two enzymatic steps and a transporter known as SLC6A8 (FIG 1 ).
  • L-arginine:glycine amidinotransferase AGAT
  • GATM glycine amidinotransferase
  • GAA is methylated by GAMT in the presence of S-adenosyl-l- methionine to form creatine.
  • GAA must exit neuronal cells and be imported through SLC6A8 or astrocytic y-aminobutyric acid transporter to cells that express GAMT 8-10 SLC6A8 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 cells 7 . This suggests that the brain relies heavily on endogenous synthesis for its creatine supply.
  • BBB blood brain barrier
  • Cerebral creatine deficiency syndromes 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.
  • 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 fluids 11-13 .
  • 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 symptoms 11 14 ' 15 .
  • 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
  • Creatine is 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 system 1-4
  • the 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.
  • CNS central nervous system
  • Creatine is a known competitive antagonist of GABAA receptors and has been recently implicated in neuromodulation 5 ’ 6 . It has also been suggested that creatine has anti-apoptotic and antioxidant properties which may participate in neuroprotection 1 ’ 2 ’ 4 .
  • AGAT is present primarily in the kidneys, though extra-renal expression is variable among species. 43 In the kidneys, it is located in the mitochondria of cells found in the proximal tubules of nephrons. 43 ’ 44 The AGAT enzyme acts at a rate limiting and tightly regulated step during the production of creatine and homoarginine. 43 ’ 46 Previous studies have shown that AGAT mutations affecting AGAT levels have been associated with chronic kidney disease (CKD). 50 Similarly, 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
  • 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 assembly 16 ’ 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 scAAV 19- 21 .
  • the small size of the GAMT gene (711 bps) allows it to be packaged into a scAAV9 vector for delivery to the CNS.
  • the present disclosure relates to nucleic acid constructs encoding a functional GAMT protein and/or a functional AGAT protein operably linked to a promoter and transcription 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.
  • 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.
  • 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.
  • GAT guanidinoacetate N -methyltransferase
  • AGAT L-arginine:glycine amidinotransferase
  • 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.
  • AGAT L-arginine:glycine amidinotransferase
  • 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.
  • 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.
  • 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.
  • 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.
  • AGAT L-arginine:glycine amidinotransferase
  • 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.
  • nucleotide sequence encoding the AGAT protein is SEQ ID NO: 15, or a functional variant thereof.
  • 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.
  • 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.
  • AGAT L-arginine:glycine amidinotransferase
  • GAMT guanidinoacetate N-methyltransferase
  • the promoter is a constitutive promoter or a tissue- or cellspecific promoter.
  • 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), human phosphoglycerate kinase 1 (PGK), cytomegalovirus immediate-early promoter (CMV), human (3-actin (hACTB), synapsin, myelin basic protein and JeT synthetic promoter.
  • CBA chicken beta actin
  • CBh CMV early enhancer
  • eF-1 a Elongation Factor 1a
  • SV40 simian virus 40 early promoter
  • PGK human phosphoglycerate kinase 1
  • CMV cytomegalovirus immediate-early promoter
  • hACTB human (3-actin (hACTB)
  • synapsin myelin basic protein and JeT synthetic promoter.
  • the nucleic acid construct comprises a sequence as set forth in SEQ ID NO: 5, or a functional variant thereof.
  • Another aspect of the disclosure is a viral vector comprising the nucleic acid construct disclosed herein.
  • the viral vector is an Adeno-Associated Virus (AAV) vector or a derivative thereof.
  • AAV Adeno-Associated Virus
  • the AAV vector is selected from the group consisting of: AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9, or a derivative thereof.
  • the viral vector is a chimeric, shuffled or capsid modified derivative of AAV.
  • compositions 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.
  • the pharmaceutical composition is formulated for intraparenchymal, intravenous or intrathecal or any other systemic method of administration.
  • 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.
