WO2024052413A1 - Vecteurs de beta-hexosaminidase - Google Patents

Vecteurs de beta-hexosaminidase Download PDF

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WO2024052413A1
WO2024052413A1 PCT/EP2023/074474 EP2023074474W WO2024052413A1 WO 2024052413 A1 WO2024052413 A1 WO 2024052413A1 EP 2023074474 W EP2023074474 W EP 2023074474W WO 2024052413 A1 WO2024052413 A1 WO 2024052413A1
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beta
mouse
nucleotide sequence
seq
hexa
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Maria Fatima Bosch Tubert
Sara MARCÓ COSTA
Gemma ELIAS BOSCH
Miguel Garcia Martinez
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Universitat Autònoma De Barcelona
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • 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
    • C12N15/86—Viral vectors
    • 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
    • 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
    • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • 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; CARE OF BIRDS, FISHES, INSECTS; 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • 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

  • aspects and embodiments described herein relate to the field of medicine, in particular to beta-hexosaminidase gene therapy for the treatment of GM2 gangliosidoses in mammals, particularly in humans.
  • GM2 gangliosidosis is a group of three neurodegenerative lysosomal storage diseases (LSD) with an autosomal recessive inheritance caused by beta-hexosaminidase deficiency (Okada & O’Brien, 1969).
  • This enzyme is a glycoprotein synthesized in the ER lumen, processed in the Golgi, and transported via the mannose-6- phosphate receptor to the lysosome (Sun et al, 2021).
  • Beta-hexosaminidase enzyme is composed of two subunits, alpha and beta, in which dimer formation is required for catalytic activity since both subunits possess an active site (Sun et al, 2021).
  • subunits alpha and beta are encoded by HEXA and HEXB genes, respectively.
  • HEXA and HEXB genes There are three isoforms of this enzyme: hexosaminidase A (HexA), a heterodimer composed of alpha and beta subunits; hexosaminidase B (HexB), a homodimer formed by two beta subunits; and hexosaminidase S (HexS), a homodimer composed of two alpha subunits (Cachon-Gonzalez et al, 2018).
  • HexA hexosaminidase A
  • HexB hexosaminidase B
  • HexS hexosaminidase S
  • Gangliosides are a group of glycosphingolipids composed of a ceramide linked to a glycan with at least one sialic acid (Schnaar, 2019). They are mainly located in caveolae-rich microdomains of the plasma membrane (Yu et al, 2011).
  • Gangliosides are responsible for several pivotal biological functions for the correct functioning of the central nervous system (CNS), such as membrane organization, neuronal differentiation, cell adhesion, cell-cell recognition, signal transduction, inflammation, and neurite outgrowth, among others (Schnaar, 2010; Zeller & Marchase, 1992; Lopez & Baez, 2018; Sonnino et al, 2018; Rubovitch et al, 2017; Regina Todeschini & Hakomori, 2008). About 5% of all brain gangliosides are GM2 gangliosides, a specific type of ganglioside (Schnaar, 2019).
  • GM2 gangliosides In association with the GM2 activator protein, GM2 gangliosides interact exclusively with the beta-hexosaminidase HexA isoform to remove the terminal N-acetylgalactosamine residue that allows GM2 ganglioside degradation (Cachon-Gonzalez et al, 2018; Sandhoff & Harzer, 2013). In Sandhoff and Tay-Sachs diseases, the beta-hexosaminidase deficiency leads to an impairment of the catabolism of GM2 ganglioside which, in turn, results in its accumulation in the endo-lysosomal compartment.
  • both Tay-Sachs and Sandhoff disease are severe, progressive neurodegenerative disorders with clinically indistinguishable phenotypes, but for subtle visceral (organomegalies) and skeletal features in Sandhoff patients (Jain et al, 2010; Venugopalan & Joshi, 2002).
  • the main characteristics are developmental regression, neurological deterioration, motor deficits such as progressive weakness leading to hypotonia, ataxia, spasticity, seizures and vision deterioration or blindness with macular cherry-red spot (Masingue et al, 2020).
  • GM2 gangliosidoses There is a broad spectrum of clinical presentations and severity in GM2 gangliosidoses mainly due to the residual enzyme activity (Sun et al, 2021 ; Cachon-Gonzalez et al, 2018). Based on the time of onset, the GM2 gangliosidoses are divided into three clinical subtypes: infantile, juvenile, and adult forms (Cachon-Gonzalez et al, 2018). In general, the later the disease occurs, the more slowly it progresses.
  • the severe infantile form begins in the first year of life with rapidly progressive diffuse neurological deterioration and a premature death within the first 5 years of life (Cachon-Gonzalez et al, 2018; Sun et al, 2021 ; Leal et al, 2020).
  • juvenile form has an onset between 2 and 10 years, while adult type show an onset after age 10.
  • Both the juvenile and adult form are more slowly progressive neurological disorders in which the clinical manifestations depend on which parts of the central nervous system are affected.
  • These patients usually die around the second decade of life or, in some cases of adult chronic forms, can survive until 60-80 years of age (Leal et al, 2020; Cachon-Gonzalez et al, 2018; Sun et al, 2021).
  • the diagnosis for these diseases begins with recognition of the clinical signs and symptoms, followed by the measurement of beta-hexosaminidase activity, the gold-standard method for diagnosis (Zhang et al, 2019; Hall et al, 2014; Lowden et al, 1973). This diagnosis could be further confirmed by a DNA-based test to identify the underlying mutation (Leal et al, 2020).
  • mice, cats, sheep, rabbits and flamingos mimic some of the biochemical and physiological characteristics of GM2 gangliosidoses (Lawson & Martin, 2016; Zeng et al, 2008; Sango et al, 1995; Phaneuf et al, 1996; Torres et al, 2010; Rahman et al, 2012; Sanders et al, 2013; Seyrantepe et al, 2018).
  • AAV adeno-associated viral vectors
  • beta-hexosaminidase isoform A (HexA) is an alpha beta (a[3) heterodimeric protein
  • efficient expression of both subunits in the same cell is important to achieve functional protein and a good therapeutic response.
  • a single AAV vector comprising both subunits may have an advantage over two-vector approaches, because an advantageous 1 :1 ratio of genes encoding the alpha and beta subunits is obtained automatically in transduced cells.
  • Single AAV vectors comprising both subunits also allow decreasing vector dose (because no double transduction of the same cell is required), which in turn results in reduced risk of capsid-triggered immunity or other toxicities.
  • IRES sequence the most used element to generate bicistronic vectors, which is an internal entry site of the ribosome to allow production of two proteins from a single mRNA (Arfi et al, 2005; Batista et al, 2010);
  • bidirectional promoter another alternative used to drive expression of two genes (HEXA and HEXB) from a single construct (the direction of expression comes from the center of the vector genome toward the inverted terminal repeats (ITR) in opposite directions) (Lahey et al, 2020); and (iii) P2A element, a self-cleaving linker to produce alpha and beta subunits from a single protein where the final product has, in the end, some amino acid residues added to the C terminal of beta subunit and a single proline in the N terminal of alpha subunit (Ornaghi etal, 2020; Woodley etal, 2019; Shaimardanova etal, 2022).
  • HexM a hybrid beta-hexosaminidase subunit which is a new variant of the human HexA alpha-subunit, incorporating critical sequences from the beta-subunit that produce a stable homodimer and promote functional interactions with the GM2 activator protein and GM2 gangliosides
  • Taluthil-Melethil et al, 2016b Tropak et al, 2016; Osmon et al, 2016; Karumuthil-Melethil et al, 2016a; Ou et al, 2020; Kot et al, 2021).
  • the present inventors have developed an improved gene therapy strategy based on a single vector encoding both the alpha and beta subunit of beta-hexosaminidase for treatment of GM2 gangliosidoses, including Tay-Sachs and Sandhoff disease.
  • the alpha and beta subunit of beta-hexosaminidase are covalently linked.
  • the gene constructs and vectors as described herein can obtain a robust and wide-spread increase in beta-hexosaminidase expression and activity in the brain, liver, and serum, and in HEK-293 cells (Examples 3, 4, 6, 7, 8, and 9).
  • this disclosure relates to a gene construct for expressing a covalently linked alpha beta (a[3) dimer of beta-hexosaminidase comprising: a. a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase; b. a nucleotide sequence encoding a peptide linker; and c. a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • a gene construct according to this disclosure is such that the nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase is positioned upstream of the nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • a gene construct according to this disclosure is such that the peptide linker is a flexible peptide linker. In some embodiments, a gene construct according to this disclosure is such that the peptide linker is a non-cleavable peptide. In some embodiments, a gene construct according to this disclosure is such that the peptide linker is not a self-cleavable peptide. In some embodiments, a gene construct according to this disclosure is such that at least 30% of the amino acid residues of the peptide linker are glycine residues.
  • a gene construct according to this disclosure is such that the nucleotide sequence encoding the peptide linker is selected from the group consisting of: a. a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that has at least 70% sequence identity with the amino acid sequence of SEQ ID NOs: 11 , 12 or 13; b. a nucleotide sequence comprising a sequence that has at least 70% sequence identity with the nucleotide sequence of SEQ ID NOs: 14, 15 or 16; and c. a nucleotide sequence which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code.
  • a gene construct according to this disclosure is such that the gene construct further comprises a promoter, preferably wherein said promotor is a constitutive promoter, more preferably a CBA promoter or a derivative thereof, even more preferably a Cbh promoter.
  • the gene construct is flanked by adeno-associated viral ITRs, preferably AAV2 ITRs.
  • a gene construct according to this disclosure is such that the nucleotide sequence encoding an alpha subunit of a betahexosaminidase and/or the nucleotide sequence encoding a beta subunit of a beta-hexosaminidase are optimized, for example codon-optimized, preferably for expression in a human cell.
  • a gene construct according to this disclosure is such that the nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase is selected from the group consisting of: a.
  • nucleotide sequence encoding a polypeptide comprising an amino acid sequence that has at least 70% sequence identity with the amino acid sequence of SEQ ID NOs: 1 or 2; b. a nucleotide sequence that has at least 70% sequence identity with the nucleotide sequence of SEQ ID NOs: 3, 4, 5, 64, 66, or 68; and c. a nucleotide sequence which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code. and/or wherein the nucleotide sequence encoding a beta subunit of a beta-hexosaminidase is selected from the group consisting of: a.
  • nucleotide sequence encoding a polypeptide comprising an amino acid sequence that has at least 70% sequence identity with the amino acid sequence of SEQ ID NOs: 6 or 7; b. a nucleotide sequence that has at least 70% sequence identity with the nucleotide sequence of SEQ ID NOs: 8, 9, 10, 65, 67, or 69; and c. a nucleotide sequence which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code.
  • Another aspect of the disclosure relates to an expression vector comprising a gene construct according to this disclosure.
  • an expression vector according to this disclosure is such that the expression vector is a viral vector, preferably an adeno-associated viral vector, more preferably an adeno-associated viral vector of serotype 1 , 2, BR1 , rhS, rh1O, PHP.B, TT or 9, most preferably an adeno-associated viral vector of serotype 9.
  • compositions comprising a gene construct and/or an expression vector according to this disclosure, optionally further comprising one or more pharmaceutically acceptable ingredients, for example selected from the group consisting of excipients, vehicles, carriers, and diluents.
  • a gene construct for use according to this disclosure, an expression vector for use according to this disclosure, or a pharmaceutical composition for use according to this disclosure is for use in the treatment of GM2 gangliosidoses, preferably wherein the GM2 gangliosidosis is selected from the group consisting of Sandhoff disease and Tay-Sachs disease.
  • a gene construct for use according to this disclosure, an expression vector for use according to this disclosure, or a pharmaceutical composition for use according to this disclosure is such that the gene construct, expression vector or pharmaceutical composition is administered by intra-CSF administration.
  • Described herein are gene constructs comprising: a. a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase; b. a nucleotide sequence encoding a peptide linker; and c. a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • elements a (a nucleotide sequence encoding an alpha subunit of a betahexosaminidase), b (a nucleotide sequence encoding a peptide linker) and c (a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase) are operably linked.
  • a description of “operably linked’’ as used herein is provided in the section “general information’’.
  • Such gene constructs are capable of expressing a covalently linked alpha beta (ap) dimer of betahexosaminidase, i.e., a fusion protein comprising an alpha subunit of a beta-hexosaminidase, a peptide linker, and a beta subunit of a beta-hexosaminidase.
  • a gene construct for expressing a covalently linked alpha beta (a ) dimer of beta-hexosaminidase comprising: a. a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase; b. a nucleotide sequence encoding a peptide linker; and c. a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • a gene construct for expressing a fusion protein comprising: a. a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase; b. a nucleotide sequence encoding a peptide linker; and c. a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • the fusion protein is then understood to comprise an alpha subunit of a beta-hexosaminidase, a peptide linker, and a beta subunit of a beta-hexosaminidase.
  • the HEXA gene (OMIM:606869; NCBI Gene ID: 3073) encodes an alpha subunit of a betahexosaminidase
  • the HEXB gene (OMIM:606873; NCBI Gene ID: 3074) encodes a beta subunit of a betahexosaminidase
  • the Hexa gene (NCBI Gene ID: 15211) encodes an alpha subunit of a betahexosaminidase
  • the Hexb gene (NCBI Gene ID: 15212) encodes a beta subunit of a betahexosaminidase.
  • Homologous genes exist in other mammalian species and can be readily identified by the skilled person using sequence databases that are commonly available in the art.
  • the skilled person also understands that genes encoding an alpha subunit of a beta-hexosaminidase and genes encoding a beta subunit of a beta-hexosaminidase, such as the HEXA, HEXB, Hexa and Hexb genes mentioned above, may give rise to different isoforms, such as different splice variants, at the mRNA and/or protein level. The number of different isoforms may vary depending on the organism. It is understood that nucleotide sequences encoding any isoforms may be suitable in the context of this disclosure.
  • a gene construct as described herein is for expression in a vertebrate, preferably a mammal (e.g., murines such as rat or mice, humans), more preferably a human.
  • a gene construct as described herein is for expression in a brain, preferably a mammalian (e.g., murine such as rat or mice, human) brain, more preferably a human brain.
  • “for expression” or “suitable for expression” may mean that the gene construct includes one or more regulatory sequences, selected on the basis of the host cells such as brain cells of the vertebrate or mammal or human to be used for expression, which is operatively linked to the nucleotide sequence to be expressed.
  • host cells to be used for expression are human or murine (such as rat or mouse) cells, preferably human cells.
  • promoter 1 may be replaced by "transcription regulatory sequence” or “regulatory sequence”. Definitions of these terms are provided in the "general information" section.
  • a “gene construct” as described herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure.
  • a “gene construct” can also be called “expression cassette” or “expression construct” or the like and refers to a gene or a group of genes, including a gene that encodes a protein of interest, which is operably linked to a regulatory sequence that controls its expression.
  • the part of this application entitled “general information” comprises more detail as to a “gene construct”. "Operably linked” as used herein is further described in the part of this application entitled “general information”.
  • a gene construct as described herein is suitable for expression in the CNS preferably in the brain of a vertebrate, preferably of a mammal, more preferably of a human.
  • a gene construct as described herein is suitable for expression in a mammalian brain, more preferably in a human or murine (such as mouse) brain.
  • a gene construct as described herein is suitable for expression in a human brain.
  • the gene construct includes one or more regulatory sequences that are capable of directing expression of the nucleotide sequence to be expressed in said brain, such as in the cortex (which includes the frontal cortex, the parietal cortex, the temporal cortex and the occipital cortex), the thalamus, the hypothalamus, the subthalamus, the epithalamus, the hippocampus, the basal ganglia (which include the striatum, globus pallidus, ventral pallidum, substantia nigra and subthalamic nucleus), the amygdala, the brain stem (which includes the midbrain, pons and medulla oblongata), and/or cerebellum.
  • the cortex which includes the frontal cortex, the parietal cortex, the temporal cortex and the occipital cortex
  • the thalamus the hypothalamus, the subthalamus, the epithalamus, the hippocampus
  • the basal ganglia which include the
  • expression of the gene construct in the brain may mean expression of the gene construct in at least one, at least two, at least three or at least four or all brain regions selected from the group consisting of the cortex, the thalamus, the hypothalamus, the subthalamus, the epithalamus, the hippocampus, the basal ganglia, the amygdala, the brain stem, and the cerebellum.
  • a gene construct as described herein is suitable for expression in the liver.
  • a gene construct as described herein is suitable for expression in the CNS (preferably the brain) and suitable for expression in the liver. While the CNS (preferably the brain) is the main target for expression, expression in the liver may increase circulating levels of beta-hexosaminidase. Without wishing to be bound by theory, this may contribute to correction of somatic pathology observed in GM2 gangliosidoses, as shown in the Examples.
