WO2019207167A1 - Therapy of sulfatase deficiencies - Google Patents

Therapy of sulfatase deficiencies Download PDF

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Publication number
WO2019207167A1
WO2019207167A1 PCT/EP2019/060980 EP2019060980W WO2019207167A1 WO 2019207167 A1 WO2019207167 A1 WO 2019207167A1 EP 2019060980 W EP2019060980 W EP 2019060980W WO 2019207167 A1 WO2019207167 A1 WO 2019207167A1
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sulfatase
human
vector
sgsh
nucleotide sequence
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PCT/EP2019/060980
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French (fr)
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Andrea Ballabio
Nicolina Cristina SORRENTINO
Alessandro FRALDI
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Fondazione Telethon
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/03—Oxidoreductases acting on sulfur groups as donors (1.8) with oxygen as acceptor (1.8.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06001—Arylsulfatase (3.1.6.1)
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06004—N-Acetylgalactosamine-6-sulfatase (3.1.6.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y310/00—Hydrolases acting on sulfur-nitrogen bonds (3.10)
    • C12Y310/01—Hydrolases acting on sulfur-nitrogen bonds (3.10) acting on sulfur-nitrogen bonds (3.10.1)
    • C12Y310/01001—N-Sulfoglucosamine sulfohydrolase (3.10.1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to improved gene therapy to treat sulfatase deficiencies, preferably via co-delivery of Sulfatase enzymes and SUMF1 protein.
  • Sulfatases are hydrolases that cleave sulfate esters from a wide range of substrates, including glycosaminoglycans, sulfolipids, and steroid sulfates ("The sulfatase gene family". Parenti, G, Meroni, G, and Ballabio, A.Curr. Opin. Genet. Dev. 1997; 7: 386-391; "Multiple sulfatase deficiency and the nature of the sulfatase family" .Hopwood, J.J and Ballabio, A.) [1].
  • At least eight inherited metabolic disorders in humans are caused by sulfatase deficiencies, resulting in impaired desulfation of sulfatase natural substrates.
  • sulfatase deficiencies resulting in impaired desulfation of sulfatase natural substrates.
  • sulfated substrates accumulate in the cells and tissues of patients, resulting in Lysosomal Storage Disorders (LSD) with consequences depending on the type and tissue distribution of the stored material.
  • LSD Lysosomal Storage Disorders
  • sulfatases include MLD (metachromatic leukodystrophy), which is characterized by the storage of sulfolipids in the central and peripheral nervous systems, and five different types of Mucopolysaccharidoses (MPSs types II, IMA, MID, IVA and VI), which are due to the accumulation of Glycosaminoglycans (GAGs) in several tissues and organs[2].
  • MLD metalchromatic leukodystrophy
  • MLD Mucopolysaccharidoses
  • IMA IMA
  • MID MID
  • IVA Mucopolysaccharidoses
  • Active sulfatases contain a unique FGIy (formylglycine) catalytic residue which derived from a post-translational modification of a cysteine precursor.
  • SUMF1 sulfatase-modifying factor 1
  • MSD multiple sulfatase deficiency
  • SUMF1 has been shown to have an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells as well as co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by diseases owing to sulfatase deficiencies or from murine models of the same diseases.
  • AAV adeno-associated virus
  • LV lentivirus
  • Mucopolysaccharidosis type IMA is one of the most common and severe forms of neurodegenerative lysosomal storage disorders (LSDs) [10, 11]. It is caused by inherited defect of the sulfamidase (SGSH), a soluble lysosomal enzyme belonging to the family of sulfatases [12] and leads to the accumulation of heparan sulfate in cell, particularly within the central nervous system (CNS). Presently, there are no treatments available to treat the CNS in M PS-111 A patients. Gene delivery aimed at correcting defective hydrolytic lysosomal defects represent the most promising replacement strategy for the CNS treatment of M PS-111 A as well as other MPSs with similar causes due to its potential for a one-time treatment [13].
  • SGSH sulfamidase
  • CNS central nervous system
  • adeno-associated viral (AAV) vectors are most commonly utilized for in vivo gene transfer because they are safe, provide significantly long transgene expression and may be generated with variable serotypes allowing efficient delivery of therapeutic genes to different target tissues [14, 15].
  • AAV adeno-associated viral
  • several therapeutic approaches have been designed and developed based on AAV-mediated gene delivery of SGSH using different route of administrations to reach the CNS [4, 5, 16-19]. Although all these approaches have demonstrated potential benefits in preclinical animal models, the effective therapeutic application of these protocols in the clinical management of MPS-IIIA patients is challenging because of the difficulty in achieving widespread distribution of the corrective enzyme in the CNS and in maintaining therapeutic threshold levels of them in targeted cells [20].
  • Enhancing the therapeutic potential of sulfamidase, along with developing tools for efficient and safe CNS targeting, may represent a novel area of intervention to improve CNS therapy in MPS- IMA patients.
  • Administration of AAVs by the cerebrospinal fluid (CSF) route of injection provides very limited access to visceral organs while at the same time allowing the circulating virus to access a large CNS surface area, a condition that could reduce cytotoxic effects of whole body exposure and may potentially limit the amount of viral vector required to efficiently transduce CNS cells [23].
  • serotype 9 has been shown to have a broad tropism, including neurons and astrocytes, when delivered either via CSF or through intravenous injection due to its capability to cross the blood-brain barrier (BBB) [24, 25].