  • CCD creatine deficiency syndrome
  • the CCD is selected from Cerebral Creatine Deficiency Syndrome 2 (CCDS2), Cerebral Creatine Deficiency Syndrome 3 (CCDS2) and secondary creatine deficiencies.
  • the viral vector or pharmaceutical composition is formulated for systemic, peritoneal, intraparenchymal, intravenous and/or intrathecal injection.
  • 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.
  • CDD Creatine Deficiency Syndrome
  • 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).
  • CDD Creatine Deficiency Syndrome
  • 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.
  • CDD Creatine Deficiency Syndrome
  • the viral vector or pharmaceutical composition is formulated for systemic, peritoneal, intraparenchymal, intravenous and/or intrathecal injection.
  • 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.
  • CK creatine kinase
  • 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.
  • 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.
  • 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.
  • 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 for qPCR 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.
  • 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.
  • WT untransfected wild type
  • 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.
  • a low dose of 1.25 ug of plasmid DNA was used.
  • 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.
  • 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 ).
  • FIG 9 is preliminary data showing intracellular creatine and GAA posttransfection of knockout GAMT cells as determined by LC-MS/MS.
  • 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 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.
  • 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.
  • 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.
  • 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 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 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 Treatment of 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 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.
  • 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 ).
  • 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.
  • 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 A significant 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 ).
  • 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 ).
  • 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 ).
  • 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 ).
  • 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 ).
  • 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 ).
  • 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 ).
  • 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 ).
  • 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 ).
  • 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
  • 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 of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • nucleic acid construct comprising a nucleotide sequence encoding a GAMT protein, operably linked to a promoter and a transcription termination site.
  • 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.
  • delivery of such nucleic acid constructs via adeno- associated viral vectors results in increased creatine content in models of AGAT-D.
  • 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.
  • nucleic acid construct of the disclosure 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.
  • 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.
  • nucleic acid encodes a GAMT protein having an amino acid sequence as set forth in SEQ ID NO: 4 or a functional variant of any thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GTM glycine amidinotransferase
  • AGAT activity refers to a protein that is known to catalyze the transfer of the amidino group of L-arginine to glycine to generate guanidinoacetate.
  • 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.
  • GAA activity refers to a protein that is known to convert GAA into creatine by donating a methyl group from adenosylmethionine to GAA.
  • nucleic acid molecule and its derivatives, as used herein, are intended to include unmodified DNA or RNA or modified DNA or RNA.
  • the nucleic 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.
  • 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.
  • polynucleotide shall have a corresponding meaning.
  • operably linked 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.
  • 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.
  • the promoter is an inducible promoter. Examples of inducible promoters include, without limitation, a tetracycline response element (TRE) (e.g.
  • the promoter is a constitutive promoter.
  • 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 early enhancer (CAG) and EF1 -HTLV.
  • the promoter is a tissue- or cellspecific promoter.
  • the promoter is a synthetic promoter such as JeT.
  • transcription termination site refers generally to a polyadenylation signal (pA) that terminates transcription of messenger RNA (mRNA).
  • pA polyadenylation signal
  • mRNA messenger RNA
  • 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).
  • 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.
  • 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.
  • 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.
  • the functional variant may also comprise conservatively substituted amino acid sequences of the sequences disclosed herein.
  • 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.
  • sequence identity 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 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.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
  • PSI- BLAST can be used to perform an iterated search which detects distant relationships between molecules.
  • the default parameters of the respective programs e.g. of XBLAST and NBLAST
  • 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.
  • a PAM120 weight residue table 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 be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • 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.
  • anneal and hybridize refer to the ability of a nucleic acid to non-covalently interact with another nucleic acid through base-pairing.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the functional variant nucleic acid sequences comprise degenerate codon substitutions or codon-optimized nucleic acid sequences.
  • degenerate codon substitution 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.
  • codon- optimized refers to a variant nucleic acid molecule comprising one or more degenerate codon substitutions that reflect the codon usage bias of a particular organism.
  • 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.