  • a gene construct according to this disclosure comprises a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase. Preferred features of such nucleotide sequences are described in this section.
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase as described herein may be derived from any gene or coding sequence encoding an alpha subunit of a beta-hexosaminidase.
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase as described herein is derived from, or comprises, the coding region (i.e., the CDS) of said gene.
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase as described herein is derived from a mammalian gene or coding sequence encoding an alpha subunit of a betahexosaminidase.
  • the HEXA gene (OMIM:606869; NCBI Gene ID: 3073) encodes an alpha subunit of a betahexosaminidase.
  • the Hexa gene (NCBI Gene ID: 15211) encodes an alpha subunit of a betahexosaminidase.
  • Homologous genes exist in other mammalian species and can be readily identified by the skilled person using sequence databases that are commonly available in the art. The skilled person also understands that genes encoding an alpha subunit of a beta-hexosaminidase, such as the HEXA and Hexa genes mentioned above, may give rise to different isoforms, such as different splice variants, at the mRNA and/or protein level. The number of different isoforms may vary depending on the organism. It is understood that nucleotide sequences encoding any isoforms may be suitable in the context of this disclosure.
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase as described herein is derived from a human or murine (such as mouse or rat) gene or coding sequence encoding an alpha subunit of a beta-hexosaminidase, preferably from a human HEXA gene or a mouse Hexa gene as described above.
  • a nucleotide sequence encoding an alpha subunit of a betahexosaminidase as described herein is derived from, or comprises, the coding region (i.e., the CDS) of a human or murine (such as mouse or rat) gene or coding sequence encoding an alpha subunit of a beta-hexosaminidase, preferably from a human HEXA gene or a mouse Hexa gene as described elsewhere.
  • the coding region i.e., the CDS
  • a human or murine such as mouse or rat
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase is optimized, for example codon-optimized, preferably for expression in a mammalian (e.g., murine such as rat or mouse, human) cell, more preferably for expression in a human cell.
  • a mammalian e.g., murine such as rat or mouse, human
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase as described herein is an optimized, preferably codon-optimized, sequence derived from a human or murine (such as mouse or rat) gene or coding sequence encoding an alpha subunit of a beta-hexosaminidase, preferably from a human HEXA gene or a mouse Hexa gene as described above.
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase as described herein is an optimized, preferably codon-optimized, sequence derived from, or comprising, the coding region (i.e., the CDS) of a human or murine (such as mouse or rat) gene or coding sequence encoding an alpha subunit of a beta-hexosaminidase, preferably from a human HEXA gene or a mouse Hexa gene as described elsewhere.
  • the coding region i.e., the CDS
  • a human or murine such as mouse or rat
  • a preferred nucleotide sequence encoding an alpha subunit of a betahexosaminidase encodes a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SEQ ID NO: 1.
  • SEQ ID NO: 1 represents the canonical amino acid sequence of the human alpha subunit of a beta-hexosaminidase.
  • a preferred nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase encodes a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SEQ ID NO: 2.
  • SEQ ID NO: 2 represents an amino acid sequence of the murine alpha subunit of a beta-hexosaminidase.
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase present in a gene construct according to the invention has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any sequence selected from the group consisting of SEQ ID NO: 3, 64, 66 and 68.
  • SEQ ID NO: 3 represents a nucleotide sequence encoding the human alpha subunit of a beta-hexosaminidase.
  • SEQ ID NOs: 64, 66, and 68 represent optimized sequences encoding the human alpha subunit of a beta-hexosaminidase.
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase present in a gene construct according to the invention has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 4-5.
  • SEQ ID NO: 4 represents a nucleotide sequence encoding the murine alpha subunit of a betahexosaminidase.
  • SEQ ID NO: 5 represents an optimized sequence encoding the murine alpha subunit of a betahexosaminidase.
  • nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase is selected from the group consisting of:
  • nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity with the amino acid sequence of any one of SEQ ID NOs: 1-2, preferably SEQ ID NO: 1 ;
  • nucleotide sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 3-5, 64, 66, or 68, preferably SEQ ID NO: 3, 64, 66 or 68, more preferably SEQ ID NO: 3, 64 or 68; (c) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code.
  • a gene construct as described herein wherein the nucleotide sequence encoding an alpha subunit of beta-hexosaminidase is optimized, for example, codon-optimized, preferably for expression in a human cell.
  • a nucleotide sequence encoding an alpha subunit of a betahexosaminidase present in a gene construct according to the invention is an optimized sequence, for example a codon optimized sequence, preferably an optimized human or murine sequence.
  • SEQ ID NO: 5 represents optimized nucleotide sequences encoding an alpha subunit of a beta-hexosaminidase with an amino acid sequence of SEQ ID NO: 2.
  • SEQ ID NOs: 64, 66, or 68 represent optimized nucleotide sequences encoding an alpha subunit of a beta-hexosaminidase with an amino acid sequence of SEQ ID NO: 1 .
  • a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase may further comprise a sequence encoding a signal peptide.
  • a preferred alpha subunit of a beta-hexosaminidase is one with a signal peptide, for example with its nascent signal peptide.
  • signal peptide is understood herein as a peptide operably linked (fused) in frame to the amino terminus of a polypeptide having biological activity and directing the polypeptide towards one or more cellular compartments, preferably to the lysosome.
  • the signal peptide is removed by signal peptidases once the polypeptide has reached its designated cellular compartment(s).
  • the signal peptide directs the alpha subunit of a beta-hexosaminidase (and thus the covalently linked alpha beta (a ) dimer) to a specific cellular compartment of a cell, preferably the lysosomal compartment.
  • a signal peptide as described herein comprises the sequence of SEQ ID NO: 57 or 58, or a sequence having up to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted, added, and/or substituted compared to SEQ ID NO: 57 or 58.
  • a signal peptide as described herein comprises the sequence SEQ ID NO: 57 or 58, or a sequence having 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 57 or 58.
  • a gene construct according to this disclosure comprises a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase. Preferred features of such nucleotide sequences are described in this section.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase as described herein may be derived from any gene or coding sequence encoding a beta subunit of a beta-hexosaminidase.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase as described herein is derived from, or comprises, the coding region (i.e., the CDS) of said gene.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase as described herein is derived from a mammalian gene or coding sequence encoding a beta subunit of a betahexosaminidase.
  • the HEXB gene (OMIM:606873; NCBI Gene ID: 3074) encodes a beta subunit of a betahexosaminidase.
  • the Hexb gene (NCBI Gene ID: 15212) encodes a beta subunit of a betahexosaminidase.
  • Homologous genes exist in other mammalian species and can be readily identified by the skilled person using sequence databases that are commonly available in the art. The skilled person also understands that genes encoding a beta subunit of a beta-hexosaminidase, such as the HEXB and Hexb genes mentioned above, may give rise to different isoforms, such as different splice variants, at the mRNA and/or protein level. The number of different isoforms may vary depending on the organism. It is understood that nucleotide sequences encoding any isoforms may be suitable in the context of this disclosure.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase as described herein is derived from a human or murine (such as mouse or rat) gene or coding sequence encoding a beta subunit of a beta-hexosaminidase, preferably a human HEXB gene or a mouse Hexb gene as described elsewhere.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase as described herein is derived from, or comprises, the coding region (i.e., the CDS) of a human or murine (such as mouse or rat) gene or coding sequence encoding a beta subunit of a beta-hexosaminidase, preferably from a human HEXB gene or a mouse Hexb gene as described elsewhere.
  • the coding region i.e., the CDS
  • a human or murine such as mouse or rat
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase is optimized, for example codon-optimized, preferably for expression in a mammalian (e.g., murine such as rat or mouse, human) cell, more preferably for expression in a human cell.
  • a mammalian e.g., murine such as rat or mouse, human
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase as described herein is an optimized, preferably codon-optimized, sequence derived from a human or murine (such as mouse or rat) gene or coding sequence encoding a beta subunit of a beta-hexosaminidase, preferably from a human /-/EXB gene or a mouse Hexb gene as described above.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase as described herein is an optimized, preferably codon-optimized, sequence derived from, or comprising, the coding region (i.e., the CDS) of a human or murine (such as mouse or rat) gene or coding sequence encoding a beta subunit of a beta-hexosaminidase, preferably from a human HEXB gene or a mouse Hexb gene as described elsewhere.
  • the coding region i.e., the CDS
  • a human or murine such as mouse or rat
  • a preferred nucleotide sequence encoding a beta subunit of a betahexosaminidase encodes a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SEQ ID NO: 6.
  • SEQ ID NO: 6 represents the canonical amino acid sequence of the human beta subunit of a beta-hexosaminidase.
  • a preferred nucleotide sequence encoding a beta subunit of a beta-hexosaminidase encodes a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SEQ ID NO: 7.
  • SEQ ID NO: 7 represents an amino acid sequence of the murine beta subunit of a beta-hexosaminidase.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase present in a gene construct according to the invention has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any sequence selected from the group consisting of SEQ ID NO: 8, 65, 67, and 69.
  • SEQ ID NO: 8 represents a nucleotide sequence encoding the human beta subunit of a beta-hexosaminidase.
  • SEQ ID NOs: 65, 67, and 69 represent an optimized sequence encoding the human beta subunit of beta-hexosaminidase.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase present in a gene construct according to the invention has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 9-10.
  • SEQ ID NO: 9 represents a nucleotide sequence encoding the murine beta subunit of a betahexosaminidase.
  • SEQ ID NO: 10 represents an optimized sequence encoding the murine beta subunit of a beta-hexosaminidase.
  • nucleotide sequence encoding a beta subunit of a beta-hexosaminidase is selected from the group consisting of:
  • nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity with the amino acid sequence of any one of SEQ ID NOs: 6-7, preferably SEQ ID NO: 6;
  • nucleotide sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 8-10, 65, 67, or 69, preferably SEQ ID NO: 8, 65, 67 or 69, more preferably 8, 65 or 69;
  • a gene construct as described herein wherein the nucleotide sequence encoding a beta subunit of beta-hexosaminidase is optimized, for example, codon-optimized, preferably for expression in a human cell.
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase present in a gene construct according to the invention is an optimized sequence, for example a codon-optimized sequence, preferably an optimized human or murine sequence.
  • SEQ ID NO: 10 represents optimized nucleotide sequences encoding a beta subunit of a beta-hexosaminidase with an amino acid sequence of SEQ ID NO: 7.
  • it has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NOs: 65, 67, or 69.
  • SEQ ID NOs: 65, 67, or 69 represent optimized nucleotide sequences encoding a beta subunit of a beta-hexosaminidase with an amino acid sequence of SEQ ID NO: 6.
  • sequence optimization and “codon optimization’’ has been provided under the section entitled
  • a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase does not comprise a sequence encoding a signal peptide.
  • a preferred beta subunit of a beta-hexosaminidase is one without a signal peptide, for example without its nascent signal peptide.
  • a gene construct according to this disclosure comprises a nucleotide sequence encoding a peptide linker. Preferred features of such peptide linkers are described in this section.
  • the length of the peptide linker as described herein is not crucial and is not particularly limited.
  • the peptide linker has a length of 6 to 50 amino acids, preferably 8 to 44 amino acids, more preferably 10 to 38 amino acids, even more preferably 11 to 35 amino acids.
  • the peptide linker has a minimum length of 6, 7, 8, 9, 10 or 11 amino acids and/or a maximum length of 50, 47, 44, 41 , 38 or 35 amino acids.
  • the peptide linker has a length of 10-14, preferably 12 amino acids. In some embodiments, the peptide linker has a length of 18-22, preferably 20 amino acids. In some embodiments, the peptide linker has a length of 31-35, preferably 33 amino acids.
  • a peptide linker as described herein is a flexible peptide linker.
  • flexible linkers are generally composed of small, non-polar (e.g., glycine) or polar (e.g., serine and threonine) amino acids, allowing them to provide flexibility and mobility of the connecting functional domains (as reviewed in Chen et al., Adv Drug Deliv Rev 2013; 65(10): 1357-1369). The flexibility thus allows for achieving the proper conformation of each subunit as required for obtaining functional fusion proteins.
  • Flexible peptide linkers such as the GS-rich linkers, are used in the synthesis of fusion proteins or peptide conjugates that are not intended to be cleaved by cellular machinery in vivo. These stable linkers covalently join functional domains together, allowing them to function as a single molecule throughout in vivo cellular processes. Therefore, in this disclosure, flexible peptide linkers are understood to be, and may be referred to as, stable flexible peptide linkers.
  • Cleavable linkers or "in vivo" cleavable linkers, such as the 2A sequences, on the other hand, are used to release separate free functional domains in vivo.
  • 2A peptides are widely used in molecular biology and genetic engineering to express multiple proteins from a single transcript. Furthermore, the 2A peptides contain functional sequences that induce efficient ribosome skipping, allowing the coordinated production of distinct proteins from a single mRNA transcript. Although a small portion of the two units could be covalently linked, the lack of flexibility of cleavable linkers hinders the proper folding of the entire protein.
  • a peptide linker as described herein preferably a flexible peptide linker as described herein, is such that at least 5%, 10%, 15%, 20%, 25% or 30% of the amino acid residues of the peptide linker are glycine residues.
  • a peptide linker as described herein, preferably a flexible peptide linker as described herein is such that at least 30% of the amino acid residues of the peptide linker are glycine residues.
  • At least one, two, three or four amino acid residues of the peptide linker, preferably of the flexible peptide linker are glycine residues.
  • a preferred number of glycine residues of the peptide linker, preferably of the flexible peptide linker, is at least four.
  • a peptide linker as described herein, preferably a flexible peptide linker as described herein is such that at least 5%, 10%, 15% or 20% of the amino acid residues of the peptide linker are serine residues.
  • a peptide linker as described herein preferably a flexible peptide linker as described herein, is such that at least 20% of the amino acid residues of the peptide linker are serine residues. In a further embodiment, at least one, two or three amino acid residues of the peptide linker, preferably of the flexible peptide linker, are serine residues. A preferred number of serine residues of the peptide linker, preferably of the flexible peptide linker, is at least three.
  • a peptide linker as described herein preferably a flexible peptide linker as described herein, is such that at least 5%, 10%, 15%, 20%, 25% or 30% of the amino acid residues of the peptide linker are glycine residues and wherein at least 5%, 10%, 15% or 20% of the amino acid residues of the peptide linker are serine residues.
  • a peptide linker as described herein, preferably a flexible peptide linker as described herein is such that at least 30% of the amino acid residues of the peptide linker are glycine residues and at least 20% of the amino acid residues of the peptide linker are serine residues.
  • At least one, two, three or four amino acid residues of the peptide linker are glycine residues and at least one, two or three amino acid residues of the peptide linker, preferably of the peptide linker, are serine residues.
  • a peptide linker as described herein preferably a flexible peptide linker as described herein, is such that at least 10%, 20%, 30%, 40% or 50% of the amino acid residues of the peptide linker are glycine or serine residues.
  • a peptide linker as described herein, preferably a flexible peptide linker as described herein is such that at least 50% of the amino acid residues of the peptide linker are glycine or serine residues.
  • At least one, two, three, four, five, six, seven, eight, nine or ten amino acid residues of the peptide linker, preferably of the flexible peptide linker, are glycine or serine residues.
  • n is the number of repeats which is preferably 2-7, more preferably 3- 6, even more preferably 4 ((GGGGS)4; SEQ ID NO: 11);
  • GSAGSAAGSGEF SEQ ID NO: 13
  • linkers derived thereof for example linkers having up to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted, added, and/or substituted.
  • flexible peptide linkers suitable for gene constructs of this disclosure include:
  • n is the number of repeats which is preferably 2-7, more preferably 3- 6, even more preferably 4 ((GGGGS)4; SEQ ID NO: 11);
  • linkers derived thereof for example linkers having up to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted, added, and/or substituted.
  • linkers suitable for gene constructs of this disclosure include:
  • GSAGSAAGSGEF SEQ ID NO: 13
  • linkers derived thereof for example linkers having up to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted, added, and/or substituted.
  • a peptide linker as described herein preferably a flexible peptide linker, is free of repetitive sequences. Without wishing to be bound by theory, this kind of linkers may be capable of avoiding undesired recombination.
  • a peptide linker as described herein is a non-cleavable peptide linker, preferably an in vivo non-cleavable peptide linker.
  • a non-cleavable peptide linker refers to a peptide linker which does not contain a protease recognition site or a protease sensitive sequence, which is not sensitive to reductive cleavage, and which is not prone to ribosome skipping.
  • an in vivo non- cleavable peptide linker refers to a peptide linker which is non-cleavable under in vivo conditions, preferably in the context of a cell such as a brain cell (including any specific brain cell type described herein). Accordingly, an in vivo non-cleavable peptide linker may be a peptide linker which is not cleavable by the endogenous machinery of a cell, e.g., by endogenous enzyme cleavage or ribosome skipping.