  • BBB blood-brain barrier
  • the inventors have previously demonstrated that AAV9 outperforms all other AAV serotypes tested in CNS transduction efficiency when administered via CSF even in large animal models [6]. Lysosomal hydrolases, including SGSH can be secreted and taken up by surrounding cells [26]. Such cross-correction capability makes gene replacement protocols potentially more effective since transduced cells (factory cells) may also correct non-transduce cells.
  • a potentiated SGSH expression cassette was generated by combining enhanced secretion efficiency of SGSH with improved SUMFl-mediated enzyme activation capacity and its therapeutic potential was explored upon intra-CSF AAV9-mediated gene delivery.
  • the present results demonstrate that by using this strategy it is possible to achieve a superior SGSH CNS bio- distribution in a large animal model (wild type pigs) and to fully rescue the CNS phenotype in a mouse model of sulfatase deficiency, as M PS-111 A.
  • the present inventors have surprisingly obtained increased enzymatic activity and CNS distribution of the sulfamidase gene through intra-cerebral spinal fluid (CSF) administration of a highly secreted version of the sulfamidase gene (eg a sulfamidase modified to include the IDS signal peptide, IDSsp) via AAV-mediated delivery and co-delivery of said modified SGSH and SUMFlproteins.
  • CSF intra-cerebral spinal fluid
  • the inventors approach was to focus on gene transfer approaches to treat the CNS in LSDs with minimal invasiveness and high CNS targeting efficiency.
  • cells expressing non-modified SGSH, or expressing modified SGSH, or expressing SUMF1 or expressing both non-modified SGSH and SUMF1 there was a striking improvement in SGSH activity by supplying exogenous modified SGSH together with SUMF1 as a bicistronic construct expressing the two proteins.
  • the construct IDSsp-SGSH-SUMFl has the highest expression levels in pigs. Further the construct IDSsp-SGSH displays less enzymatic activity than the IDSsp-SGSH-SUMFl construct and the SGSH activity induced by the SGSH-SUMF construct is lower than the one induced by the IDSsp-SGSH-SUMFl construct.
  • the vector is structurally different than prior vectors in that it incorporates both the modified SGSH and SUMF1 genes in a single expression cassette under the control of a single promoter.
  • the two genes are co-delivered as one vector, rather than as single gene vectors.
  • the bicistronic vector is superior either to modified SGSH alone, or to a combination of single gene vectors for modified SGSH and SUMF1.
  • the present vector would be used to deliver the modified SGSH and SUMF1 genes to human brain so as to increase SGSH brain distribution and SGSH activity for the treatment of CNS pathology in sulfatase deficiency. While a preferred embodiment of the present invention involves co-delivery of a modified sulfatase, preferably SGSH, and SUMF1 to the brain via an adeno-associated viral vector, it is also anticipated that other delivery mechanisms, including retro viral, adeno viral, herpes simplex viral, cytomegaloviral, could also be used.
  • Methods for formulating pharmaceutical compositions or carriers for the bicistronic constructs or vectors disclosed herein are well known in the art (e.g. U.S. Pat. Nos.
  • Non-viral delivery vehicles could be used as well, including approaches that utilize liposomes, polylysine carrier complexes, or naked DNA (1992, Proc. Natl. Acad. Sci. USA 89:6099-6103; Zhu et al., Systemic gene expression after intravenous DNA delivery into adult mice, (1993) Science 261:209-211; Yoshimura et al., (1992) Nucleic Acids Research 20: 3233-3240).
  • Methods for combination therapy involving chemotherapy and gene therapy are well known (e.g. U.S. Pat. Nos. 6,054,467; 5,747,469).
  • the inventors have used the cytomegalovirus promoterto achieve expression of modified SGSH and SUMF1, but other ubiquitous promoters could be used as well, including the Rous Sarcoma Virus promoter, and SV40 promoter, CAG promoter (hybrid of the chicken b-actin promoter and CMV enhancer), as well as glial cell specific promoters, for example GFAP (Glial fibrillar Acid promoter).
  • the vector could be used in combination with already existing therapies for sulfatase deficiencies, including enzyme replacement therapy and small molecule therapy, wherein small molecules include molecular tweezers such as those described in PCT Publication No: W02010102248.
  • the present invention would also include variants of modified SGSH and SUMF1 (such as mutated or truncated forms of these enzyme activators) that retain the SGSH activity of wild-type SGSH or that retain the sulfatase-modifying activity of SUMF1, eg variants and/or fragments retaining activity, eg functional variants and/or functional fragments thereof.
  • a fundamental aspect of the present invention is the co-delivery of two factors within one vector, a sulfatase gene product and a SUMF1 gene product. Conveniently, co-delivery may be obtained via expression of a bi-cistronic construct encoding the two gene products with an IRES element inserted between the two.
  • the sequence encoding the sulfatase gene is placed upstream (eg 5') to the SUMF1 coding sequence, since the coding sequence following the promoter is expressed at a higher rate compared to the second sequence.
  • the construct is composed of: promoter- sp-Sulfatase- IRES-SUMF1.
  • the two factors may be expressed from one construct via 2A-peptide-mediated cleavage, eg P2a multi-gene expression system, as described in Wang et al., Scientific Reports volume5, Article number: 16273 (2015). It is anticipated that all types of human sulfatase deficiencies, irrespective of their endogenous sulfatase and SUMF1 status, would be amenable to this approach. Preferred embodiments would be the treatment of M PS-111 A via co-delivery of modified SGSH and SUMF1.
  • the present invention relates to novel viral vectors comprising a construct encoding for a partially modified sulfatase, preferably a sulfamidase, whose signal peptide is substituted with an exogenous signal peptide; preferably the IDS (Iduronate 2-Sulfatase) signal peptide (IDSsp- Sgsh), alternatively, the hAAT signal peptide may be used as described in WO2012085622; the construct is a bicistronic construct further encoding SUMF1.