  • the nucleic acid construct disclosed herein comprises a codon-optimized or degenerate nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct disclosed herein comprises a codon-optimized or degenerate nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct disclosed herein comprises a codon-optimized or degenerate nucleotide sequence of SEQ ID NO: 5.
  • 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.
  • 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.
  • 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.
  • nucleic acid molecule which contains a single gene or protein.
  • the nucleic acid molecule is a monocistronic nucleic acid molecule.
  • the monocistronic nucleic acid molecule encodes a GAMT protein.
  • the monocistronic nucleic acid molecule encodes a AGAT protein.
  • bicistronic refers to a nucleic acid molecule which contains two genes or proteins.
  • the nucleic acid molecule is a bicistronic nucleic acid molecule.
  • the bicistronic nucleic acid molecule encodes a GAMT protein and a AGAT protein.
  • the bicistronic nucleic acid molecule contains two promoters.
  • the bicistronic nucleic acid molecule contains one promoter.
  • two or more polypeptides encoded by a polynucleotide described herein can be separated by an intervening sequence encoding a linker polypeptide.
  • the linker is a cleavage-susceptible linker.
  • the polypeptides of interest are expressed as fusion proteins linked by a cleavage-susceptible linker polypeptide.
  • cleavage susceptible linker polypeptide(s) are expressed as fusion proteins linked by a cleavage-susceptible linker polypeptide.
  • the bicistronic nucleic acid molecule contains a linker.
  • the linker is P2A.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a frans-acting gene regulatory protein binds the enhancer in order to affect transcriptional activity of the associated promoter.
  • 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).
  • viral constructs may include sequences that facilitate incorporation of the nucleic acid into a viral particle and/or integration into the host genome.
  • the viral construct may include inverted terminal repeats (ITRs) for example from an AAV such as AAV9, or other viral sequences.
  • ITRs inverted terminal repeats
  • 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.
  • 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.
  • 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 peptide that 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.
  • 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.
  • the viral vector is an AAV vector or a derivative thereof.
  • the AAV vector is selected from the group consisting of AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, or a derivative thereof.
  • AAV derivative 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.
  • 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.
  • the AAV vector is a chimeric, shuffled or capsid modified derivative of AAV. and Kits
  • 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’s Pharmaceutical Sciences (2003 - 20 th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • pharmaceutically acceptable means compatible with the treatment of animals, in particular, humans.
  • 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.
  • an active compound or substance such as a nucleic acid construct or viral vector described herein
  • 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.
  • 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, 18 th Ed. 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.
  • 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.
  • 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 pharmaceutical compositions 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.
  • 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.
  • the secondary creatine deficiencies include chronic kidney disease, chronic liver disease, cancer, muscle disorders, myalgic encephalomyelitis/chronic fatigue syndrome, and neurodegenerative conditions.
  • Muscle disorders include, but are not limited to, for example fibromyalgia.
  • Neurodegenerative disorders include, but are not limited to, Parkinson’s disease and Alzheimer’s disease.
  • 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.
  • 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.
  • 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.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • 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.
  • 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.
  • 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.
  • CCDS2 Cerebral Creatine Deficiency Syndrome 2
  • GAMT-D guanidinoacetate methyltransferase deficiency
  • 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.
  • 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.
  • CCDS3 Cerebral Creatine Deficiency Syndrome 3
  • AGAT-D L-arginine:glycine amidinotransferase deficiency
  • GAAA guanidinoacetic acid
  • 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).
  • CCDS3 Cerebral Creatine Deficiency Syndrome 3
  • CCDS2 Cerebral Creatine Deficiency Syndrome 2
  • 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.
  • 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.
  • treating 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.
  • 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.
  • “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.
  • prevention 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.
  • the term “subject” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.
  • the term “subject” includes mammals that have been diagnosed with a CDDs, optionally the CCD is CCDS2 or CCDS3.
  • the subject is a mammal.
  • the subject is human.
  • the term “subject” refers to a human having, or suspected of having a CDDs, optionally the CCD is CCDS2 or CCDS3.