  • a peptide linker as described herein is not a cleavable peptide linker, preferably not an in vivo cleavable peptide linker.
  • a cleavable peptide linker refers to a peptide linker which contains a protease recognition site ora protease sensitive sequence, which is sensitive to reductive cleavage, or which is prone to ribosome skipping.
  • an in vivo cleavable peptide linker refers to a peptide linker which is cleavable under in vivo conditions, preferably in the context of a cell such as a brain cell (including any specific brain cell type described herein). Accordingly, an in vivo cleavable peptide linker may be a peptide linker which is cleavable by the endogenous machinery of a cell, e.g., by endogenous enzyme cleavage or ribosome skipping.
  • a gene construct as described herein wherein the peptide linker is not a self-cleaving peptide.
  • a peptide linker as described herein is not a P2A self-cleaving peptide, e.g., a P2A self-cleaving peptide as described in Woodley et al. Mol Ther 2019; 12:47-57.
  • a description of “self-cleaving peptide’’ has been provided under the section entitled “general information’’.
  • a preferred nucleotide sequence encoding a peptide linker encodes a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with any one of SEQ ID NOs: 11-13.
  • SEQ ID NO: 11 represents an amino acid sequence of a 20 aa long glycine-rich (GGGGS) 4 flexible peptide linker.
  • SEQ ID NO: 12 represents an amino acid sequence of a 20 aa long glycine-rich (GGGG
  • SGGSSGGSSGSETPGTSESATPESSGGSSGGSS represents an amino acid sequence of a 33 aa long flexible peptide linker (Anzalone et al, 2019).
  • SEQ ID NO: 13 represents an amino acid sequence of a 12 aa long flexible peptide linker (Waldo et al, 1999).
  • a nucleotide sequence encoding a peptide linker present in a gene construct according to the invention has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%,
  • nucleotide sequence encoding a peptide linker is selected from the group consisting of:
  • nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity with the amino acid sequence of any one of SEQ ID NOs: 11-13;
  • nucleotide sequence comprising a sequence that has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 14-16;
  • a gene construct as described herein comprises a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • Such a gene construct is capable of expressing a covalently linked alpha beta (ap) dimer of betahexosaminidase, i.e., a fusion protein comprising an alpha subunit of a beta-hexosaminidase, a peptide linker, and a beta subunit of a beta-hexosaminidase.
  • a gene construct for expressing a fusion protein comprising: a. a nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase; b. a nucleotide sequence encoding a peptide linker; and c. a nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • the fusion protein is then understood to comprise an alpha subunit of a beta-hexosaminidase, a peptide linker, and a beta subunit of a beta-hexosaminidase, all of which are described elsewhere herein.
  • fusion proteins that can be encoded by any of the gene constructs described herein, are also an aspect of this disclosure.
  • a preferred nucleotide sequence encoding a covalently linked alpha beta (a ) dimer of beta-hexosaminidase encodes a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with any one of SEQ ID NOs: 17-19.
  • SEQ ID NO: 17 represents an amino acid sequence of a murine covalently linked (L1 linker) alpha beta (ap) dimer of betahexosaminidase.
  • SEQ ID NO: 18 represents an amino acid sequence of a murine covalently linked (L2 linker) alpha beta (ap) dimer of beta-hexosaminidase.
  • SEQ ID NO: 19 represents an amino acid sequence of a murine covalently linked (L3 linker) alpha beta (ap) dimer of beta-hexosaminidase.
  • a nucleotide sequence encoding a covalently linked alpha beta (ap) dimer of betahexosaminidase present in a gene construct according to the invention has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 20-22.
  • SEQ ID NO: 20 represents a nucleotide sequence encoding a murine covalently linked (L1 linker) alpha beta (a[3) dimer of beta-hexosaminidase.
  • SEQ ID NO: 21 represents a nucleotide sequence encoding a murine covalently linked (L2 linker) alpha beta (a ) dimer of betahexosaminidase.
  • SEQ ID NO: 22 represents a nucleotide sequence encoding a murine covalently linked (L3 linker) alpha beta (ap) dimer of beta-hexosaminidase.
  • a preferred nucleotide sequence encoding a covalently linked alpha beta (ap) dimer of beta-hexosaminidase encodes a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SEQ ID NO: 70.
  • SEQ ID NO: 70 represents an amino acid sequence of a human covalently linked (L1 linker) alpha beta (ap) dimer of beta-hexosaminidase.
  • a nucleotide sequence encoding a covalently linked alpha beta (ap) dimer of betahexosaminidase present in a gene construct according to the invention has at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 71-74.
  • SEQ ID NO: 71 represents a nucleotide sequence encoding a human covalently linked (L1 linker) alpha beta (ap) dimer of beta-hexosaminidase.
  • SEQ ID NOs: 72-74 represent optimized nucleotide sequences encoding a human covalently linked (L1 linker) alpha beta (ap) dimer of betahexosaminidase.
  • a gene construct according to this disclosure is such that expression of the gene construct, optionally expression of the gene construct in a cell (preferably a brain cell), results in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of covalently linked alpha beta (ap) dimer of beta-hexosaminidase, i.e., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of fusion protein.
  • a gene construct according to this disclosure is such that expression of the gene construct, optionally expression of the gene construct in a cell (preferably a brain cell), does not result in a detectable level of covalently linked alpha beta (ap) dimer of beta-hexosaminidase, i.e., of fusion protein.
  • detection of the covalently linked alpha beta (ap) dimer, i.e., of the fusion protein may be performed by any suitable method known to the person skilled in the art, e.g., methods to measure expression as described in the section "general information", preferably by a Western blot assay, such as a Western blot assay as described in the examples section.
  • a gene construct as described herein wherein the nucleotide sequence encoding an alpha subunit of beta-hexosaminidase and/or the nucleotide sequence encoding a beta subunit of a beta-hexosaminidase are optimized, for example, codon-optimized, preferably for expression in a human cell.
  • the nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase is positioned upstream of the nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • ‘upstream’’ refers to a location within the gene construct which is toward the 5’ end of the polynucleotide from a specific reference point.
  • a gene construct as described herein wherein the nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase is positioned upstream of the nucleotide sequence encoding a peptide linker, and wherein the nucleotide sequence encoding the peptide linker is positioned upstream of the nucleotide sequence encoding a beta subunit of a beta-hexosaminidase.
  • a nucleotide sequence encoding a peptide linker is positioned between the nucleotide sequence encoding an alpha subunit of a beta-hexosaminidase and the nucleotide sequence encoding a beta subunit of a beta- hexosaminidase.
  • An alpha subunit of a beta-hexosaminidase and a beta subunit of a beta-hexosaminidase encoded by the nucleotide sequences described herein exert at least a detectable level of an activity of a beta-hexosaminidase as known to a person of skill in the art.
  • a covalently linked alpha beta (a[3) dimer of beta-hexosaminidase as described herein maintains beta-hexosaminidase function.
  • Betahexosaminidase catalyzes the hydrolysis of terminal N-acetyl-D-hexosamine residues in N-acetyl-p-D- hexosaminides. Means and methods to measure this activity are commonly known in the art, for example as described in the Examples section. In the context of this disclosure, an activity of a beta-hexosaminidase can also be to normalize GM2 ganglioside accumulation of GM2 gangliosidosis patients as described in more detail later herein.
  • An activity of a beta-hexosaminidase can also be to normalize lysosomal distension, lysosomal homeostasis, autophagy, myelinization and neuroinflammation in the CNS of GM2 gangliosidosis patients.
  • An activity of a beta-hexosaminidase can also be to normalize cholesterol in liver and GAG storage in several peripheral tissues and to normalize lysosomal homeostasis in the liver of GM2 gangliosidosis patients.
  • An activity of a beta-hexosaminidase can also be to improve general locomotor and exploratory activity, as well as motor coordination, mobility, and disease progression in GM2 gangliosidosis patients.
  • betahexosaminidase can also be to improve survival of GM2 gangliosidosis patients. These activities could be assessed by methods known to a person of skill in the art, for example by using enzymatic assays or behavioral test (such as the open field test, the righting reflex test, the mesh test, the hindlimb clasping test or the rotarod test).
  • the nucleotide sequence encoding a covalently linked alpha beta (a ) dimer of betahexosaminidase as described herein is operably linked to a promoter.
  • a gene construct as described herein further comprises a promoter.
  • the promoter is a constitutive promoter. A description of “promoter” has been provided under the section entitled “general information”.
  • a promoter as used herein encompasses derivatives of promoters and should exert at least an activity of a promoter as known to a person of skill in the art (especially when the promoter sequence is described as having a minimal identity percentage with a given SEQ ID NO).
  • a promoter described as having a minimal identity percentage with a given SEQ ID NO should control transcription of the nucleotide sequence to which it is operably linked as assessed in an assay known to a person of skill in the art.
  • such assay could involve measuring expression of the transgene. Expression may be assessed as described under the section entitled “general information” or as shown in the Examples.
  • a constitutive promoter as described herein is selected from the group consisting of a CAG promoter, a CMV promoter, a Cbh promoter, a mini-CMV promoter, a chicken beta-actin promoter (CBA), a rous-sarcoma-virus (RSV) promoter, an elongation factor 1 alpha (EF1 alpha) promoter, an early growth response factor-1 (Egr-1) promoter, an Eukaryotic Initiation Factor 4A (elF4A) promoter, a ferritin heavy chainencoding gene (FerH) promoter, a ferritin heavy light-encoding gene (FerL) promoter, a glyceraldehyde-3- phosphate dehydrogenase (GAPDH) promoter, a GRP78 promoter, a GRP94 promoter, a heat-shock protein 70 (hsp70) promoter, an ubiquitin B promoter, a SV40
  • Derivatives of promoters as described herein comprise promoters that have been mutated as to differentiate the directed expression of the transgenes operably linked to said promoters as compared to the non-mutated promoters, which can be increased or decreased, preferably increased.
  • Methods of mutating nucleotide sequences are known to the skilled person and can comprise any of introduction of single nucleotide polymorphisms, nucleotide insertions and nucleotide deletions.
  • CBA promoters and their derivatives are particularly useful for expression of gene constructs in the CNS.
  • promoters can also encompass promoters that have been shortened (by nucleotide deletions) or elongated (by nucleotide insertions) compared to their wild-type sequences, with shortened promoters being preferred.
  • the constitutive promoter is a chicken beta-actin (CBA) promoter or a derivative thereof.
  • a gene construct as described herein further comprises a promoter, wherein said promotor is a constitutive promoter, preferably a CBA promoter or a derivative thereof, more preferably a Cbh promoter.
  • a CBA promoter (for mammalian expression) comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 23, or a sequence having at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith.
  • the CBA promoter or a derivative thereof is a novel hybrid form of the CBA promoter (Cbh). Accordingly, in more preferred embodiments, the constitutive promoter is a Cbh promoter. In some embodiments, a Cbh promoter or a derivative thereof is suitable for promoting the expression of genes in mammals.
  • a (mammalian) Cbh promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 24.
  • Additional sequences may be present in a gene construct as described herein.
  • exemplary additional sequences suitable for gene constructs described herein include inverted terminal repeats (ITRs) and an SV40 polyadenylation (polyA) signal (SEQ ID NOs: 25-27).
  • ITRs inverted terminal repeats
  • polyA polyadenylation
  • “ITRs” is intended to encompass one 5’ITR and one 3’ITR, each being derived from the genome of an AAV.
  • Preferred ITRs are from AAV2 and are represented by SEQ ID NO: 25 (5’ ITR) and SEQ ID NO: 26 (3’ ITR).
  • SEQ ID NO: 25 5’ ITR
  • SEQ ID NO: 26 3’ ITR
  • a gene construct as described herein wherein the gene construct is flanked by adeno- associated viral ITRs.
  • said adeno-associated viral ITRs are AAV2 ITRs.
  • a gene construct as described herein is flanked by adeno-associated viral ITRs, preferably ITRs are AAV2 ITRs.
  • the AAV2 ITRs are represented by SEQ ID NO: 25 (5’ ITR) and SEQ ID NO: 26 (3’ ITR).
  • a gene as described herein which further comprises a polyA sequence.
  • said polyA sequence is a SV40 polyA sequence.
  • the SV40 polyA sequence is represented by SEQ ID NO: 27.
  • nucleotide sequences may be operably linked to the nucleotide sequence(s) encoding a covalently linked alpha beta (a ) dimer of beta-hexosaminidase, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like.
  • the additional sequences are operably linked to the nucleotide sequences described elsewhere.
  • any of the nucleotide sequences described herein may be operably linked with each other within the gene construct of the disclosure.
  • a gene construct of the disclosure comprises a Cbh promoter.
  • the gene construct further includes 5’ and 3’ flanks of inverted terminal repeats (ITRs) derived from the genome of an AAV, preferably from AAV2.
  • the gene construct further includes a polyA sequence, preferably SV40 polyA.
  • such gene construct has the nucleotide sequence of SEQ ID NOs:75-84, preferably SEQ ID NOs: 75-77, or 81-84, more preferably SEQ ID NOs: 81-84, even more preferably SEQ ID NOs: 82 or 84, or a sequence having at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith.
  • such gene construct has the nucleotide sequence of SEQ ID NOs: 81-84, preferably SEQ ID NOs: 82 or 84, except that the L1 linker is replaced with another peptide linker as described herein, for example the L2 or L3 linker as described herein.
  • the level of sequence identity or similarity as used herein is preferably 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.
  • Gene constructs described herein can be placed in expression vectors.
  • an expression vector comprising a gene construct as described in any of the preceding embodiments.
  • expression vector includes non-viral and viral vectors.
  • Suitable expression vectors may be selected from any genetic element which can facilitate transfer of genes or nucleic acids between cells, such as, but not limited to, a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc.
  • a suitable expression vector may also be a chemical vector, such as a lipid complex or naked DNA.
  • naked DNA refers to a nucleic acid molecule that is not contained within a viral particle, bacterial cell, or other encapsulating means that facilitates delivery of nucleic acid into the cytoplasm of the target cell.
  • a naked nucleic acid can be associated with standard means used in the art for facilitating its delivery of the nucleic acid to the target cell, for example to facilitate the transport of the nucleic acid through the alimentary canal, to protect the nucleic acid from stomach acid and/or nucleases, and/or serve to penetrate intestinal mucus.
  • the expression vector is a viral expression vector.
  • a description of “viral expression vector” or “viral vector” in short has been provided under the section entitled “general information”.
  • a viral vector may be a viral vector selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.
  • An adenoviral vector is also known as an adenovirus derived vector
  • an adeno-associated viral vector is also known as an adeno-associated virus derived vector
  • a retroviral vector is also known as a retrovirus derived vector
  • a lentiviral vector is also known as a lentivirus derived vector.
  • a preferred viral vector is an adeno-associated viral vector.
  • the expression vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.
  • the expression vector is an adeno-associated viral vector. A description of “adeno-associated viral vector” has been provided under the section entitled “general information”.
  • the viral vector is an adeno-associated vector (AAV) selected from the group consisting of AAV of serotype 1 (AAV1), AAV of serotype 2 (AAV2), AAV of serotype 3 (AAV3), AAV of serotype 4 (AAV4), AAV of serotype 5 (AAV5), AAV of serotype 6 (AAV6), AAV of serotype 7 (AAV7), AAV of serotype 8 (AAV8), AAV of serotype 9 (AAV9), AAV of serotype rh10 (AAVrhI O), AAV of serotype rh8 (AAVrh8), AAV of serotype Cb4 (AAVCb4), AAV of serotype rh74 (AAVrh74), AAV of serotype DJ (AAVDJ), AAV of serotype 2.5 (AAV2.5), AAV of serotype BR1 (AAV-BR1), AAV of serotype PHP.B (AAV) a
  • Serotypes AAV1 , AAV2, AAV-BR1 , AAV- PHP.B, AAV-PHP.eB, AAV-TT, AAVrh8, AAVrhIO and AAV9 are advantageous in the context of achieving brain expression of hexosaminidase.
  • the vector is an AAV of serotype 9, PHP.B, TT, or rh10.
  • the vector is AAV1 , AAV2, AAV-BR1 , AAVrh8, or AAV9.
  • the vector is an AAV of serotype 9. This serotype is demonstrated in the examples to be especially advantageous for use as an expression vector according to the invention.
  • AAV expression vectors described herein may also be denoted as recombinant AAV or rAAV vectors.
  • the expression vector is an AAV9 and comprises a gene construct comprising a nucleotide sequence encoding a covalently linked alpha beta (a[3) dimer of beta-hexosaminidase, operably linked to a Cbh promoter.