  • the bicistronic construct is obtained via insertion of an IRES element between the two coding sequences.
  • the construct is a multigene construct encoding the sulfamidase and SUMF1 as a multi-gene expression system.
  • the viral vector is an AAV9 or AAV1.
  • the viral vector is administered by intra-CSF delivery.
  • the present inventors obtained a novel therapeutic tool which is an improvement over prior therapeutics, able to increase the enzymatic activity and the CNS distribution of the SGSH enzyme in an MPS-IIIA mouse model.
  • the advantages of the constructs of the invention are improved biodistribution of therapeutic gene to the CNS.
  • the construct of the invention can be used at lower doses since it has a lower toxicity.
  • intra-CNS particularly intra-CSF
  • administration of a sulfatase gene improves sulfatase secretion derived from IDS signal peptide, combined to co expression with SUMF1 gene product which improves activation of the sulfamidase enzyme.
  • the strategy of the present invention is based on the construction of a chimeric sulfatase, in particular a sulfamidase (the sulfatase enzyme which is deficient in MPS-IIIA), optimized with an amino-acid sequence to the N -terminus of the protein which confers to the modified sulfamidase the capability to be highly secreted.
  • the signal peptide of the human lduronate-2-sulfatase (IDS) gene is fused to the human sulfatase derived amino acid sequence deprived of its signal peptide, as reported in Sorrentino et al. (EMBO Mol Med (2013) 5, 675-690 and in WO2012085622).
  • Said chimeric sulfamidase is optionally co-expressed with the SUMF 1 (sulfatase-modifying factor 1) protein, which is an essential factor for sulfatase activities.
  • SUMF 1 sulfatase-modifying factor 1
  • Sulfatase deficiencies of the present invention include sulfatase deficiencies with a neurological involvement, eg MLD (metachromatic leukodystrophy), MSD, and MPS (mucopolysaccharidosis) II, 111 A, HID, IVA. Sulfatase deficiencies of the present invention may not include MSD.
  • Said sulfatase deficiencies are due to deficiency of the following factors: MLD (metachromatic leukodystrophy) due to the deficiency of arylsulfatase A (ARSA NC_000022.11,; 607574), galactosamine (N-acetyl)-6-sulfatase (GALNS NC_000016.10, 611458) for MPS IVA and glucosamine (N-acetyl)-6-sulfatase (GNS NC_000012.12; 252940) for MPS HID, iduronate 2- sulfatase (IDS;OMIM ref.300823), for MPS II, MPS IMA (SGSH, 605270).
  • MLD metalchromatic leukodystrophy
  • GALNS NC_000016.10, 611458 galactosamine
  • GALNS NC_000016.10, 611458 galactosamine
  • the present invention provides sulfatase and SUMF1 expressed from the same construct, wherein the sulfatase has an exogenous signal peptide with an increased secretion efficiency compared to the native protein.
  • the present invention in particular provides a nucleotide sequence encoding a modified sulfatase and sulfatase-modifying factor 1 (SUMF1), said modified sulfatase comprising in the N-terminal-C-terminal sequence order: a) an amino acid sequence of an exogenous signal peptide, preferably the signal peptide of the human lduronate-2-sulfatase (IDS) or the hAAT signal peptide, and b) an amino acid sequence of a human sulfatase said human sulfatase being deprived of its signal peptide, wherein said nucleotide sequence optionally comprises a sequence of an IRES element between the sequence encoding for said modified sulfatase and said sulfatase-modifying factor 1 (SUMF1).
  • SUMF1 modified sulfatase and sulfatase-modifying factor 1
  • the signal peptide of the human lduronate-2-sulfatase is preferably characterized by the sequence MPPPRTGRGLLWLGLVLSSVCVALG (SEQ ID NO: 14).
  • the signal peptide of hAAT is preferably characterized by the sequence MPSSVSWGILLLAGLCCLVPVSLA (SEQ ID NO: 15).
  • the sulfatase-modifying factor 1 consists of SEQ ID No 13 or a functional fragment thereof or a functional variant thereof, more preferably said functional variant has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID No. 13 and functions as a sulfatase- modifying factor.
  • said functional fragment is at least 374 amino acid long a nd functions as a sulfatase-modifying factor.
  • the expression "functions as a sulfatase-modifying factor” indicates that its function consists in the post-translational activation of all sulfatases in the endoplasmic reticulum.
  • the product of SUM F1 gene oxidises a cysteine residue located within a consensus CxPxR sequence shared by all sulfatases to generate a formylglycine. This cysteine oxidation is absolutely required by sulfatases to exert their enzymatic activity.
  • the human sulfatase is selected from the group consisting of: human N- sulfoglucosamine sulfohydrolase (SGSH), human arylsulfatase A (ARSA), human arylsulfatase B (ARSB), human arylsulfatase D (ARSD human arylsulfatase G (ARSG), human galactosa mine (N- acetyl)-6-sulfate sulfatase (GALNS), human glucosamine (N-acetyl)-6-sulfatase (GNS), human steroid sulfatase (microsomal), isozyme S (STS), human sulfatase 1 (SULF1), human sulfatase 2 (SULF2), human sulfatase modifying factor 2 (SU MF2) or a functional fragment thereof or a functional variant thereof, preferably the human sulfoglu
  • the human sulfatase is the human sulfamidase (SGSH) or a functional fragment thereof or a functional variant thereof.
  • SGSH human sulfamidase
  • the human sulfatase is not SUM F1.