  • 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.
  • administered 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.
  • 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.
  • 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.
  • the nucleic acid constructs or viral vectors described herein may be administered by parenteral administration and the pharmaceutical compositions formulated accordingly.
  • 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 - 20 th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • 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.
  • Gl gastrointestinal
  • 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.
  • 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.
  • 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.
  • an effective amount or “therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • 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.
  • 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.
  • 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.
  • 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 is required.
  • 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.
  • 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).
  • IMDM Iscove Modified Dulbecco Medium
  • FBS dialyzed fetal bovine serum
  • 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.
  • 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).
  • IMDM Iscove Modified Dulbecco Medium
  • FBS dialyzed fetal bovine serum
  • 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
  • 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 folding 32 .
  • ITR inverted terminal repeats
  • 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.
  • kit protocol QIAprep Spin Miniprep Kit, Qiagen
  • 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 folding 32 . Additionally, the GATM-GAMT vector contained a peptide 2A sequence (P2A), derived from a picornavirus, between the GATM and GAMT sequences.
  • P2A peptide 2A sequence
  • 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 in transfection. 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).
  • 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.
  • 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 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’-
  • 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 2.5x10 11 vector genomes (vg) per mouse.
  • the syringe was inserted 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNALater Solution Invitrogen
  • Quantitative Polymerase Chain Reaction [00166] 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.
  • 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.
  • 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 practices 37 .
  • LaminB2 (Lrnnb2) GGACCCAAGGACTACCTCAAGGG AGGGCACCTCCATCTCGGAAAC
  • Ubiquitin-C (Ubc) GCCCAGTGTTACCACCAAGA CCCATCACACCCAAGAACA
  • 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.
  • 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.
  • RIPA 1X radioimmunoprecipitation assay
  • Protein concentration was determined using the Pierce BCA Protein Assay Kit following the manufacturer protocol (Thermo Fisher, 23225). [00170] 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 ProteinTM KaleidoscopeTM 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).
  • the secondary antibody Goat anti-rabbit IgG, HRP, A27036
  • 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)
  • RIPA 1X radioimmunoprecipitation assay
  • PMSF phenylmethylsulphonyl fluoride
  • 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.
  • the secondary antibody Goat anti-rabbit IgG, HRP, A27036
  • Example 2 An In vitro study showed restored GAMT expression and intracellular creatine content in a treated cellular model of GAMT-D.
  • GAMT protein expression is restored following transfections of cellular models of GAMT-D with plasmids carrying the designed GAMT construct.
  • 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’ AACq values 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.
  • 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 and to 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).
  • 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.
  • 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 mice 40 .
  • 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 gene delivery 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.
  • Example 4 An in vitro study showed increased intracellular creatine content in a treated cellular model of AG AT-D and GAMT-D.
  • 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).
  • a bicistronic plasmid demonstrated creatine production (FIG 15A) without GAA accumulation (FIG 15C).
  • both the monocistronic and bicistronic plasmids increased creatine (FIG 15B) and completely reduced GAA levels (FIG 15D).
  • Example 5 An in vivo dose study showed increased creatine levels in GAMT knockout mice in long-term
  • 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).
  • Example 6 An in vivo dose study shows reduced GAA accumulation in GAMT knockout mice in a dose dependent manner
  • GAA accumulation was also reduced in the lumbar and cervical spinal cord for all doses as compared to knockout controls (FIG 20).
  • GAA accumulation was significant reduced at only the high dose as compared to untreated knockout controls (FIG 22A).
  • FIGG 22B, C no significant differences in GAA accumulation were observed (FIG 22B, C).
  • FIGG 22B it was noted that in the liver the GAA accumulation at the high dose was significantly lower than the medium dose
  • Example 7 An in vivo study shows improved muscular strength in mice treated with SCAAV9.GAMT
  • mice treated with the high dose demonstrated improved muscular strength on the mesh test as compared to untreated knockout controls at the 13-week endpoint.