  • the gene construct further includes 5’ and 3’ flanks of inverted terminal repeats (ITRs) derived from the genome of an AAV, preferably from AAV2.
  • the gene construct further includes a polyA sequence, preferably SV40 polyA.
  • such expression vector comprises a gene construct having the nucleotide sequence of SEQ ID NOs: 75-84, preferably SEQ ID NOs: 75-77 or 81- 84, more preferably SEQ ID NOs: 81-84, even more preferably SEQ ID NOs: 82 or 84, or a sequence having at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith.
  • rAAV recombinant AAV
  • the methods generally involve (a) the introduction of the AAV genome comprising the gene construct to be expressed into a cell, (b) the presence or introduction of an AAV helper construct in the cell, wherein the helper construct comprises the viral functions missing from the AAV genome and, optionally, (c) the introduction of a helper virus into the host cell.
  • All components for AAV vector replication and packaging need to be present, to achieve replication and packaging of the AAV genome into AAV vectors. These typically include AAV cap proteins, AAV rep proteins and, optionally, viral proteins upon which AAV is dependent for replication. Rep and cap regions are well known in the art, see e.g., Chiorini et al. (1999, J.
  • the AAV cap and rep proteins may derive from the same AAV serotype, or they can derive from a combination of different serotypes, preferably they derive from the serotypes described elsewhere herein.
  • the viral proteins upon which AAV is dependent for replication may derive from any virus, such as an herpes simplex viruses (such as HSV types 1 and 2), a vaccinia virus, an adeno-associated virus or an adenovirus, preferably from an adenovirus.
  • the producer cell line is transfected transiently with the polynucleotide of the invention (comprising the expression cassette flanked by ITRs) and with construct(s) that encode(s) rep and cap proteins and provide(s) helper functions.
  • the cell line supplies stably the helper functions and is transfected transiently with the polynucleotide of the invention (comprising the expression cassette flanked by ITRs) and with construct(s) that encode(s) rep and cap proteins.
  • the cell line supplies stably the rep and cap proteins and the helper functions and is transiently transfected with the polynucleotide of the invention.
  • the cell line supplies stably the rep and cap proteins and is transfected transiently with the polynucleotide of the invention and a polynucleotide encoding the helper functions.
  • the cell line supplies stably the polynucleotide of the invention, the rep and cap proteins and the helper functions.
  • the recombinant AAV (rAAV) genome present in a rAAV vector comprises at least the nucleotide sequences of the inverted terminal repeat regions (ITRs) of one of the AAV serotypes (preferably the ones of serotype AAV2 as disclosed herein), or nucleotide sequences substantially identical thereto or nucleotide sequences having at least 60%, 70%, 80%, 90%, 95% or 99% identity thereto, and a nucleotide sequence encoding a covalently linked alpha beta (ap) dimer of beta-hexosaminidase (under control of a suitable regulatory element) inserted between the two ITRs.
  • a vector genome generally requires the use of flanking 5’ and 3’ ITR sequences to allow for efficient packaging of the vector genome into the rAAV capsid.
  • the complete genome of several AAV serotypes and corresponding ITRs has been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No.2, p1309-1319, incorporated herein by reference). They can be either cloned or made by chemical synthesis as known in the art, using for example an oligonucleotide synthesizer as supplied e.g., by Applied Biosystems Inc. (Fosters, CA, USA) or by standard molecular biology techniques.
  • the ITRs can be cloned from the AAV viral genome or excised from a vector comprising the AAV ITRs.
  • the ITR nucleotide sequences can be either ligated at either end to the nucleotide sequence comprising one or more genes using standard molecular biology techniques, or the AAV sequence between the ITRs can be replaced with the desired nucleotide sequence.
  • the rAAV genome as present in a rAAV vector does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV.
  • This rAAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g., gfp) or a gene encoding a chemically, enzymatically, or otherwise detectable and/or selectable product (e.g., lacZ, aph, etc.) known in the art.
  • the rAAV genome as present in said rAAV vector further comprises a promoter sequence operably linked to the nucleotide sequence encoding a covalently linked alpha beta (ap) dimer of beta-hexosaminidase.
  • a suitable 3’ untranslated sequence may also be operably linked to the nucleotide sequence encoding a covalently linked alpha beta (a ) dimer of beta-hexosaminidase.
  • Suitable 3’ untranslated regions may be those naturally associated with the nucleotide sequence or may be derived from different genes, such as for example the SV40 polyadenylation signal (SEQ ID NO: 27).
  • the introduction into a producer cell can be carried out using standard virological techniques, such as transformation, transduction and transfection. Most vectors do not replicate in the producer cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Green, Molecular Cloning. A Laboratory Manual, 4 th Edition (2012), Cold Spring Harbor Laboratory Press (incorporated herein by reference), and in Metzger et al (1988) Nature 334: 31-36 (incorporated herein by reference).
  • suitable expression vectors can be expressed in, yeast, e.g., S.
  • a cell may thus be a prokaryotic or eukaryotic producer cell.
  • a cell may be a cell that is suitable for culture in liquid or on solid media.
  • the producer cells are cultured under standard conditions known in the art to produce the assembled AAV vectors which are then purified using standard techniques such as polyethylene glycol precipitation or CsCI gradients (Xiao et al. 1996, J. Virol. 70: 8098-8108, incorporated herein by reference). Residual helper virus activity can be inactivated using known methods, such as for example heat inactivation.
  • a host cell transduced or transfected with any of the gene constructs or expression vectors described herein is a brain cell, preferably a brain cell of a mammal.
  • a host cell transduced or transfected with any of the gene constructs or expression vectors described herein is a murine brain cell (such as a brain cell of a rat or mouse) or a human brain cell, preferably of a mouse or a human, more preferably of a human.
  • a host cell as described herein is an isolated host cell.
  • transduction is preferably used.
  • the transduced host cell may or may not comprise the packaging components of the viral vectors.
  • "Host cell” or “target cell” refers to the cell into which the DNA delivery takes place, such as the brain cells of a mammalian subject as described elsewhere herein.
  • AAV vectors in particular are able to transduce both dividing and non-dividing cells.
  • composition comprising a gene construct as described herein and/or an expression vector as described herein, optionally further comprising one or more pharmaceutically acceptable ingredients.
  • a composition may be called a gene therapy composition.
  • the composition is a pharmaceutical composition.
  • ‘pharmaceutically acceptable ingredients’ include pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and/or excipients.
  • the one or more pharmaceutically acceptable ingredients may be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents, and excipients, preferably selected from the group consisting of excipients, vehicles, carriers, and diluents.
  • Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and/or excipients may for instance be found in Remington: The Science and Practice of Pharmacy, 23rd edition. Elsevier (2020), incorporated herein by reference.
  • a further compound may be present in a composition of the invention.
  • Said compound may help in delivery of the composition.
  • Suitable compounds in this context are: compounds capable of forming complexes, nanoparticles, micelles and/or liposomes. It is understood that these compounds are capable of delivering gene constructs and expression vectors as described herein, complexed or trapped in a vesicle or liposome, through a cell membrane. Many of these compounds are known in the art.
  • Suitable compounds comprise polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives; synthetic amphiphiles (SAINT-18); lipofectinTM, DOTAP.
  • PEI polyethylenimine
  • PECs polypropyleneimine or polyethylenimine copolymers
  • SAINT-18 synthetic amphiphiles
  • lipofectinTM DOTAP
  • compositions as described herein can be formulated in viral genome (vg) dosage units to contain an amount of viral genomes that is in the range of about 10 A 9 - 10 A 16 vg, preferably 10 A 10 - 10 A 16 vg, more preferably 10 A 11 - 10 A 15 vg, even more preferably 10 A 12 - 10 A 14 vg.
  • gene constructs, expression vectors and compositions as described herein for use in therapy.
  • gene constructs, expression vectors and compositions as described herein are for use as a medicament.
  • gene constructs, expression vectors, and compositions as described herein are provided for use in the treatment of GM2 gangliosidoses. Complications of GM2 gangliosidoses may also be encompassed.
  • GM2 gangliosidoses are a group of three neurodegenerative lysosomal storage diseases characterized by a beta-hexosaminidase deficiency. This beta-hexosaminidase enzyme is composed of two subunits, alpha and beta, in which dimer formation is required for catalytic activity. Mutations in the alpha subunit lead to Tay-Sachs disease, while mutations in the beta subunit lead to Sandhoff disease. Accordingly, in preferred embodiments, gene constructs, expression vectors, and compositions as described herein are provided for use in the treatment of Sandhoff disease or Tay-Sachs disease.
  • a method of treatment of GM2 gangliosidoses comprising administering a gene construct, an expression vector and/or a composition as described herein.
  • administering a gene construct, an expression vector or a composition means administering to a subject such as a subject in need thereof.
  • a therapeutically effective amount of a gene construct, an expression vector or a composition is administered.
  • a gene construct, an expression vector or a composition as described herein for the manufacture of a medicament for the treatment of GM2 gangliosidoses, preferably Sandhoff disease or Tay-Sachs disease.
  • a gene construct, an expression vector or a composition as described herein for the treatment of GM2 gangliosidoses, preferably Sandhoff disease or Tay-Sachs disease.
  • an effective amount or therapeutically (and/or prophylactically) effective amount may be administered.
  • an “effective amount’’ is an amount sufficient to exert beneficial or desired results.
  • a “therapeutically effective amount’’ is an amount that, when administered to a subject in need thereof, is sufficient to exert some therapeutic effect as described herein, such as, but not limited to, increased betahexosaminidase activity, normalization of GM2 in the cerebral cortex as well as cholesterol accumulation in the cortex and cerebellum, normalization of lysosomal distension, lysosomal homeostasis, autophagy, myelinization and neuroinflammation in the CNS, normalization of cholesterol and GAG storage in several peripheral tissues and normalization of lysosomal homeostasis in the liver, improved general locomotor and exploratory activity, as well as motor coordination, mobility and disease progression, and improved survival compared to an untreated subject.
  • the therapeutically effective amount is a vg dosage unit of 10 A 9 - 10 A 16 vg, preferably 10 A 10 - 10 A 16 vg, more preferably 10 A 11 - 10 A 15 vg, even more preferably 10 A 12 - 10 A 14 vg. It is understood that this effective amount is expressed as the total dose per subject.
  • the human brain volume is around 1200 cm A 3.
  • these doses correspond with about 0.83x10 A 6 - 0.83x10 A 13 vg, preferably 0.83x10 A 7 - 0.83x10 A 13 vg, more preferably 0.83x10 A 8 - 0.83x10 A 12 vg, even more preferably 0.83x10 A 9 - 0.83x10 A 11 vg when expressed per ml of brain.
  • the gene constructs, expression vectors and composition may be administered to a subject, such as a subject in need thereof.
  • the subject (in need) can be a healthy, asymptomatic or partially symptomatic subject.
  • the subject (in need) may also suffer from or be at risk for developing any of the symptoms, diseases, and conditions described herein.
  • the subject (in need) may be a subject inflicted with any of the symptoms, diseases, and conditions described herein.
  • the therapy and/or treatment and/or medicament may involve expression of a covalently linked alpha beta (a ) dimer of beta-hexosaminidase in the CNS, preferably the brain, and/or transduction of the CNS, preferably the brain.
  • expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and/or the thalamus and/or the subthalamus and/or the epithalamus and/or the cortex and/or the hippocampus and/or the basal ganglia and/or the amygdala and/orthe cerebellum and/or the brain stem.
  • expression of the gene construct in the brain may mean expression of the gene construct in at least one or at least two or at least three or at least four or all brain regions selected from the group consisting of the hypothalamus and/or the thalamus and/or the subthalamus and/orthe epithalamus, and/orthe cortex and/or the hippocampus and/orthe basal ganglia and/or the amygdala and/orthe cerebellum and/orthe brain stem.
  • expression in the CNS and/or the brain and/or the hypothalamus and/or the thalamus and/or the subthalamus and/or the epithalamus and/or the cortex and/or the hippocampus and/orthe basal ganglia and/or the amygdala and/or the cerebellum and/or the brain stem may mean specific expression in the CNS and/or the brain and/or the hypothalamus and/or the thalamus and/or the subthalamus and/or the epithalamus and/orthe cortex and/or the hippocampus and/or the basal ganglia and/orthe amygdala and/or the cerebellum and/or the brain stem.
  • the therapy and/or treatment and/or medicament may involve expression of a covalently linked alpha beta (ap) dimer of beta-hexosaminidase in the liver.
  • involving the expression of a gene construct’ may be replaced by “causing the expression of a gene construct’’ or “inducing the expression of a gene construct’’ or “involving transduction’’ or the like.
  • a treatment or a therapy or a use or the administration of a medicament as described herein does not have to be repeated.
  • a treatment or a therapy or a use or the administration of a medicament as described herein may be repeated each year or each 2, 3, 4, 5, 6, 7, 8, 9 or 10, including intervals between any two of the listed values, years.
  • the subject treated may be a vertebrate, preferably a mammal, such as a rodent (preferably mice, rats), or a human. In preferred embodiments, the subject treated is a human.
  • a gene construct and/or an expression vector and/or a composition and/or a medicament as described herein preferably exhibits at least one, at least two, at least three, or all of the following effects:
  • GM2 gangliosidoses preferably Sandhoff disease or Tay-Sachs disease (as described herein);
  • GM2 gangliosidoses preferably Sandhoff disease or Tay- Sachs disease (as described herein).
  • a gene construct and/or an expression vector and/or a composition and/or a medicament as described herein preferably exhibits at least one, at least two, at least three, or all of the following effects: normalization of GM2 and cholesterol accumulation; normalization of lysosomal distension, lysosomal homeostasis, autophagy, myelinization and neuroinflammation in the CNS; and/or normalization of cholesterol and GAG storage in several peripheral tissues and a normalization of lysosomal homeostasis in the liver; and/or improvement of general locomotor and exploratory activity, as well as motor coordination, mobilty and disease progression; and/or increase in survival.
  • Alleviating a symptom of GM2 gangliosidoses may mean that a symptom of GM2 gangliosidoses (e.g., developmental regression, neurological deterioration, motor deficits and vision deterioration) is improved or decreased or that the progression of a typical symptom has been slowed down in an individual, in a cell, tissue or organ of said individual as assessed by a physician.
  • a decrease or improvement of a typical symptom may mean a slowdown in progression of symptom development or a complete disappearance of symptoms. Symptoms, and thus also a decrease in symptoms, can be assessed using a variety of methods, to a large extent the same methods as used in diagnosis of GM2 gangliosidoses, including clinical examination and routine laboratory tests.
  • Laboratory tests may include both macroscopic and microscopic methods, molecular methods, radiographic methods such as X-rays, Magnetic Resonance Imaging, biochemical methods, immunohistochemical methods and others.
  • Beta-hexosaminidase levels and activity could be assessed using techniques known to a person of skill in the art, for example as done in the experimental part. This diagnostic could be further confirmed by a DNA-based test to identify the underlying mutation causing the GM2 gangliosidosis.
  • “decrease” means at least a detectable decrease (respectively a detectable improvement) using an assay known to a person of skill in the art, such as assays as carried out in the experimental part.
  • the decrease may be a decrease of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%.
  • the decrease may be seen after at least one week, one month, six months, one year or more of treatment using a gene construct and/or an expression vector and/or a composition of the invention.
  • the decrease is observed after a single administration.
  • the decrease is observed for a duration of at least one week, one month, six months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years or more, preferably after a single administration.
  • Improving a parameter may mean that the value of a typical parameter associated with GM2 gangliosidoses (e.g., beta-hexosaminidase expression and/or activity) is improved in an individual or in a cell, tissue, or organ of said individual, as assessed by a physician.
  • improvement of a parameter may be interpreted as to mean that said parameter assumes a value closer to the value displayed by a healthy individual.
  • the improvement of a parameter may be seen after at least one week, one month, six months, one year or more of treatment using a gene construct and/or an expression vector and/or a composition of the invention. Preferably, the improvement is observed after a single administration.
  • the improvement is observed for a duration of at least one week, one month, six months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years or more, preferably after a single administration.
  • a gene construct and/or an expression vector and/or a composition as described herein is preferably able to alleviate a symptom or a parameter or a characteristic of GM2 gangliosidoses, preferably Sandhoff disease or Tay-Sachs disease, in a patient or a cell, tissue or organ of said patient if after at least one week, one month, six months, one year or more of treatment using a gene construct and/or an expression vector and/or a composition of the invention, said symptom or parameter or characteristic has decreased (e.g. is no longer detectable or has slowed down), as described herein.
  • GM2 gangliosidoses preferably Sandhoff disease or Tay-Sachs disease
  • a gene construct and/or an expression vector and/or a composition as described herein may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing GM2 gangliosidoses, preferably Sandhoff disease or Tay-Sachs disease, and may be administered in vivo, ex vivo or in vitro.