  • the natural signal peptide is underlined above.
  • the modified sulfamidase has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ. I D No. 12 and functions with improved sulfamidase activity (e.g. measured by enzymatic activity assay) and/or secretion (e.g. the modified version of sgsh show an increased secretion efficiency respect to the native version of sulfamidase).
  • nucleotide sequence has at least 75 % ,80%, 85%, 90%, 95%, 99% identity to SEQ. ID No. 4.
  • the present invention provides a vector comprising the nucleic acid as defined above.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector, an adeno- associated virus vector, an adenoviral vector, a retroviral vector, a polio viral vector, a murine Maloney-based viral vector, an alpha viral vector, a pox viral vector, a herpes viral vector, a vaccinia viral vector, a baculoviral vector, or a parvoviral vector.
  • the adeno- associated virus is AAV9, AAV1, AAVSH19, AAV2/7, AAVPHP.B.
  • the invention also provides the nucleotide sequence as defined above or the vector as defined above in combination with a carrier, preferably the carrier is a lipid or a polypeptide, preferably a liposome, a polylysine carrier complex.
  • a carrier preferably the carrier is a lipid or a polypeptide, preferably a liposome, a polylysine carrier complex.
  • the invention further provides the nucleic acid as defined above or the vector as defined above for medical use, preferably for gene therapy.
  • the invention further provides the nucleic acid as defined above or the vector as defined above for use in the treatment of a sulfatase deficiency with a neurological involvement, preferably of a lysosomal sulfatase deficiency.
  • the sulfatase deficiency is selected from the group consisting of: metachromatic leukodystrophy (MLD), mucopolysaccharidosis MPS type IMA, mucopolysaccharidosis MPS type MID, mucopolysaccharidosis MPS type IVA, mucopolysaccharidosis MPS type II, preferably the sulfatase deficiency is MPS type IMA.
  • MLD metachromatic leukodystrophy
  • MPS type IMA mucopolysaccharidosis MPS type IMA
  • mucopolysaccharidosis MPS type MID mucopolysaccharidosis MPS type IVA
  • mucopolysaccharidosis MPS type II preferably the sulfatase deficiency is MPS type IMA.
  • the vector as defined above is administered at a dose of from 0,5 x 10 12 - 1 x 10 13 GC/kg, preferably 1 x 10 12 GC/kg to 10 x 10 12 GC/kg, more preferably 4.5 xlO 12 GC/Kg.
  • the invention also provides the nucleic acid or the vector for use as defined above in combination with a further therapeutic agent, preferably the further therapeutic agent is selected from the group consisting of: enzyme replacement therapy and small molecule therapy.
  • Small molecule therapy of particular interest for the present invention is therapy based on molecular tweezers, compounds described for instance in PCT Publication No: W02010102248.
  • Particularly preferred molecular tweezers are of general formulas
  • XI and X2 are both 0; A alone, or A combined with XI, forms a substituent selected from the group consisting of phosphate, hydrogen phosphate, alkylphosphonate, arylphosphonate, alkylphosphamide, arylphosphamide, sulfate, hydrogen sulfate, alkylcarboxylate, and .
  • g alone, or B combined with X2 forms a substituent selected from the group consisting of phosphate, hydrogen phosphate, alkylphosphonate, arylphosphonate, alkylphosphamide, arylphosphamide, sulfate, hydrogen sulfate, alkylcarboxylate and .
  • eac h 0 f Rl, R2, R3, and R4 is, independently, selected from the group consisting of H, Cl, Br, I, OR, NR2, N02, C02H, and C02R5, wherein R5 is alkyl, aryl or H, or Rl and R2 combine to form an aliphatic or aromatic ring, and/or R3 and R4 combine to form an aliphatic or aromatic ring.
  • Preferred molecular tweezers are (TW1/CLR01), of formula:
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid as defined above or the nucleotide sequence as defined above or the vector as defined above and pharmaceutically acceptable diluents and/or excipients and/or carriers.
  • composition further comprising a therapeutic agent, preferably the therapeutic agent is selected from the group consisting of: enzyme replacement therapy and small molecule therapy.
  • the pharmaceutical composition is administered through a route selected from the group consisting of: intra cerebral spinal fluid (CSF), intrathecal, parenteral, intralesional, intraperitoneal, intramuscular, intratumoral, subcutaneous, intraventricular, intra cisterna magna, lumbar, intracranial, intraspinal, intravenous, topical, nasal, oral, ocular, or any combination thereof.
  • CSF cerebral spinal fluid
  • the present invention also provides a nucleotide sequence encoding for a modified sulfatase, said modified sulfatase comprising in the N-terminal-C-terminal sequence order: a) an amino acid sequence of an exogenous signal peptide, preferably the signal peptide of the human lduronate-2-sulfatase (IDS) or the hAAT signal peptide, and b) an amino acid sequence a human sulfatase said human sulfatase being deprived of its signal peptide for medical use, wherein said nucleotide sequence is administered intra-CSF.
  • an exogenous signal peptide preferably the signal peptide of the human lduronate-2-sulfatase (IDS) or the hAAT signal peptide
  • IDDS human lduronate-2-sulfatase
  • hAAT signal peptide hAAT signal peptide
  • the human sulfatase is selected from the group consisting of: human N- sulfoglucosamine sulfohydrolase (SGSH), human arylsulfatase A (ARSA), human arylsulfatase B (ARSB), human arylsulfatase D (ARSD), human arylsulfatase E (chondrodysplasia punctata 1) (ARSE), human arylsulfatase F (ARSF), human arylsulfatase G (ARSG), human arylsulfatase family, member H (ARSH), human arylsulfatase family, member I (ARSI), human arylsulfatase family, member J (ARSJ), human arylsulfatase family, member K (ARSK), human galactosamine (N-acetyl)-6-sulfate sulfatase (GALNS), human SGSH),
  • the human sulfatase is the human sulfamidase.