  • the mice treated with the high dose were not significantly different than control heterozygous control mice (FIG 25A). Th same effect was seen in both male and female mice (FIGs 25B,C).
  • SEQ ID NO: 1 Optimized Human GAMT Coding Sequence
  • SEQ ID NO: 4 GAMT Protein Sequence (translation of SEQ ID Nos: 1 and 2)
  • SEQ ID NO: 5 Murine GAMT Coding Sequence
  • CTTCTA GAGGAGGAAGAGTGCTGGAGGTGGGATTCGGCATGGCTATTGCTGCCTCTA
  • SEQ ID NO: 6 Murine G AMT AAV Vector GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCG
  • SEQ ID NO: 9 JeT promoter + Kozak sequence
  • SEQ ID NO: 10 Poly A Region
  • SEQ ID NO: 12 Optimized Human AGAT-P2A-GAMT protein sequence
  • SEQ ID NO: 15 Optimized Human AGAT (G T ) Coding Sequence
  • SEQ ID NO: 16 Optimized Human AGAT Protein Sequence
  • SEQ ID NO: 17 Optimized Human AGAT -P2A- GAMT full viral vector sequence
  • SEQ ID NO: 18 Optimized Human AGAT (GATM) Full Viral Vector Sequence
  • Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.
  • Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.
  • Mcguire DM Gross MD
  • Van Pilsum JF Van Pilsum JF
  • Towle HC Repression of rat kidney L- arginine:glycine amidinotransferase synthesis by Cr at a pretranslational level. J Biol Chem 259: 12034-12038, 1984.
  • Ostojic SM Diagnostic and Pharmacological Potency of Creatine in Post-Viral Fatigue Syndrome. Nutrients. 2021 Feb 4;13(2):503.

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Abstract

La présente invention concerne des constructions d'acides nucléiques pour l'expression de la guanidinoacétate méthyltransférase (GAMT) et/ou l'expression de la L-arginine:glycine amidinotransférase (AGAT), et des vecteurs viraux contenant lesdites constructions utiles pour le traitement de syndromes de déficit en créatine (CDD). L'invention concerne également des méthodes et des utilisations des vecteurs de l'invention pour le traitement de CCD.
PCT/CA2023/051522 2022-11-14 2023-11-14 Compositions et méthodes de traitement de déficits en créatine Ceased WO2024103166A1 (fr)

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Citations (2)

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US20190241633A1 (en) * 2016-05-04 2019-08-08 Curevac Ag Rna encoding a therapeutic protein
CA3132840A1 (fr) * 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique humaine 2 et procedes de regulation accordable

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US20190241633A1 (en) * 2016-05-04 2019-08-08 Curevac Ag Rna encoding a therapeutic protein
CA3132840A1 (fr) * 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique humaine 2 et procedes de regulation accordable

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Title
BAKER STEVEN ANDREW; GAJERA CHANDRESH R.; WAWRO ADAM M.; CORCES M. RYAN; MONTINE THOMAS J.: "GATM and GAMT synthesize creatine locally throughout the mammalian body and within oligodendrocytes of the brain", BRAIN RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 1770, 19 August 2021 (2021-08-19), NL , XP086767681, ISSN: 0006-8993, DOI: 10.1016/j.brainres.2021.147627 *
KHOJA SUHAIL, LAMBERT JENNA; NITZAHN MATTHEW; ELIAV ADAM; ZHANG YUCHEN; TAMBOLINE MIKAYLA; LE COLLEEN T.; NASSER ERAM; LI YUNFENG;: "Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities", MOLECULAR THERAPY- METHODS & CLINICAL DEVELOPMENT, NATURE PUBLISHING GROUP, GB, vol. 25, 1 June 2022 (2022-06-01), GB , pages 278 - 296, XP093173970, ISSN: 2329-0501, DOI: 10.1016/j.omtm.2022.03.015 *

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