  • Said gene construct and/or expression vector and/or composition may be directly or indirectly administered to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing GM2 gangliosidoses, preferably Sandhoff disease or Tay-Sachs disease, and may be administered directly or indirectly in vivo, ex vivo or in vitro.
  • a gene construct and/or an expression vector and/or a composition may be administered by different administration modes.
  • An administration mode may be intravenous, intramuscular, intraperitoneal, intranasal, subcutaneous, intraarticular, intra-adipose tissue, oral, intrahepatic, intrasplanchnic, intraductal, intra-ear, intracranial, intraparenchymal, intrathecal, intracerebroventricular, intracerebral, hippocampal, striatal administration, ophthalmic administration, administration via the cerebrospinal fluid (CSF) and/or administration via the cisterna magna.
  • CSF cerebrospinal fluid
  • a more preferred administration mode is intracranial, intraparenchymal, intrathecal, intracerebroventricular, intracerebral, hippocampal, striatal administration, administration via the cisterna magna and/or administration via the CSF.
  • An even more preferred administration mode is intra-CSF administration, intracerebroventricular, intrathecal administration and/or administration via the cisterna magna.
  • a gene construct as described herein is for use in therapy, wherein the gene construct is administered by intra-CSF.
  • a gene construct and/or an expression vector and/or a composition of the invention may be directly or indirectly administered using suitable means known in the art. Improvements in means for providing an individual or a cell, tissue, or organ of said individual with a gene construct and/or an expression vector and/or a composition of the invention are anticipated, considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect of the invention.
  • a gene construct and/or an expression vector and/or a composition can be delivered as is to an individual, a cell, tissue or organ of said individual. Depending on the disease or condition, a cell, tissue or organ of said individual may be as earlier described herein. When administering a gene construct and/or an expression vector and/or a composition of the invention, it is preferred that such gene construct and/or an expression vector and/or a composition is dissolved in a solution that is compatible with the delivery method.
  • a therapeutically effective dose of a gene construct and/or an expression vector and/or a composition as mentioned above is preferably administered in a single and unique dose, hence avoiding repeated periodical administration.
  • Treating” or “treatment” as used herein may include delaying (e.g., delaying progression or delaying deterioration), preventing, ameliorating, or curing. Accordingly, throughout this disclosure, “treating” and the like may be replaced with “treating, delaying, ameliorating or curing” and the like.
  • treating may be understood to treat a disease, condition or symptom as compared to said disease, condition, or symptom prior to administering the composition.
  • “treating” as used herein may be understood to treat a disease, condition or symptom as compared to said disease, condition or symptom in a subject inflicted with the same disease, condition or symptom receiving a placebo.
  • the occurrence of any of the effects described elsewhere herein may be compared in the same way.
  • Beta-hexosaminidase also named beta-N-acetylhexosaminidase, (EC 3.2.1.52) is a lysosomal enzyme composed of dimers of alpha- and/or beta-subunits.
  • the alpha and beta subunits are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the lysosome.
  • each subunit contains an amino-terminal signal peptide that is subsequently cleaved by signal peptidase.
  • Beta-hexosaminidase catalyzes the hydrolysis of terminal N-acetyl- D-hexosamine residues in N-acetyl-[3-D-hexosaminides. Means and methods to measure this activity are commonly known in the art, for example as described in the Examples section. As used herein and unless explicitly stated otherwise, “beta-N-acetylhexosaminidase”, and “beta-hexosaminidase” and the like refer to the mammalian enzyme beta-hexosaminidase. As stated elsewhere herein, three isoforms of the betahexosaminidase exist.
  • the hexosaminidase A (HexA) is composed of an alpha and a beta subunit
  • the hexosaminidase B (HexB) is composed of two beta subunits
  • the hexosaminidase S (HexS) is composed of two alpha subunits.
  • the alpha and beta subunits are encoded by the HEXA and HEXB genes, respectively, and in mice the alpha and beta subunits are encoded by the Hexa and Hexb genes, respectively, as described in more detail elsewhere herein.
  • total beta-hexosoaminidase activity in a cell or tissue is determined by the sum of the activities of all three isoforms. Accordingly, as used herein and unless explicitly stated otherwise, “total beta-hexosaminidase activity’’ and “beta-HEXO” and the like refer to the activity of HexA, HexB, and HexS summed together.
  • beta-hexosaminidase A’ encompasses full-length molecules, variants, isoforms, and fragments that retain enzymatic activity against HexA substrates, such as GM2 ganglioside.
  • the term also encompasses natural and engineered molecules identical or substantially identical to these sequences.
  • the term “HexA expression constructs’’ or “HexA expression vectors’’ refers to constructs encoding both the alpha and beta subunits of betahexosaminidase.
  • beta-hexosaminidase deficiency or “hexosaminidase deficiency’ or the like refers to reduced expression orfunction of hexosaminidase compared to normal levels for sex and age matched subjects. Deficiencies may be the result of genetic mutations or other molecular events that impair transcription, translation, post-translational modification, sub-cellular localization, dimerization, or enzymatic function of the hexosaminidase alpha and beta subunits. The severity of hexosaminidase deficiency may vary across subjects and may or may not result in clinical symptoms associated with lysosomal storage disorders.
  • lysosomal storage disease refers to a group of diseases that are caused by a lack of enzymes that normally serve as catalyst for the breakdown of substances in the cells of the body. These enzymes are found in sac-like structures in cells called lysosomes. Lysosomes act as the “recycling center” of the cell, breaking down molecules into simple products for the cell to use to build new material. The lack of certain enzymes causes an accumulation within the cell of the substance that the enzyme would normally help eliminate. Abnormal storage causes inefficient functioning and damage of the body's cells, which can lead to serious health problems.
  • self-cleaving peptide refers to a peptide sequence that is associated with a cleavage activity that occurs between two amino acid residues within the peptide sequence itself. For example, in P2A peptides, cleavage occurs between the proline (P) and glycine (G) in the C-terminal of the peptide resulting in the peptide located upstream of the 2A peptide to have extra amino acids on its C-terminal end while the peptide located downstream the 2A peptide will have an extra P on its N-terminal end.
  • P proline
  • G glycine
  • ribosomal skip mechanism This cleavage occurs through a ‘ribosomal skip mechanism’ during translation wherein normal peptide bond formation between the P and G residue is impaired, without affecting the translation of the rest of the peptide.
  • ribosomal skip mechanisms are well known in the art and are known to be used by several viruses for the expression of several proteins encoded by a single messenger RNA.
  • central nervous system or “CNS” refers to the part of the nervous system that comprises the brain and the spinal cord, to which sensory impulses are transmitted and from which motor impulses pass out, and which coordinates the activity of the entire nervous system.
  • brain refers to the central organ of the nervous system and consists of the cerebrum, the brain stem, and the cerebellum. It controls most of the activities of the body, processing, integrating, and coordinating the information it receives from the sense organs, and making decisions as to the instructions sent to the rest of the body.
  • hypothalamus refers to a region of the forebrain below the thalamus which coordinates both the autonomic nervous system and the activity of the pituitary, controlling body temperature, thirst, hunger, and other homeostatic systems, and involved in sleep and emotional activity.
  • Hiippocampus belongs to the limbic system, and plays important roles in the consolidation of information from short-term memory to long-term memory, and in spatial memory that enables navigation. The hippocampus is located under the cerebral cortex (allocortical) and in primates in the medial temporal lobe.
  • cortex or “cerebral cortex”, as used herein, is the outer layer of neural tissue of the cerebrum of the brain, in humans and other mammals. It plays a key role in memory, attention, perception, awareness, thought, language, and consciousness. It includes the frontal cortex, the parietal cortex, the temporal cortex, and the occipital cortex. “Cerebellum”, as used herein, refers to a major feature in the hindbrain of all vertebrates. In humans, it plays an important role in motor control. It may also be involved in some cognitive functions such as attention and language as well as in regulating fear and pleasure responses. “Thalamus” as used herein, refers to a walnutsized structure located in the forebrain of all vertebrates.
  • Subthalamus as used herein, is a structure located between the thalamus and the midbrain. It contains the subthalamic nucleus and functions in the regulation of movements controlled by skeletal muscles.
  • Epithalamus refers to a small structure which is located behind the third ventricle and dorsal and caudal to the thalamus. It acts as a connection between the limbic system and other parts of the brain.
  • the basal ganglia refer to a collection of subcortical nuclei, such as the striatum, globus pallidus, ventral pallidum, substantia nigra and subthalamic nucleus, in the brains of all vertebrates. These ganglia are primarily responsible for motor control, as well as other roles such as motor learning, executive functions and behaviors, and emotions.
  • the “amygdala” as used herein, is a complex structure of cells nested in the middle ofthe brain, adjacent to the hippocampus. It plays a key role in the fight-or-flight response, emotion, and memory.
  • the “brain stem” as used herein, refers to a stalk-like portion of the brain which connects the brain to the spinal cord. It includes the midbrain, pons, and medulla oblongata, and mainly controls subconscious body functions, such as breathing and maintaining the heart rate.
  • a nucleic acid molecule such as a nucleic acid molecule encoding a covalently linked alpha beta (a[3) dimer of beta-hexosaminidase, is represented by a nucleic acid or nucleotide sequence which encodes a protein fragment or a polypeptide or a peptide or a derived peptide.
  • a covalently linked alpha beta (a ) dimer of beta-hexosaminidase protein fragment or a polypeptide or a peptide or a derived peptide are represented by an amino acid sequence.
  • each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct as identified herein by a given sequence identity number is not limited to this specific sequence as disclosed.
  • Each coding sequence as identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.
  • nucleotide sequence that encodes an amino acid sequence that has at least 60%, 70%, 80%, 90%, 95% or 99% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.
  • Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.
  • Another preferred level of sequence identity or similarity is 70%.
  • Another preferred level of sequence identity or similarity is 80%.
  • Another preferred level of sequence identity or similarity is 90%.
  • Another preferred level of sequence identity or similarity is 95%.
  • Another preferred level of sequence identity or similarity is 99%.
  • Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given
  • Each non-coding nucleotide sequence i.e., of a promoter or of another regulatory region
  • a nucleotide sequence comprising a nucleotide sequence that has at least 60% sequence identity or similarity with a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: A as example).
  • a preferred nucleotide sequence has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: A.
  • such non-coding nucleotide sequence such as a promoter exhibits or exerts at least an activity of such a non-coding nucleotide sequence such as an activity of a promoter as known to a person of skill in the art.
  • sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • Similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Identity and “similarity” can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated herein by reference.
  • Sequence identity and “sequence similarity’ can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman- Wunsch) which aligns the sequences optimally overthe entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman).
  • a global alignment algorithm e.g., Needleman- Wunsch
  • sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman).
  • Sequences may then be referred to as “substantially identical’’ or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water using default parameters) share at least a certain minimal percentage of sequence identity (as described below).
  • a global alignment is suitably used to determine sequence identity when the two sequences have similar lengths.
  • local alignments such as those using the Smith- Waterman algorithm, are preferred.
  • EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps.
  • EMBOSS water uses the Smith-Waterman local alignment algorithm.
  • the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).
  • nucleic acid and protein sequences of some embodiments of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.
  • search can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, incorporated herein by reference.
  • Gapped BLAST can be utilized as described in Altschul et al. , (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference.
  • BLASTx and BLASTn the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov/.
  • conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below. Alternative conservative amino acid residue substitution classes :
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg, Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.
  • the term "gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g., an mRNA) in a cell, operably linked to suitable regulatory regions (e.g., a promoter).
  • a gene will usually comprise several operably linked fragments, such as a promoter, a 5 1 leader sequence, a coding region and a 3 -nontranslated sequence (3'-end) e.g., comprising a polyadenylation- and/or transcription termination site.
  • the coding region of a gene also known as the coding sequence (CDS), is the portion of a gene that codes for protein.
  • a chimeric or recombinant gene is a gene not normally found in nature, such as a gene in which for example the promoter is not associated in nature with part or all of the transcribed DNA region. "Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e., which is capable of being translated into a biologically active protein or peptide.
  • a "transgene” is herein described as a gene or a coding sequence or a nucleic acid molecule (i.e., a molecule encoding a covalently linked alpha beta (a ) dimer of beta-hexosaminidase) that has been newly introduced into a cell, i.e., a gene that may be present but may normally not be expressed or expressed at an insufficient level in a cell.
  • ‘insufficient’’ means that although said beta-hexosaminidase dimer is expressed in a cell, a condition and/or disease as described herein could still be developed. In this case, the invention allows the over-expression of a beta-hexosaminidase.
  • the transgene may comprise sequences that are native to the cell, sequences that naturally do not occur in the cell and it may comprise combinations of both.
  • a transgene may contain sequences coding for a beta-hexosaminidase and/or additional proteins as earlier identified herein that may be operably linked to appropriate regulatory sequences for expression of the sequences coding for a beta-hexosaminidase in the cell.
  • the transgene is not integrated into the host cell’s genome.
  • promoter or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
  • a ‘‘ubiquitous promoter’’ is active in substantially all tissues, organs, and cells of an organism.
  • any expression vector comprising any of the gene construct as described herein, wherein the covalently linked alpha beta (a[3) dimer of beta-hexosaminidase nucleotide sequence has been replaced by a nucleotide sequence encoding for GFP, can be produced.
  • Cells transduced as described herein can then be assessed for fluorescence intensity according to standard protocols.
  • Promoters that are capable of initiating transcription in brain cells, whilst still allowing for any leaky expression in other (maximum five, six, seven or eight) organs and parts of the body, are advantageous.
  • a ‘‘regulator” or ‘‘transcriptional regulator” is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis. In some embodiments, all of the nucleotide sequences described in this document may be operably linked with each other within the gene construct of this disclosure.
  • protein or “polypeptide” or ‘‘amino acid sequence” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure, or origin.
  • amino acids or “residues” are denoted by three-letter symbols.
  • a residue may be any protein
  • Gene constructs as described herein could be prepared using any cloning and/or recombinant DNA techniques, as known to a person of skill in the art, in which a nucleotide sequences encoding said covalently linked alpha beta (a[3) dimer of beta-hexosaminidase are expressed in a suitable cell, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al. , "Current Protocols in Molecular Biology", (2003, supra) and in Sambrook and Green (2012, supra)-, both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J.A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).
  • expression vector or “vector” or ‘‘delivery vector’ generally refers to a tool in molecular biology used to obtain gene expression in a cell, for example by introducing a nucleotide sequence that is capable of effecting expression of a gene or a coding sequence in a host compatible with such sequences.
  • An expression vector carries a genome that is able to stabilize and remain episomal in a cell.
  • a cell may mean to encompass a cell used to make the construct or a cell wherein the construct will be administered.
  • a vector is capable of integrating into a cell's genome, for example through homologous recombination or otherwise.
  • a nucleic acid or DNA or nucleotide sequences encoding a covalently linked alpha beta (a ) dimer of beta-hexosaminidase is incorporated into a DNA construct capable of introduction into and expression in an in vitro cell culture.
  • a DNA construct is suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, (e.g. , Sf9), yeast, fungi, or other eukaryotic cell lines.
  • a DNA construct prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment.
  • the term ‘‘operably linked’’ has already been described herein.
  • a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
  • DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide.
  • a DNA sequence that is operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame.
  • enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.
  • an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment.
  • suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g., Sambrook and Green, 2012, supra).
  • a transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognized by the host.
  • the selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Green, 2012, supra).
  • An expression vector includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment.
  • the replication system is only functional in the cell that is used to make the vector (bacterial cell as E. Coli).
  • Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Green (2012, supra) and in Metzger et al. (1988) Nature 334: 31-36.
  • suitable expression vectors can be expressed in, yeast, e.g., S.
  • Cerevisiae e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli.
  • a cell may thus be a prokaryotic or eukaryotic host cell.
  • a cell may be a cell that is suitable for culture in liquid or on solid media.
  • a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal.
  • a viral vector or a viral expression vector or a viral gene therapy vector is a vector that comprises a gene construct as described herein.
  • a viral vector or viral expression vector or a viral gene therapy vector is a vector that is suitable for gene therapy.
  • Vectors that are suitable for gene therapy are described in Anderson 1998, Nature 392: 25-30; Walther and Stein, 2000, Drugs 60: 249-71 ; Kay et al., 2001 , Nat. Med. 7: 33-40; Russell, 2000, J. Gen. Virol. 81 : 2573-604; Amado and Chen, 1999, Science 285: 674-6; Federico, 1999, Curr. Opin. Biotechnol.10: 448-53; Vigna and Naldini, 2000, J. Gene Med. 2: 308-16; Marin et al. , 1997, Mol. Med.