  • the modified sulfamidase has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID No. 12.
  • nucleotide sequence has at least 75 % ,80%, 85%, 90%, 95%, 99% identity to SEQ. ID No. 1.
  • the present invention also provides a vector comprising the above nucleic acid or nucleotide sequence for medical use, wherein said vector is administered intra-CSF.
  • the vector is a viral vector
  • the viral vector is a lentiviral vector, an adeno- associated virus vector, an adenoviral vector, a retroviral vector, a polio viral vector, a murine Maloney-based viral vector, an alpha viral vector, a pox viral vector, a herpes viral vector, a vaccinia viral vector, a baculoviral vector, or a parvoviral vector
  • the adeno- associated virus is AAV9, AAV1, AAVSH19, AAVPHP.B.
  • nucleotide sequence is inserted in a vector, preferably a viral vector, still preferably an adeno-associated vector.
  • a vector is administered intra-CSF.
  • medical use is for the treatment of the treatment of a sulfatase deficiency with neurological impairment, preferably of a lysosomal sulfatase deficiency.
  • the sulfatase deficiency is selected from the group consisting of: metachromatic leukodystrophy (MLD), mucopolysaccharidosis MPS type II, mucopolysaccharidosis MPS type IMA, mucopolysaccharidosis MPS type MID, mucopolysaccharidosis MPS type IVA, preferably the sulfatase deficiency is MPS type MIA.
  • MLD metachromatic leukodystrophy
  • MPS type II mucopolysaccharidosis MPS type II
  • mucopolysaccharidosis MPS type IMA mucopolysaccharidosis MPS type MID
  • mucopolysaccharidosis MPS type IVA mucopolysaccharidosis MPS type IVA
  • Sulfatases (EC 3.1.6. )-are enzymes of the esterase class that catalyze the hydrolysis of sulfate esters. These may be found on a range of substrates, including steroids, carbohydrates and proteins. Sulfate esters may be formed from various alcohols and amines. In the latter case the resultant N-sulfates can also be termed sulfamates.
  • human sulfatase refers to a well-defined class of enzymes. Sulfatases are a conserved gene family having the following defined functional representatives in the human genome assembly of April 2003:
  • ARSA Homo sapiens arylsulfatase A
  • transcript variant 2 mRNA.
  • ARSB Homo sapiens arylsulfatase B (ARSB), transcript variant 1, mRNA. (J05225)
  • ARSD Homo sapiens arylsulfatase D (ARSD), mRNA.
  • ARSG Homo sapiens arylsulfatase G (ARSG), transcript variant 1, mRNA.
  • GALNS Homo sapiens galactosamine (N-acetyl)-6-sulfate sulfatase (GALNS), mRNA.
  • GNS Homo sapiens glucosamine (N-acetyl)-6-sulfatase (GNS), mRNA. (NM_002076.4)
  • IDS Homo sapiens iduronate 2-sulfatase (IDS), transcript variant 1, mRNA. (NG_011900).
  • SGSH Homo sapiens N-sulfoglucosamine sulfohydrolase (SGSH), mRNA. (U30894)
  • SULF1 Homo sapiens sulfatase 1 (SULF1), transcript variant 4, mRNA.
  • SULF1 Homo sapiens sulfatase 1 (SULF1), transcript variant 4, mRNA.
  • SULF2 Homo sapiens sulfatase 2 (SULF2), transcript variant 1, mRNA.
  • SUMF1 Homo sapiens sulfatase modifying factor 1 (SUMF1), transcript variant 1, mRNA. (AB448737)
  • SUMF2 Homo sapiens sulfatase modifying factor 2 (SUMF2), transcript variant 5, mRNA.
  • arylsulfatase A EC 3.1.6.8 (ASA), a lysosomal enzyme which hydrolyzes cerebroside sulfate;
  • arylsulfatase B EC 3.1.6.12 (ASB) which hydrolyzes the sulfate ester group from N- acetylgalactosamine 4-sulfate residues of dermatan sulfate;
  • arylsulfatase C (ASD) and E (ASE); steryl-sulfatase EC 3.1.6.2 (STS), a membrane bound enzyme which hydrolyzes 3-beta-hydroxy steroid sulfates;
  • iduronate 2-sulfatase EC 3.1.6.13 IDS
  • a lysosomal enzyme that hydrolyzes the 2-sulfate groups from iduronic acids in dermatan sulfate and heparan sulfate;
  • N-acetylgalactosamine-6-sulfatase EC 3.1.6.4, which hydrolyzes the 6-sulfate groups of the N-acetyl-D-galactosamine of chondroitin sulfate and D-galactose 6-sulfate units of keratan sulfate;
  • N-sulfoglucosamine sulfohydrolase EC 3.10.1.1 the lysosomal enzyme that hydrolyses N- sulfo-D-glucosamine into glucosamine and sulfate;
  • glucosamine-6-sulfatase EC 3.1.6.14 (G6S), which hydrolyzes the N-acetyl-D-glucosamine 6-sulfate units of heparan sulfate and keratan sulfate;
  • N-sulfoglucosamine sulfohydrolase EC 3.10.1.1 the lysosomal enzyme that hydrolyses N- sulfo-D-glucosamine into glucosamine and sulfate;
  • Sulfatase deficiencies of the present invention include sulfatase deficiencies with a neurological involvement, eg MLD (metachromatic leukodystrophy), MSD (Multiple Sulfatase Deficiency), MPS (mucopolysaccharidosis) II, IMA, MID, IVA.