  • a particularly suitable gene therapy vector includes an adenoviral and adeno-associated virus (AAV) vector. These vectors infect a wide number of dividing and non-dividing cell types including synovial cells and liver cells. The episomal nature of the adenoviral and AAV vectors after cell entry makes these vectors suited for therapeutic applications, (Russell, 2000, J. Gen. Virol. 81 : 2573-2604; Goncalves, 2005, Virol J. 2(1):43; incorporated herein by reference) as indicated above. AAV vectors are even more preferred since they are known to result in very stable long-term expression of transgene expression (up to 9 years in dog (Niemeyer et al, Blood.
  • AAV vectors are even more preferred since they are known to result in very stable long-term expression of transgene expression (up to 9 years in dog (Niemeyer et al, Blood.
  • adenoviral vectors are modified to reduce the host response as reviewed by Russell (2000, supra).
  • Method for gene therapy using AAV vectors are described by Wang et al., 2005, J Gene Med. March 9 (Epub ahead of print), Mandel et al., 2004, Curr Opin Mol Ther. 6(5):482-90, and Martin et al., 2004, Eye 18(11):1049-55, Nathwani et al, N Engl J Med. 2011 Dec 22;365(25):2357-65, Apparailly et al, Hum Gene Ther. 2005 Apr;16(4):426-34; all of which are incorporated herein by reference.
  • a suitable gene therapy vector includes a retroviral vector.
  • a preferred retroviral vector for application in the present invention is a lentiviral based expression construct. Lentiviral vectors have the ability to infect and to stably integrate into the genome of dividing and non-dividing cells (Amado and Chen, 1999 Science 285: 674- 6, incorporated herein by reference). Methods for the construction and use of lentiviral based expression constructs are described in U.S. Patent No.'s 6,165,782, 6,207,455, 6,218,181 , 6,277,633 and 6,323,031 and in Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16); all of which are incorporated herein by reference.
  • Suitable gene therapy vectors include an adenovirus vector, a herpes virus vector, a polyoma virus vector or a vaccinia virus vector.
  • Adeno-associated virus vector AAV vector
  • AAV vector Adeno-associated virus vector
  • AAV vector Adeno-associated virus vector
  • AAV virus AAV virus
  • AAV virion AAV viral particle
  • AAV particle used as synonyms herein, refer to a viral particle composed of at least one capsid protein of AAV (preferably composed of all capsid protein of a particular AAV serotype) and an encapsulated polynucleotide of the AAV genome.
  • the particle comprises a heterologous polynucleotide (i.e., a polynucleotide different from a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell) flanked by AAV inverted terminal repeats, then they are typically known as an "AAV vector particle” or "AAV viral vector” or "AAV vector”.
  • AAV refers to a virus that belongs to the genus Dependovirus family Parvoviridae.
  • the AAV genome is approximately 4.7 Kb in length, and it consists of single strand deoxyribonucleic acid (ssDNA) that can be positive or negative detected.
  • the invention also encompasses the use of double stranded AAV also called dsAAV or scAAV.
  • the genome includes inverted terminal repeats (ITR) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap.
  • ITR inverted terminal repeats
  • ORFs open reading frames
  • the frame rep is made of four overlapping genes that encode proteins Rep necessary for AAV lifecycle.
  • the frame cap contains nucleotide sequences overlapping with capsid proteins: VP1 , VP2 and VP3, which interact to form a capsid of icosahedral symmetry (see Carter and Samulski, 2000, and Gao et al, 2004, incorporated herein by reference).
  • a preferred viral vector or a preferred gene therapy vector is an AAV vector.
  • An AAV vector as used herein preferably comprises a recombinant AAV vector (rAAV vector).
  • a “rAAV vector” as used herein refers to a recombinant vector comprising part of an AAV genome encapsidated in a protein shell of capsid protein derived from an AAV serotype as explained herein.
  • Part of an AAV genome may contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype, such as AAV1 , AAV2, AAV3, AAV4, AAV5 and others.
  • ITR inverted terminal repeats
  • Preferred ITRs are those of AAV2 which are represented by sequences comprising, consisting essentially of, or consisting of SEQ ID NO: 25 (5’ ITR) and SEQ ID NO: 26 (3’ ITR).
  • the invention also preferably encompasses the use of a sequence having at least 80% (or at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity with SEQ ID NO: 25 as 5’ ITR and a sequence having at least 80% (or at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least
  • Protein shell comprised of capsid protein may be derived from any AAV serotype.
  • a protein shell may also be named a capsid protein shell.
  • rAAV vector may have one or preferably all wild type AAV genes deleted but may still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue, and packaging of AAV virions.
  • the ITR sequences may be wild type sequences or may have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with wild type sequences or may be altered for example by insertion, mutation, deletion, or substitution of nucleotides, as long as they remain functional.
  • functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be infected or target cell.
  • a capsid protein shell may be of a different serotype than the rAAV vector genome ITR.
  • a nucleic acid molecule represented by a nucleic acid sequence of choice is preferably inserted between the rAAV genome or ITR sequences as identified above, for example an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3’ termination sequence.
  • Said nucleic acid molecule may also be called a transgene.
  • AAV helper functions generally refers to the corresponding AAV functions required for rAAV replication and packaging supplied to the rAAV vector in trans.
  • AAV helper functions complement the AAV functions which are missing in the rAAV vector, but they lack AAV ITRs (which are provided by the rAAV vector genome).
  • AAV helper functions include the two major ORFs of AAV, namely the rep coding region and the cap coding region or functional substantially identical sequences thereof. Rep and Cap regions are well known in the art, see e.g., Chiorini et al. (1999, J. of Virology, Vol 73(2): 1309-1319) or US 5,139,941 , incorporated herein by reference.
  • the AAV helper functions can be supplied on an AAV helper construct.
  • Introduction of the helper construct into the host cell can occur e.g., by transformation, transfection, or transduction prior to or concurrently with the introduction of the rAAV genome present in the rAAV vector as identified herein.
  • the AAV helper constructs of the invention may thus be chosen such that they produce the desired combination of serotypes for the rAAV vector’s capsid protein shell on the one hand and for the rAAV genome present in said rAAV vector replication and packaging on the other hand.
  • AAV helper virus provides additional functions required for AAV replication and packaging.
  • Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2) and vaccinia viruses.
  • the additional functions provided by the helper virus can also be introduced into the host cell via plasmids, as described in US 6,531 ,456 incorporated herein by reference.
  • Transduction refers to the delivery of a covalently linked alpha beta (a[3) dimer of beta-hexosaminidase into a recipient host cell by a viral vector.
  • transduction of a target cell by a rAAV vector of the invention leads to transfer of the rAAV genome contained in that vector into the transduced cell.
  • Home cell or “target cell’’ refers to the cell into which the DNA delivery takes place, such as the muscle cells of a subject.
  • AAV vectors are able to transduce both dividing and non-dividing cells.
  • Expression may be assessed by any method known to a person of skill in the art. For example, expression may be assessed by measuring the levels of transgene expression in the transduced tissue on the level of the mRNA orthe protein by standard assays known to a person of skill in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry analysis of protein-derived peptides or ELISA.
  • Expression may be assessed at any time after administration of the gene construct, expression vector or composition as described herein. In some embodiments herein, expression may be assessed after 1 week, 2 weeks, 3 weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9, weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, or more.
  • CNS- and/or brain-specific expression refers to the preferential or predominant (at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher or more) expression of a covalently linked alpha beta (a ) dimer of betahexosaminidase, in the CNS and/or brain as compared to other organs or tissues.
  • Other organs or tissues may be the liver, adipose tissue, skeletal muscle, pancreas, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, and others.
  • Sequence optimization refers to the processes employed to modify an existing coding sequence, or to design a coding sequence, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence.
  • An example of sequence optimization is codon optimization. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. For example, to suit the codon preference of mammalians, preferably of murine, canine, or human expression hosts. Optimization, such as codon optimization, can also eliminate elements that potentially impact negatively RNA stability and/or translation (e. g.
  • optimized sequences show at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase in gene expression, transcription, RNA stability and/or translation compared to the original, not codon-optimized sequence.
  • Intra-CSF administration means direct administration into the CSF, located in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain. Intra- CSF administration can be performed via intra-cisterna magna, intracerebroventricular or intrathecal administration.
  • intra-cisterna magna administration means administration into the cisterna magna, an opening of the subarachnoid space located between the cerebellum and the dorsal surface of the medulla oblongata.
  • ‘intracerebroventricular administration” means administration into the either of both lateral ventricles of the brain.
  • intrathecal administration involves the direct administration into the CSF within the intrathecal space of the spinal column.
  • intraparenchymal administration means local administration directly into any region of the brain parenchyma.
  • intranasal administration means administration by way of the nasal structures.
  • intravenous administration refers to direct administration into a vein, typically by injection.
  • intramuscular administration means direct administration a muscle.
  • intra-adipose tissue administration involves direct administration into adipose tissue.
  • intraperitoneal administration means administration into the peritoneum (or body cavity).
  • intracranial administration involves administration in the adipose tissue below the skin.
  • intraarticular administration refers to direct administration into a joint.
  • intrahepatic administration involves the direct administration to the liver, predominantly via a hepatic vein or artery.
  • intrasplanchnic administration means administration to the splanchninc administration.
  • intracranial administration refers to administration into the skull. Intracranial administration, therefore, also encompasses the administration to any brain region which is accessible after penetration of the skull. For example, intracranial administration also includes intracerebral administration.
  • striatal administration involves direct administration into the striatum or corpus striatum.
  • ophthalmic administration refers to direct administration to the eyes.
  • intracerebral administration means direct administration into the cerebrum.
  • hippocampal administration involves administration into the hippocampus.
  • oral administration refers to a route of administration where a substance is taken through the mouth.
  • intra-ear administration involves direct administration into the ear.
  • intraductal administration refers to administration within the duct of a gland.
  • gene constructs, expression vectors and compositions according to the invention are administered as a single dose.
  • the term ‘‘effective amount” or ‘‘pharmaceutically effective amount” or ‘‘therapeutically effective amount” or “prophylactically effective amount” of a composition is a quantity sufficient to achieve or maintain a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated, e.g., a GM2 gangliosidosis.
  • the amount of a composition of the invention administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity, and type of disease.
  • an effective amount is the amount sufficient to cause a decrease in the severity of symptoms associated with the disorder.
  • an effective amount is the amount sufficient to delay the onset of or decrease the likelihood of onset of GM2 gangliosidoses.
  • the verb "to consist of may be replaced by "to consist essentially of meaning that a composition as described herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the verb "to consist of” may be replaced by "to consist essentially of meaning that a method as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention.
  • a gene construct is understood to represent one or more gene constructs.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • At least a particular value means that particular value or more.
  • “at least 2” is understood to be the same as “2 or more” i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 etc.
  • the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.
  • the word "about” or “approximately” when used in association with a numerical value preferably means that the value may be the given value (of 10) more or less 10%, preferably 5%, more preferably 1 % of the value.
  • the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
  • FIG. 1 Design of the AAV constructs.
  • the AAV constructs used in the present patent application were Null, L1 AB mouse (SEQ ID NO: 75), L2 AB mouse (SEQ ID NO: 76), L3 AB mouse (SEQ ID NO: 77), L1 BA mouse (SEQ ID NO: 78), L2 BA mouse (SEQ ID NO: 79), L3 BA mouse (SEQ ID NO: 80), Hexa+Hexb (SEQ ID NOs: 85-86), P2A AB mouse (SEQ ID NO: 87), L1 AB human noh (SEQ ID NO: 81), L1 AB human GA (SEQ ID NO: 82), L1 AB human IDT (SEQ ID NO:83), L1 AB human NV (SEQ ID NO:84), and P2A BA mouse (SEQ ID NO:88) under the control of the ubiquitous promoter Cbh (a short version of the widely used CAG promoter composed of the cytomegalovirus (CMV) early enhancer
  • All expression cassettes also included the polyA sequence from SV40 (SEQ ID NO: 27).
  • Null construct is a noncoding plasmid carrying the Cbh promoter and the SV40 polyA sequence, but no transgene.
  • L1 AB, L2 AB and L3 AB are three different mouse constructs encoding first the optimized murine Hexa and then the optimized murine Hexb coding-sequence fused with a short linker: L1 , L2 or L3.
  • L1 AB noh, L1 AB GA, L1 AB IDT, and L1 1 B NV constructs have the same design as their corresponding mouse constructs, except that they comprise the non-optimized or optimized human HEXA and HEXB coding sequences.
  • L1 BA, L2 BA and L3 BA are three different mouse constructs encoding first the optimized murine Hexb and then the optimized murine Hexa coding-sequence fused with a short linker: L1 , L2 or L3.
  • Hexa+Hexb constructs are two mouse constructs, one of them consists of the optimized murine Hexa coding-sequence while the other consists of the optimized murine Hexb coding-sequence.
  • P2A AB construct is a mouse construct composed of the optimized murine Hexa and Hexb coding-sequence fused with the self-cleaving linker P2A.
  • FIG. 1 HEK293 cell transfection of plasmids encoding both Hexa and Hexb fused with three different short linkers.
  • HEK293 cells were transfected with 0.8 pg of plasmid encoding the following mouse constructs: L1 AB mouse, L2 AB mouse, L3 AB mouse, L1 BA mouse, L2 BA mouse, and L3 BA mouse (SEQ ID NOs: 75-80). Histograms depict total beta-hexosaminidase activity (i.e.
  • FIG. 7 Restoration of lysosomal homeostasis in the CNS following an intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Percentage of WT activity of several lysosomal enzymes analyzed in brain extracts from 4-month-old male and female wild-type (healthy) mice, untreated Sandhoff mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of either control vectors (Null) or AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse, L2 AB mouse, and L3 AB mouse) into the CSF at 1 month of age.
  • a short linker L1 AB mouse, L2 AB mouse, and L3 AB mouse
  • GALNS alphaN- acetylgalactosamine-6 sulfatase
  • GUSB beta-glucuronidase
  • HGSNAT heparan-alpha-glucosaminide N- acetyltransferase
  • NAGLU alpha-N-acetylglucosaminidase
  • SGSH N-sulphoglucosamine sulphohydrolase
  • Figure 9 Recovery of myelinization process in the CNS after an intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice.
  • Results are expressed as mean + SEM.
  • n 4-7 animals/group. *P ⁇ 0.05, **P ⁇ 0.01 , and ***P ⁇ 0.001 vs. Null
  • Figure 10 Correction of astrogliosis after an intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Immunostaining with an antibody specific for the astrocyte marker GFAP in different brain regions of 4-month-old male and female wild-type (healthy) mice, untreated Sandhoff mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of either control vectors (Null) or AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • Null control vectors
  • AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • Results are expressed as mean + SEM. ***P ⁇ 0.001 vs. Null.
  • FIG. 13 Intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice corrects secondary storage pathology in peripheral tissues.
  • FIG. 14 Intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice restores hepatic lysosomal homeostasis.
  • Activity as % of WT, of N-sulphoglucosamine sulphohydrolase (SGSH) and heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) in liver extracts obtained from 4-month-old male and female wild-type (healthy) mice, untreated Sandhoff mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of either control vectors (Null) or AAV9 vectors encoding both HexA and HexB genes fused with a short linker (L1 AB mouse, L2 AB mouse, and L3 AB mouse) into the CSF at 1 month of age.
  • Results are expressed as mean + SEM.
  • n 4-6 animals/group. *P ⁇ 0.05, **P
  • FIG. 15 Normalization of behavioral deficits after intra-CSF administration of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice.
  • the Open-Field test was performed in 4-month-old naive-tested male and female wild-type (healthy) mice, untreated Sandhoff mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of either control vectors (Null) or AAV9 vectors encoding both Hexa and Hexb genes (L1 AB mouse, L2 AB mouse, and L3 AB mouse) into the CSF at 1 month of age. Data corresponds to the locomotor activity recorded during the first 3 minutes.
  • Figure 16 Correction of motor coordination, mobility, and disease progression deficits after intra-CSF administration of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Motor coordination, mobility, and disease progression was assessed with the following tests: righting reflex, mesh, hindlimb clasping and rotarod tests. All tests were performed in 4-month-old male and female wild-type (healthy) mice, untreated Sandhoff mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of either control vectors (Null) or AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse, L2 AB mouse, and L3 AB mouse) into the CSF at 1 month of age.
  • a short linker L1 AB mouse, L2 AB mouse, and L3 AB mouse
  • Males: P ⁇ 0.0001 for WT, L1 AB mouse, and L3 AB mouse vs. Null; P 0.8776 for Sandhoff vs. Null.
  • Females: P ⁇ 0.0001 forWT, L1 AB mouse, and L3 AB mouse vs. Null; P 0.7626 for Sandhoff vs. Null.