  • MLD metal-chromatic leukodystrophy
  • MSD Multiple Sulfatase Deficiency
  • MPS micopolysaccharidosis II
  • IMA Multiple Sulfatase Deficiency
  • MPS micopolysaccharidosis type 111 A
  • SGSH sulfamidase
  • heparan sulfates an enzyme involved in the stepwise degradation of large macromolecules called heparan sulfates.
  • FIG. 1 Sulfamidase activity in cultured glial cells.
  • Cultured glia cells derived from C57BL/6 mice were transfected with SGSH, AATsp-SGSH, IDSspSGSH constructs. Both cells and 24 h conditioned media were collected and assayed for sulphamidase activity. The secretion efficiency has been indicated as percentage of the SGSH activity in the medium/ activity in the pellet and the medium.
  • FIG. 1 Sulfamidase specific activity in the CNS of pigs injected with AAV9 vectors carrying different SGSH expression cassettes
  • A WT pigs at P60 of age were injected via cisterna magna with 4.5X1012 GC/Kg of AAV9 encoding different human SGSH expression cassettes under the CMV promoter: SGSH WT, IDSspSGSH (SGSH bearing the alternative IDS signal peptide), SGSH- IRES-SUMF1 (bicistronic cassette encoding both SGSH and SUMF1 proteins), IDSspSGSH-IRES- SUMF1 (bicistronic cassette encoding both IDSspSGSH and SUMF1 proteins).
  • FIG. 3 Sulfamidase activity in MPS-IIIA mice injected with AAV9 vectors carrying different SGSH expression cassettes
  • P60 MPS-IIIA mice were intra-CSF injected (via lateral ventricle administration: ICV) with 4.5x1012 GC/Kg of AAV9 encoding under the CMV promoter the following expression cassettes: GFP, SGSH WT, IDSspSGSH, or IDSspSGSH-IRES-SUMFl.
  • the brain and the first region of the spinal cord of treated mice was divided in five slices (A-E) covering the main representative area of the CNS (A: olfactory bulb and prefrontal cortex, B: frontal cortex, lateral septum and basal ganglia regions, C: parietal cortex, hippocampus, striatum, thalamus, D: occipital cortex, pons, hippocampus; E: cerebellum, medulla oblongata, cervical region of spinal cord).
  • A olfactory bulb and prefrontal cortex
  • B frontal cortex, lateral septum and basal ganglia regions
  • C parietal cortex, hippocampus, striatum, thalamus
  • D occipital cortex, pons, hippocampus
  • E cerebellum, medulla oblongata, cervical region of spinal cord.
  • FIG. 4 CNS transduction in M PS-111 A mice injected with AAV9 bearing IDSspSGSH-IRES-SUMFl transgene (A) P60 M PS-111 A mice were ICV injected with 4.5x1012 GC/Kg of AAV9 encoding either IDSspSGSH and SUMF1 or GFP.
  • Five different slices covering the main CNS regions (A-E; as described in the figure 3) were collected at both 1-month (ETP) and 7-months (LTP) after injection. Sulfamidase activity was measured in these areas and expressed as percentage of the activity found in age-matched WT untreated mice. N 6-7 animals for each group. Data represent mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • FIG. 5 Sulfamidase protein quantitation in the brain of M PS-111 A mice injected with AAV9 bearing the IDSspSGSH-IRES-SUMFl transgene.
  • Sulfamidase protein was immuno-quantified by ELISA and expressed as ng of SGSH/mg protein in the five CNS slices (A-E; as described in the supplementary figure 1) of the indicated experimental groups of mice at ETP and LTP.
  • FIG. 6 Rescue of storage pathology, inflammation and memory impairment in MPS-IIIA mice injected with AAV9 encoding IDSspSGSH-IRES-SUMFl.
  • D Neuroinflammation was evaluated at LTP in M PS-111 A mice injected with AAV9 encoding IDSspSGSH-IRES-SUMFl by immunostaining with anti-GFAP (astrogliosis) in paraffin sections from frontal cortex, parietal cortex and lateral septum. Age-matched WT and M PS-11 IA mice injected with AAV9 encoding GFP were used as controls.
  • FIG. 7 Liver transduction in MPS-IIIA mice injected with AAV9 bearing IDSspSGSH-IRES- SUMFl transgene.
  • FIG. 8 Assessment of exploratory activity in M PS-I I IA mice injected with AAV9 bea ring I DSspSGSH-I RES-SUM Fl transgene.
  • M PS-I IIA mice and relative controls (WT) were tested at 6 and 9 months of age in the open field test.
  • I DSsp human iduronate-2-sulfatase signal peptide
  • Reverse Bglll SGSH G AAG AT CTT C ACAG CT C ATTGTG (SEQ I D NO: 18)
  • the PCR product was digested with Notl/Bglll restriction enzymes and cloned into the pAAV2.1- CMV-GFP by replacing the GFP protein.
  • the IDSspSGSH cassette to use for the production of the IDSspSGSH-IRES-SUMFl cassette was amplified from the p3xFLAG-CMV-IDSspSGSH by the following oligos:
  • Reverse Xbal SGSH TG CTCT AG AT C ACAG CT CATTGTG (SEQ ID NO: 20)
  • the PCR product was digested with Notl/Xbal restriction enzymes and cloned into the pAAV2.1- CMV-SGSH-IRES-SUMF1, already developed in Fraldi el al paper [4], by replacing the SGSH protein.
  • the resulting plasmids were used to generate the correspondent AAV serotype 9 (AAV2/8) viral vectors according to the protocols established by AAV Vector Core of TIGEM institute.
  • rAAV administration and CNS samples collection was carried out according to the Sorrentino el al. paper [6].
  • the dorsal area of the neck was trimmed and surgically prepared, and the puncture of the cisterna magna was performed.
  • One ml of CSF was collected before the injection in order to analyze it as a preinjection physiological standard.
  • the dose of 4,5 x 10 12 GC/Kg of viral vector in a volume range from 1.5 to 2.8 ml was injected slowly to avoid a sudden increase in intracranial pressure.
  • Piglets were then placed in Trendelenburg position for 2 minutes in order to help the injected compound to spread toward the more rostral parts of the CNS. Animals were then monitored until complete recovery.
  • mice Homozygous mutant (sgsh-/-; phenotypically M PS-111 A affected) and WT C57BL/6 mice were used [7], [4], [9]. After being collected, the brain samples were harvested and stored frozen until use. To collect brain, mice were perfused with PBS (pH 7.4), and to prepare brain samples for Sulphamidase assays standard capillary depletion protocols were used according to the Fraldi et al. protocols as published [4]. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Cardarelli Hospital in Naples and authorized by the Italian Ministry of Health. In detail, M PS-111 A mice at 2 months of age were anesthetized with ketamine/medetomidine. The AAV vectors were injected with a dosage of 5.4X10 12 GC/Kg bilaterally into the lateral ventricles.
  • mice Mice were anesthetized by were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed on a stereotaxic instrument with a motorized stereotaxic injector. A midline incision was made to expose the bregma. A hole in the skull was made by a drill (anteroposterior +2.18 mm, mediolateral 0,6 mm, dorsoventral -1.7 mm). rAAV9 vectors 5.4 x 1012 GC/Kg were injected in a volume of 10 ul into the lateral ventricles at a rate of 1 mI/min. After allowing the needle to remain in place for 5 min, the needle was slowly raised at a rate of 0.1 cm/min.
  • Pigs Animals were euthanized 1 month after injection with a single bolus (0.3 ml/kg) of Tanax and total body perfusion with Dulbecco's phosphate-buffered saline was started. After median sternotomy, the right atrium was opened and the left ventricle was infused with 500 ml of warm Dulbecco's phosphate-buffered saline (+38 °C) and 1,000 ml of cold Dulbecco's phosphate- buffered saline (+4 °C); blood ejected from the right atrium was drained using a surgical aspirator. As far as CNS samples, collected tissues were the whole brain and cervical region of spinal cord.
  • mice were euthanized at 1, 6 and 7 months by injection with ketamine and xylazine before blood and CSF collection. CSF was collected by glass capillary inserted into the cisterna magna. For tissue collection, mice were intraca rdially perfused with PBS (pH 7,4) and brains were removed, divided into halves and fixed for further analysis.
  • the right half was sliced in five slices (A-E) and frozen in liquid nitrogen, the left half was sliced in two coronally anterior and posterior parts fixed in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin.
  • a coronally slice derived from left half of brain was fixed in 4% paraformaldehyde, 25% glutaraldehyde in phosphate buffer for plastic embedding.
  • the left lateral lobe of the liver from the mice were fixed in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin, and the right medial lobe of the liver of the mice was frozen in liquid nitrogen.
  • SGSH activity Twenty main regions covering the entire CNS of injected pigs were dissected and homogenized with Tissue Lyser using 10 volumes of H20 mQ (700 mI). In mice 5 different slices covering the entire brain and liver samples were homogenized separately with Tissue Lyser using 8 volumes of H20 mQ. The SGSH activity was assayed by a 4-methylumbelliferone-derived fluorogenic substrate (4- MU; Moscerdam Substrates), following established protocols [5].
  • the plate was then incubated with the HRP substrate, TMB peroxidase substrate (Bio-Rad, Hercules, California, USA #1721066) for 30 min at 37°C.
  • This enzyme-substrate reaction was stopped using a stop buffer (2N of H2S04) and the absorbance of each well was measured at the absorbance wavelength of 450 nm (Lml) with a reference wavelength of 655 nm (Lm2) using a microplate reader (GloMax ® -Multi+ Detection System, Promega, Madison, Wisconsin, USA).
  • the concentrations of HNS in samples were calculated using the HNS calibration curve in the same plate.
  • Genomic DNA was extracted from mouse brain samples using a DNeasy Blood and Tissue Extraction kit (Qjagen, Valencia, CA). DNA concentration was determined by using a Nanodrop.
  • Real-time PCR was performed on 100 ng of genomic DNA using a LightCycler SYBR green I system (Roche, Almere, The Netherlands). Amplification was run on a LightCycler 96 device (Roche) with standard cycles.
  • the primers forward SGSH (5' CATCCACTTTGCCTTTCTCTCCA 3', SEQ ID No.: 21) and reverse SGSH (5' T CAAAG CCTCCGT CAT CCG C 3', SEQ ID No.: 22) were used.
  • a standard curve was generated, using the corresponding AAV vector plasmid pAAV2.1CMV-IDSspSGSH- IRES-SUMF1.
  • Brain samples and liver samples were lysed in water by Tissue Lyser equipment. The lysates were then digested with proteinase K and extracts were clarified by centrifugation and filtration. GAG levels in brain extracts were determined using Blyscan sulfated glycosaminoglycan kit (Biocolor, Carrickfergus, UK) with chondroitin 4-sulfate as the standard.
  • Immunohistochemical experiments were performed in 5micron paraffin sections with BondRX Stainer following the standard protocol.