  • FIG. 20 Survival study after an intra-CSF administration of AAV9 vectors encoding Hexa and Hexb genes in Sandhoff mice.
  • Males: P ⁇ 0.0001 for WT, P2A AB mouse, and Hexa+Hexb vs. Null; P 0.8776 for Sandhoff vs. Null.
  • Females: P ⁇ 0.0001 forWT, P2A AB mouse, and Hexa+Hexb vs. Null; P 0.7626 for Sandhoff vs. Null.
  • FIG. 23 HEK293 cell transfection of plasmids encoding both HEXA and HEXB fused with a peptide linker.
  • FIG. 24 HEK293 cell transfection of plasmids encoding both Hexa and Hexb fused with three different short linkers.
  • HEK293 cells were transfected with 4 pg of plasmid encoding the following mouse constructs: L1 AB mouse (SEQ ID NO: 75), L2 AB mouse (SEQ ID NO: 76), L3 AB mouse (SEQ ID NO: 77), Hexa+Hexb (SEQ ID NOs: 85-86), P2A AB mouse (SEQ ID NO: 87) and P2A BA mouse (SEQ ID NO: 88).
  • Figure 25 Long-term increased HexA activity in the CNS after intra-CSF gene transfer of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Determination of HexA activity in different brain sections (l-V) of 8-month-old male and female wild-type (healthy) mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • a short linker L1 AB mouse and L3 AB mouse
  • Figure 26 Long-term correction of secondary lipid storage in the CNS following an intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Representative images of filipin staining to detect unesterified cholesterol in cerebral cortex and cerebellum from 8-month-old male wildtype (healthy) mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • a short linker L1 AB mouse and L3 AB mouse
  • Sandhoff mice received a total dose of 1x10 A 11 vg/mouse of control vectors (Null) into the CSF at 1 month of age and were euthanized at 4 months of age.
  • n 3 animals/group. Scale bars, 25 pm.
  • Figure 27 Long-term normalization of CNS lysosomal compartment size after an intra-CSF gene transfer of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Evaluation of the size of lysosomal compartment by LIMP2 immunostaining in different brain regions of 8-month-old male and female wild-type (healthy) mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • a short linker L1 AB mouse and L3 AB mouse
  • Figure 28 Long-term restoration of lysosomal homeostasis in the CNS following an intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Percentage of WT activity of several lysosomal enzymes analyzed in brain extracts from 8-month-old male and female wild-type (healthy) mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • a short linker L1 AB mouse and L3 AB mouse
  • Restoration of altered activities of alpha-N-acetylgalactosamine- 6 sulfatase (GALNS), beta-glucuronidase (GUSB), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), alpha-N-acetylglucosaminidase (NAGLU) and N-sulphoglucosamine sulphohydrolase (SGSH) in all treated Sandhoff mice. Results are expressed as mean + SEM. n 5 animals/group. *P ⁇ 0.05, **P ⁇ 0.01 and
  • Figure 30 Long-term correction of microglial infiltration after an intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Quantification by qPCR of CD68 expression, a marker of microglia, in section I and V, the most rostral and caudal regions, respectively, of the brain in 8-month-old male and female wild-type (healthy) mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • a short linker L1 AB mouse and L3 AB mouse
  • FIG 32 Long-term intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice restores hepatic lysosomal homeostasis.
  • SGSH N-sulphoglucosamine sulphohydrolase
  • Figure 33 Long-term normalization of behavioral deficits after intra-CSF administration of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice.
  • the Open-Field test was performed in 8- month-old naive-tested male and female wild-type (healthy) mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of AAV9 vectors encoding both Hexa and Hexb genes (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age.
  • Sandhoff mice received a total dose of 1x10 A 11 vg/mouse of control vectors (Null) into the CSF at 1 month of age.
  • Figure 34 Long-term correction of mobility and disease progression deficits after intra-CSF administration of AAV9 vectors encoding both Hexa and Hexb genes in Sandhoff mice. Mobility and disease progression was assessed with the righting reflex test in 8-month-old male and female wild-type (healthy) mice and Sandhoff mice that received a total dose of 1x10 A 11 vg/mouse of AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker (L1 AB mouse and L3 AB mouse) into the CSF at 1 month of age. Sandhoff mice received a total dose of 1x10 A 11 vg/mouse of control vectors (Null) into the CSF at 1 month of age.
  • L1 AB mouse and L3 AB mouse short linker
  • L1 AB mouse gene construct (SEQ ID NO:75) L2 AB mouse gene construct (SEQ ID NO:76) L3 AB mouse gene construct (SEQ ID NO:77) L1 BA mouse gene construct (SEQ ID NO:78) L2 BA mouse gene construct (SEQ ID NO:79) L3 BA mouse gene construct (SEQ ID NO:80) Hexa single gene construct (SEQ ID NO:85) Hexb single gene construct (SEQ ID NO:86) P2A AB mouse gene construct (SEQ ID NO:87) P2A BA mouse gene construct (SEQ ID NO:88) L1 AB human noh gene construct (SEQ ID NO: 81) L1 AB human GA gene construct (SEQ ID NO:82) L1 AB human IDT gene construct (SEQ ID NO:83) L1 AB human NV gene construct (SEQ ID NO:84)
  • HEK293 Human embryonic kidney 293 (HEK293) cells were cultured at 37°C under humidified 5% CO 2 in Dulbecco’s Modified Eagle Medium (DMEM high glucose, Thermofisher Scientific) containing 10% Gibco fetal bovine serum (FBS) (ThermoFischer Scientific). The day before transfection, HEK293 cells were seeded in 6- or 24-well plates at a cell density of 1.2x10 A 6 or 2.25-2.5x10 A 5 cells/well in 2 or 1 ml/well of total volume, respectively. Prior to transfection, cells were assessed for correct cell confluence (90%) at a bright field microscope. Cells were transfected using Lipofectamine® 2000 (ThermoFisher Scientific), following the protocol provided by the manufacturer.
  • DMEM Modified Eagle Medium
  • FBS Gibco fetal bovine serum
  • plasmid DNA and 10 or 2 pl/well of Lipofectamine® 2000 were each diluted in 250 or 50 pl OptiMEM prepared for 6- or 24-well plates, respectively.
  • Mixed reagents were incubated for 5 minutes.
  • the Lipofectamine mix was added to the DNA mix, then gently mixed, and incubated for 20 minutes priorto drop-wise addition to wells (500 or 100 pl/well to 6- or 24 well plates, respectively).
  • Each plasmid (pAAV-Cbh-omHexa-L1-omHexb (SEQ ID NO:28), pAAV-Cbh-omHexa-L2-omHexb (SEQ ID NO:29), pAAV- Cbh-omHexa-L3-omHexB (SEQ ID NO: 30), pAAV-Cbh-omHexb-L1-omHexA (SEQ ID NO: 31), pAAV-Cbh- omHexb-L2-omHexA (SEQ ID NO:32), pAAV-Cbh-omHexb-L3-omHexa (SEQ ID NO:33), pAAV-Cbh-omHexa- P2A-omHexb (SEQ ID NO:36), pAAV-Cbh-omHexb-P2A-omHexa (SEQ ID NO:59),
  • a mutant C57B129SF2/J HexB-deficient mouse (Sandhoff) in which a neomycin resistance cassette was inserted into and disrupted exon 13 of the Hexb gene was purchased from The Jackson Laboratory (Sango et al, 1995). This targeted mutation resulted in no detectable functional protein. Sandhoff and healthy control mice were inbred from heterozygous founders.
  • a mutant C57B129SF2/J HexA-deficient mouse (Tay-Sachs) in which a neomycin resistance cassette was inserted into and disrupted exon 8 of the Hexa gene was purchased from The Jackson Laboratory (Sango et al, 1995). This targeted mutation resulted in neither detectable RNA transcript nor functional protein. Tay-Sachs and healthy control mice were inbred from heterozygous founders.
  • Single-stranded AAV vectors of serotype 9 were produced by triple transfection of HEK293 cells according to standard methods (Ayuso et al, 2010). Cells were cultured to 80% confluence in roller bottles (RB) (850 cm 2 , flat; CorningTM, Sigma-Aldrich Co., Saint Louis, MO, US) in DMEM supplemented with 10% FBS and then cotransfected by calcium phosphate method with: 1) a plasmid carrying the expression cassette flanked by the AAV2 ITRs (SEQ ID NOs: 25-26); 2) a helper plasmid carrying the AAV2 rep gene and the AAV of serotype 9 cap gene; and 3) a plasmid carrying the adenovirus helper functions.
  • Transgenes used were: a) the murine optimized Hexa and Hexb coding-sequence fused with a short linker (L1 - SEQ ID NOs: 11 and 14 , L2 - SEQ ID NOs: 12 and 15 or L3 - SEQ ID NOs: 13 and 16); b) the murine optimized Hexa and Hexb coding-sequence fused with a self-cleaving linker (P2A); c) the murine optimized Hexa coding-sequence (SEQ ID NO: 5); d) the murine optimized Hexb coding-sequence (SEQ ID NO: 10); e) the human HEXA coding-sequence (SEQ ID NO: 3); f) the human coding HEXB coding-sequence (SEQ ID NO:8); g) the human optimized HEXA codingsequence (SEQ ID NOs: 64, 66, 68); h) the human
  • each cassette was driven by the ubiquitous promoter Cbh (a short version of the widely used CAG promoter composed of the cytomegalovirus (CMV) early enhancer, chicken beta-actin promoter and a hybrid intron) (SEQ ID NO: 24).
  • Cbh a short version of the widely used CAG promoter composed of the cytomegalovirus (CMV) early enhancer, chicken beta-actin promoter and a hybrid intron
  • the expression cassette also included the polyA sequence from SV40 (SEQ ID NO: 27).
  • AAV vectors were purified with an optimized method based on a polyethylene glycol precipitation step and two consecutive cesium chloride (CsCI) gradients.
  • CsCI cesium chloride
  • This second-generation CsCI- based protocol reduced empty AAV capsids and DNA and protein impurities dramatically (Ayuso et al, 2010).
  • Purified AAV vectors were dialyzed against PBS, filtered, and stored at -80°C. Titers of viral genomes were determined by quantitative PCR following the protocol described for the AAV2 reference standard material using linearized plasmid DNA as standard curve (Lock et al, 2010). The vectors were constructed according to molecular biology techniques well known in the art.
  • mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and the skin of the posterior part of the head, from behind the ears to approximately between the scapulae, was shaved. Mice were held in prone position, with the head at a slightly downward inclination. A 2-mm rostro-caudal incision was made to introduce a Hamilton syringe at an angle of 45-55° into the cisterna magna, between the occiput and the C1-vertebra and 5 pl of vector dilution was administered.
  • mice were dosed with the same number of vector genomes/mouse irrespective of body weight (1x10 A 11 vg/mouse).
  • the mouse brain volume is about 0.45 cm A 3, so this dose corresponds to about 2.22 x 10 A 11 vg per ml of brain.
  • mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Blood was extracted by cardiac puncture and, afterwards, animals were transcardially perfused with 10 ml of PBS. This procedure cleared blood from tissues thus removing all traces of circulating beta-hexosaminidase. Then, the entire brain and multiple somatic tissues were collected. The encephalon was longitudinally divided into two hemispheres (left and right), and each half was then coronally sectioned in five regions (I to V), section I being the most frontal and section V the most caudal. All samples were either snap frozen in liquid nitrogen and stored at -80°C or immersed in formalin or 4%PFA for subsequent histological analyses.
  • Total beta-hexosaminidase (total beta-HEXO) was assayed in 0.2 pg of protein or 1 pl of medium at pH 4.5 for 1 h at 37°C with 5 mM 4-MUG (Sigma).
  • HexA was assayed in 0.2 pg of protein, 1 pl of medium or 0.4 pl of serum at pH 4.4 for 1 h at 37°C with 5 mM 4-MUGS (Sigma).
  • Heparan acetylCoA:alpha-Glucosaminide N-Acetyltransferase (heparan-alpha-glucosaminide N- acetyltransferase; HGSNAT) activity was assayed in 30 pg of protein extracts incubated with 3 mM 4- methylumbelliferyl-beta-D-glucosamine (Moscerdam substrates) supplemented with 12 mM Acetyl coenzyme A (Sigma) for 17h at 37°C.
  • N-sulfoglucosamine sulfohydrolase (SGSH) was assayed in a two-step protocol.
  • the first step consisted of the incubation of 30 pg of tissue protein extract with 10 mM of 4-MU-alphaGlcNS for 17 hours at 47°C.
  • the second incubation was carried out in the presence of 10 U/ml of alpha-glucosidase (Sig ma-Ald rich) in 0.2% BSA for 24 hours at 37°C.
  • Alpha-N-acetylgalactosamine-sulfate (NAGLU) activity was assayed in 30 pg of tissue protein extract incubated with 2.5 nmol/l 4-methylumbelliferyl-alpha-d-N-acetyl- glucosaminide (MU-alphaGIcNAc, Moscerdam Substrates) for 3 h at 37°C.
  • MU-alphaGIcNAc 4-methylumbelliferyl-alpha-d-N-acetyl- glucosaminide
  • GALNS N-acetylgalactosamine-6-sulfatase activity was assayed in 10 pg of total protein with a first incubation step with 10 mM 4- methylumbelliferyl beta-D-galactopyranoside-6-sulfate (Toronto Research Chemicals) for 17h at 37°C followed by a second incubation with beta-galactosidase (Sigma) for 2h at 37°C.
  • beta-glucuronidase GUSB
  • activity was assayed in 15 pg of protein incubated for 1 h with 2 mM 4-methylumbelliferyl-beta-D-glucuronide (Sigma) at pH 4.8 and 37°C.
  • tissue were fixed for 12-24 h in formalin, embedded in paraffin and sectioned. Tissue sections were incubated overnight at 4°C with rabbit anti-LIMP2 antibody (NB400; Novus Biologicals) and rabbit anti-GFAP antibody (Ab6673; Abeam) and subsequently incubated with biotinylated goat anti-rabbit antibody (31820; Vector Laboratories), used as secondary antibody.
  • rabbit anti-LIMP2 antibody NB400; Novus Biologicals
  • rabbit anti-GFAP antibody Ab6673; Abeam
  • biotinylated goat anti-rabbit antibody 31820; Vector Laboratories
  • LIMP2 and GFAP signals were amplified by incubating sections with ABC-Peroxidase staining kit (Thermo Scientific), visualized using 3,3-diaminobenzidine (Sigma-Aldrich) as a chromogen, and counterstained with hematoxylin. Brightfield images were obtained with an optical microscope (Eclipse 90i; Nikon). LIMP2 and GFAP signals were quantified with the NIS-Elements Advanced Research 2.20 software in 3-4 images of each brain region (original magnification, 20X) per animal, using the same signal threshold settings for all images. Then, the percentage of positive area was calculated, i.e. , the area, in pixels, with a positive signal over the total tissue area in the image.
  • tissues were fixed overnight in 4% PFA. Afterwards, tissues were dehydrated with 30% sucrose in PBS at 4°C for 48h for cryoprotection, subsequently embedded in Optimal Cutting Temperature (OCT) compound and kept at -80°C until processing. Brains were then cryosectioned at -15°C using a cryostat microtome to 14 pm thickness. For filipin staining, tissue sections were rehydrated in PBS, and incubated with filipin complex diluted in PBS to a working concentration of 0.05 mg/mL for 2h at room temperature in dark.
  • OCT Optimal Cutting Temperature
  • GM2 immunohistochemistry tissue sections were rehydrated in PBS, incubated overnight at 4°C with mouse anti-GM2 antibody (AMS A2576; Amsbio) and subsequently incubated with biotinylated horse anti-mouse antibody (BA2000; Vector Laboratories), used as secondary antibody.
  • GM2 signal was visualized as previously described herein for immunohistochemical detection of LIMP2 and GFAP. Fluorescence (filipin) and bright-field (GM2) images were obtained with an optical microscope (Eclipse 90i; Nikon).
  • Proteins from medium of HEK-293 cells were separated by 10% wt/vol sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and incubated overnight at 4°C with the following antibodies anti-rabbit anti-HEXA (PAA195MuO1 , Cloud-Clone Corp) and anti-rabbit anti-HEXB (PAA637MuO1 , Cloud-Clone Corp).
  • SDS sodium dodecyl sulfate
  • PVDF polyacrylamide gel electrophoresis
  • RNA analysis Total RNA was purified from brain and liver homogenized in Tripure Isolation Reagent (Roche) with RNeasy Mini kit (Qiagen). To eliminate the residual DNA, total RNA was treated with DNAsel (Qiagen). RNA was quantified in a NanoDrop ND-1000 spectrophotometer (NanoDrop). cDNA was synthesized with a Transcriptor First Strand cDNA Synthesis kit (Roche). Real-time quantitative PCR was performed in a QuantstudioTM 5 (Thermofisher) using Lightcycler 480 SyBr Green I Master Mix (Roche).
  • lipid extraction For lipid extraction, ⁇ 100 mg of tissue was homogenized in 7.5 ml chloroform-methanol (2:1 vol/vol) and then 1.5 ml 0.05% H 2 SO 4 was added to the mixture. After an overnight at 4°C to separate the organic from the aqueous phase, 1 ml of the organic phase was recovered and mixed with 1 ml of X-100 Triton 1 % in chloroform. After evaporation at 90°C, 1 ml of chloroform was added and then evaporated, this process was performed twice. In the end, extracted lipids were resuspended in 500 pl of milliQ water. Total cholesterol determination was quantified in lipid extracts spectrophotometrically using an enzymatic assay (ABX Pentra Cholesterol CP, Horiba) in a Pentra 400 Analyzer (Horiba).
  • Tissue samples were weighed and then digested with proteinase K. The resultant extracts were clarified by centrifugation and filtration. GAG content was determined in tissue extracts with the Blyscan sulfated glycosaminoglycan kit (Biocolor), using chondroitin 4-sulfate as standard. GAG content was normalized to wet tissue weight.
  • the open field test was performed between 9:00 am and 2:00 pm, to minimize influence of circadian cycles. Briefly, animals were placed in the lower right corner of a brightly lit chamber (41x41x30 cm) divided in three squared concentric regions (center, 14x14cm; periphery, 27x27cm; and border, 41x41 cm). Exploratory behavior and general activity were recorded during the first three minutes using a video-tracking system (SmartJunior v3, Panlab).
  • Righting reflex was used to assess mobility.
  • a mouse was placed on its back on a tabletop (supine position) and the time elapsed forthe animal to right itself through 180° (all 4 paws were right on the floor) was measured, with a maximum of 10s allowed. The test was performed 3 times on each mouse and the mean of all three trials was used for analysis.
  • mice were tested in the mesh test.
  • the mouse was placed right in the center of a wire mesh that then was rotated 180° to an inverted position (over the course of about 2 s) with the head of the mouse declining first.
  • the mouse was held 40 cm above a soft padded surface, and the latency to hang upside down from the wire mesh was recorded to a maximum time of 60 s.
  • Mice were given three trials, spaced out by 5 min, and the mean of all three trials was recorded for analysis. If animals did not explore during the trial, they were not included in the data analysis.
  • mice were tested on an accelerating rotarod (Rotarod LE8200; Panlab), spinning at 4 RPM. Lane width, 50 mm; rod diameter, 30 mm. Before the trial, mice were gently placed on the rod with the speed set at 4 rpm and trained to remain on the rod. After training, mice were given three consecutive trials of 90 seconds in which the rod accelerated from 4-40 rpm in a 5-minute interval. Between trials, animals rest for 90 seconds. The next day, mice took 3 more trials on the rod. Latencies to fall from the rod were recorded and the maximum latency of all trials was used for analysis.
  • the ubiquitous promoter Cbh (SEQ ID NO: 24) (a short version of the widely used CAG promoter composed of the cytomegalovirus (CMV) early enhancer, chicken beta-actin and a hybrid intron) together with the polyA sequence from SV40 (SEQ ID NO: 27) were chemically synthetized (GeneArt; Invitrogen). Afterwards, the Cbh+SV40 was excised by Notl digestion and then cloned inside the Notl restriction site of the AAV backbone plasmid pAAV-MCS (multicloning site) (AmpR).
  • CMV cytomegalovirus
  • SV40 SEQ ID NO: 27
  • the pAAV-MCS plasmid was previously generated and contained the ITRs from the AAV2 genome (SEQ ID NOs: 25-26), as well as the multicloning site for cloning the coding sequences (CDS) of interest. After this cloning, the resulting plasmid was named pAAV-Cbh-SV40 ( Figure 1A).
  • L1 AB, L2 AB and L3 AB mouse constructs SEQ ID NOs: 75-77
  • a 3’ fragment of the optimized murine Hexa CDS omHexa
  • a 5’ fragment of the optimized murine Hexb CDS omHexb
  • L1 , L2 or L3 - SEQ ID NOs: 11-16 short linkers
  • CDS were cloned inside the pAAV-Cbh-om/-/exa-P2A-om/-/exb- SV40 plasmid flanked by Afel and BbvCI restriction sites at 5’ and 3’ ends, respectively.
  • the resulting plasmid was named pAAV-Cbh-om/-/exa-L1-om/-/exb-SV40 (SEQ ID NO: 28), pAAV-Cbh-om/-/exa-L2-om/-/exb- SV40 (SEQ ID NO: 29) and pAAV-Cbh-omHexa-L3-omHexb-SV40 (SEQ ID NO: 30) ( Figure 1 B).
  • L1 BA, L2 BA and L3 BA mouse constructs SEQ ID NOs: 78-80
  • a 3’ fragment of the optimized murine Hexb CDS omHexb
  • a 5’ fragment of the optimized murine Hexa CDS omHexa was fused with one of the short linkers (L1 , L2 or L3 - SEQ ID NOs: 11-16) and used as starting material and chemically synthetized for this purpose (GeneArt; Invitrogen).
  • CDS were cloned inside the pAAV-Cbh-om/-/exb-P2A-om/-/exa-SV40 plasmid (previously generated) flanked by Alel and Avril restriction sites at 5’ and 3’ ends, respectively.
  • the 3’ fragment of the optimized murine Hexb CDS and the 5’ fragment of the optimized murine Hexa CDS fused with one of the short linkers (L1 , L2 or L3) was excised by Alel/Avrl I digestion and then cloned between the Alel and Avril restriction sites at 5’ and 3’ ends, respectively.
  • the optimized murine HexB CDS and then the optimized murine Hexa CDS fused with one of the short linkers (L1 , L2 or L3) was excised by Alel/Avrl I digestion and then cloned between the Alel and Avril restrictions sites of the AAV backbone plasmid pAAV-Cbh-om/-/exb-P2A- om/-/exa-SV40 (AmpR).
  • the resulting plasmids was named pAAV-Cbh-om/-/exb-L1-om/-/exa-SV40 (SEQ ID NO: 31), pAAV-Cbh-om/-/exb-L2-om/-/exa-SV40 (SEQ ID NO: 32) and pAAV-Cbh-omHexb-L3-omHexa-SV40 (SEQ ID NO: 33) ( Figure 1 C).
  • the optimized murine either Hexa or Hexb CDS were used as starting material and chemically synthetized for this purpose (GeneArt; Invitrogen). Either of these CDS was cloned inside the pAAV-Cbh-SV40 plasmid flanked by Nhel and BamHI restriction sites at 5’ and 3’ ends, respectively.
  • Optimized murine either Hexa or Hexb was excised by Nhel/BamHI digestion and then cloned between the Nhel and BamHI restriction sites of the AAV backbone plasmid pAAV-Cbh-SV40 (AmpR).
  • the resulting plasmids were named pAAV-Cbh-om/-/exa-SV40 (SEQ ID NO: 34) or pAAV-Cbh-om/-/exb-SV40 (SEQ ID NO: 35) ( Figure 1 D).
  • the optimized murine Hexa and Hexb CDS fused with the self-cleaving linker P2A was used as starting material and chemically synthetized for this purpose (GeneArt; Invitrogen).
  • This CDS was cloned inside the pAAV-Cbh-SV40 plasmid flanked by Nhel and BamHI restriction sites at 5’ and 3’ ends, respectively.
  • the optimized murine Hexa and Hexb CDS fused with the self-cleaving linker P2A was excised by Nhel/BamHI digestion and then cloned between the Nhel and BamHI restrictions sites of the AAV backbone plasmid pAAV-Cbh-SV40 (AmpR).
  • the resulting plasmid was named pAAV-Cbh-omHexa-P2A-omHexb-SV40 (SEQ ID NO; 36) ( Figure 1 E).
  • L1 AB noh, L1 AB GA, L1 AB IDT, and L1 AB NV human constructs SEQ ID NOs: 81-84
  • the nonoptimized and GA-, IDT- and NV-optimized human HEXA and HEXB CDS fused with a short linker L1 - SEQ ID NOs: 11-14
  • This CDS was cloned inside the pAAV-Cbh-SV40 plasmid flanked by Nhel and BamHI restriction sites at 5’ and 3’ ends, respectively.
  • HEXA-L1-HEXB The non-optimized and GA-, IDT- and NV-optimized human HEXA and HEXB CDS fused with a short linker (HEXA-L1-HEXB) was excised by Nhel/BamHI digestion and then cloned between the Nhel and BamHI restrictions sites of the AAV backbone plasmid pAAV-Cbh-SV40 (AmpR).
  • the resulting plasmid was named pAAV-Cbh-nohHEXA-L1-nohHEXB-SV40 (SEQ ID NO; 60), pAAV-Cbh-ohHEXA-L1-ohHEXB-SV40 GA (SEQ ID NO; 61), pAAV-Cbh-ohHEXA-L1-ohHEXB-SV40 IDT (SEQ ID NO; 62) or pAAV-Cbh-ohHEXA-L1- ohHEXB-SV40 NV (SEQ ID NO; 63) ( Figure 1 B).
  • the optimized murine Hexb and Hexa CDS fused with the self-cleaving linker P2A was used as starting material and chemically synthetized for this purpose (GeneArt; Invitrogen).
  • This CDS was cloned inside the pAAV-Cbh-SV40 plasmid flanked by Nhel and BamHI restriction sites at 5’ and 3’ ends, respectively.
  • AAV9-Cbh-SV40, AAV9-Cbh-omHexa-SV40, AAV9-Cbh-omHexb-SV40, AAV9-Cbh-omHexa-P2A-omHexb- SV40, AAV9-Cbh-om/-/exa-L1-om/-/exb-SV40, AAV9-Cbh-omHexa-L2-omHexb-SV40, AAV9-Cbh-omHexa-L3- omHexb-SV40, AAV9-Cbh-omHexb-L1-omHexa-SV40, AAV9-Cbh-omHexb-L2-omHexa-SV40 and AAV9-Cbh- om/-/exb-L3-om/-/exa-SV40 were generated by helper virus-free transfection of HEK293 cells using
  • Cells were cultured to 80% confluence in roller bottles (RB) (Corning) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs; 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described (Ayuso et al, 2010). Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • Example 4 Therapeutic efficacy after intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes (L1 AB mouse, L2 AB mouse and L3 AB mouse) in Sandhoff mice
  • the endo-lysosomal compartment is an organelle also involved in autophagy; therefore, the perturbation of lysosomal distension and dysfunction could lead to an altered autophagic efflux.
  • LC3B-I I/LC3B-I a ratio of the cytosolic form (LC3B-I) versus the covalently linked to a lipid in the phagosome membrane (LC3B-I) was assessed by Western-blot.
  • Hepatic overexpression of HexA and HexB genes resulted in increased secretion of HexA activity into the bloodstream with values several folds higher than those documented in WT littermates ( Figure 12).
  • hepatic and circulating HexA activity were almost undetectable in 4-month-old untreated and Null-injected Sandhoff mice.
  • the levels of HexA activity were considerably lower in females than in males, reproducing observation made in other studies in liver (Haurigot et al, 2013; Ribera et al, 2015; Roca et al, 2017; Motas et al, 2016).
  • Sandhoff disease is a neurodegenerative disorder that mainly affects the CNS together with a regress in developmental milestones (Regier, 2016).
  • the juvenile and late-onset forms presented gait disturbance, incoordination and imbalance; while infantile forms showed a developmental arrest, exaggerated startle response, and hypotonia (Regier, 2016; Bley 2011 Pediatrics).
  • To further characterize the behavioral, locomotor and coordination alterations in Sandhoff mouse model a battery of non-invasively behavioral tests (Open Field, Rotarod, Mesh, Righting reflex and Hindlimb clasping tests) was performed.
  • Example 5 Therapeutic efficacy after intra-CSF delivery of two AAV9 vectors encoding Hexa or Hexb (Hexa+Hexb) or AAV9 vectors encoding both Hexa and Hexb genes fused with the self-cleaving linker P2A (P2A AB) in Sandhoff mice
  • a total dose of either 2x10 A 11 vg/mouse for the single Hexa and Hexb AAV9 vectors (1x10 A 11 vg/mouse for each vector) or 1x10 A 11vg/mouse for P2A AB mouse AAV9 vector was administered into the cisterna magna of 1-month-old Sandhoff mice. These animals were then analyzed 3 months after treatment. Age-matched Sandhoff animals injected into the cisterna manga with a total dose of 1x10 A 11 vg/mouse of AAV9-Cbh-Null vectors were used as control. Healthy wild-type and untreated Sandhoff mice served as additional control groups.
  • the designed constructs used the Cbh ubiquitous promoter, the optimized murine Hexa and Hexb genes and the polyA sequence that were exactly the same as our previous studies with a covalently linked betahexosaminidase with a short flexible linker (L1 , L2 or L3).
  • Sandhoff mice were treated in the same conditions (at 1 month of age with the same dose (1x10 A 11 vg/animal)) and followed up to 3 months posttreatment.
  • Example 6 Increased beta-hexosaminidase after intra-CSF delivery of AAV9 vectors encoding Hexa and Hexb genes (L1 AB mouse and L3 AB mouse construct) in Tay-Sachs mice
  • Example 7 Increased beta-hexosaminidase in HEK293 cells after transfection with plasmids encoding both HEXA and HEXB genes fused with a short linker
  • the "GA”, “IDT” and “NV” optimized sequences of the human HEXA gene are represented by SEQ ID NO: 64, 66 and 68.
  • the "GA”, “IDT” and “NV” optimized sequences of the human /-/EXB gene are represented by SEQ ID NO: 65, 67, and 69.
  • Example 4 The therapeutic effects described in Example 4 were also observed in Sandhoff mice 7 months after intra-CSF delivery of AAV9 vectors encoding both Hexa and Hexb genes fused with a short linker L1 or L3 (L1 AB mouse and L3 AB mouse) under the control of the ubiguitous promoter Cbh a total dose of 1x10 A 11 vg/mouse.
  • Hepatic overexpression of HexA and HexB genes also resulted in increased secretion of HexA activity, at long term, into the bloodstream with values several folds higherthan those documented in WT littermates (Figure 31).
  • the levels of HexA activity were considerably lower in females than in males, reproducing observation made in other studies in liver (Haurigot et al, 2013; Ribera et al, 2015; Roca et al, 2017; Motas et al, 2016).
  • This gender difference is due to certain AAV serotypes are more efficient at transducing the liver of male rodents (Ruzo et al, 2012a, 2012b; David off et al, 2003).
  • Gm2 gangliosidoses Clinical features, pathophysiological aspects, and current therapies Int J Mol Sci 21 : 1-27 doi: 10 3390/ijms21176213 [PREPRINT]
  • Tay-Sachs disease generalized absence of a beta-D-N-acetylhexosaminidase component Science 165: 698-700
  • Torres PA Zeng BJ, Porter BF, Alroy J, Horak F, Horak J & Kolodny EH (2010) Tay-Sachs disease in Jacob sheep Mol Genet Metab 101 : 357-363
  • Venugopalan P & Joshi SN (2002) Cardiac involvement in infantile Sandhoff disease J Paediatr Child Health 38: 98-100
  • SEQ ID NO: 5 Optimized nucleotide sequence of Mus musculus alpha subunit of beta-hexosaminidase
  • SEQ ID NO: 10 Optimized nucleotide sequence of Mus musculus beta subunit of beta-hexosaminidase
  • SEQ ID NO: 1 Amino acid sequence of peptide linker L1 : GGGGSGGGGSGGGGSGGGGS
  • SEQ ID NO: 12 Amino acid sequence of peptide linker L2: SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
  • SEQ ID NO: 16 Nucleotide sequence of peptide linker L3: GGCAGCGCCGGCAGCGCCGCCGGCAGCGGCGAGTT C
  • SEQ ID NO: 64 GA optimized nucleotide sequence of Homo sapiens alpha subunit of beta hexosaminidase
  • SEQ ID NO: 65 GA optimized nucleotide sequence of Homo sapiens beta subunit of beta hexosaminidase

Abstract

L'invention concerne une construction génique comprenant une séquence nucléotidique codant pour une sous-unité alpha d'une bêta-hexosaminidase, une séquence nucléotidique codant pour un lieur peptidique, et une séquence nucléotidique codant pour une sous-unité bêta d'une beta-hexosaminidase. Les aspects et les modes de réalisation ci-décrits peuvent être utilisés dans le traitement des gangliosidoses à GM2, y compris les maladies de Tay-Sachs et de Sandhoff.
PCT/EP2023/074474 2022-09-07 2023-09-06 Vecteurs de beta-hexosaminidase WO2024052413A1 (fr)

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