  • the primary antibodies used were mouse anti-Human SGSH (Shire 2C7) 1:25 for immunofluorescence and 1:500 for IHC, rabbit anti-LAMPl (ab24170 Abeam), rabbit anti-GFAP (ab7260Abcam), rabbit anti-NeuN (abl77487).
  • 5-micron paraffin- embedded tissue sections were incubated overnight at 4°C with rabbit anti-GFAP (Z0334; Dako Cytomation).
  • the detection system including secondary antibodies used for immunohistochemistry was Bond Polymer refine kit (Leica, DS9800) for the visualization of SGSH and LAMP1 signals.
  • the secondary antibody used for the detection of GFAP and IBAI signal was biotinylated universal antibody of Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).
  • the GFAP and IBAI stained slides were scanned with Hamamatsu Nanozoomer 2.0-RS scanner and viewed with NDP.view2. Total number of GFAP and IBAI positive signals were counted using the cell-counter program (ImageJ software) with a fixed threshold.
  • Behavioral tests were carried out in a behavioral testing room maintained under constant light, temperature, and humidity. The mice were tested during daylight hours (between 9am and 6pm). Before testing, animals were habituated to the testing room for at least 30 min. The same groups of animals were tested at 6 and 9 months of age. The inventors performed the open field which in previous studies they have found to be impaired in adult M PS-11 IA mice. Additionally, the inventors tested them in the contextual fear conditioning task, which allows to evaluate animal ability to learn and remember a Pavlovian association between a mild food-shock and a specific context.
  • Open field task was performed as previously described [5]. Mice were placed in the middle of a Plexiglas arena with a masonite base (43 x 32 x 40 cm). Animals were left free to explore the device for 10 min. The distance travelled (m), the immobility time (s) and line crossing, were recorded using a video camera (Panasonic WV-BP330) hanging over the arena that was connected to a video-tracking system (Any-Maze, Stoelting, USA).
  • Contextual fear conditioning Each mouse was trained in a conditioning chamber (30 cm x 24 cm x 21 cm; Ugo Basile) that had a removable grid floor and waste pan.
  • the grid floor contained 36 stainless steel rods (3-mm diameter) spaced 8 mm center to center.
  • the shock intensity was 0.5 mA with a duration of 2 sec and it was presented for three times and was associated to a context.
  • mice were tested without foot- shock but with the same context. Freezing behavior was defined as complete lack of movement, except for respiration and scored with a video-tracking system (ANY-MAZE, Stoelting, USA).
  • Example 1 IN VITRO study - Testing of IDSspSGSH cassette in glia cells
  • the inventors transfected primary cultured glia derived from C57BL/6 mice with a vector expressing the IDSspSGSH cDNA of SEQ. ID No. 1 (pAAV2.1-CMV-IDSspSGSH, SEQ. ID No.7) and compared it to two other constructs: one bearing the unmodified version of SGSH of SEQ SEQ ID No. 2 (pAAV2.1-CMV-SGSH, SEQ ID No. 5) and a construct carrying the human a-antitrypsin (hAATAATspSGSH cDNA of SEQ ID No.
  • the secretion experiment was started one day after transfection, by adding the conditioned media to the primary transfected glia cells. 24h after secretion, the enzymatic activity in both the pellet and the medium of glia cells was evaluated, demonstrating that the IDS sp replacement strongly increased the secretion rate of ISDsp-modified sulfamidase compared to wild-type sulfamidase (Fig.l). The secretion efficiency has been indicated as percentage of the SGSH activity in the medium/ activity in the pellet and the medium.
  • the IDSspSGSH construct showed increased enzymatic activity in the medium associated with improved secretion efficiency in glia cells transfected with the IDSspSGSH compared to cells transfected with the other constructs, demonstrating the capability of the IDSspSGSH protein to be functional and highly secreted in the highly relevant in vitro model of primary glia cells.
  • Example 2 Usage of a SGSH expression cassette with enhanced enzyme secretion and activation improved enzyme bio-distribution in the CNS of pigs upon intra-CSF AAV9- mediated gene delivery
  • the inventors generated a bi-cistronic SGSH expression cassette containing a chimeric SGSH bearing the iduronate sulfatase signal peptide (IDSsp) and the gene encoding SUMF1 (IDSspSGSH-IRES-SUMFl).
  • IDSspSGSH-IRES-SUMFl the gene encoding SUMF1
  • WT pigs were intra CSF injected via the cisterna magna with 4.5xl0 12 GC/Kg of AAV9 encoding IDSspSGSH-IRES-SUMFl under the CMV promoter.
  • AAV9-SGSH WT SGSH
  • IDSsp-modified SGSH AAV9-IDSspSGSH
  • bicistronic cassette containing SUMF1 WT SGSH
  • Example 3 AAV9-mediated intra-CSF delivery of IDSspSGSH-IRES-SUMFl resulted in a strong and sustained SGSH activity in the brain of Sgsh -/- mice
  • the inventors then performed an efficacy study in a larger cohort of M PS-111 A mice in which AAV9 encoding IDSspSGSH-IRES-SUMFl was delivered by ICV. Following one month (early time point; ETP) or seven months (late time point; LTP) after injection the enzyme biodistribution and the pathological phenotype were analysed. Consistently with previous results (Figure 3) a significant increase of SGSH activity in the brain of injected animals with peaks of 35% of WT levels was observed at ETP (Figure 4A). Importantly, such increased activity was sustained at LTP (25% of WT levels) ( Figure 4A).
  • Example 4 Rescue of storage pathology, lysosomal enlargement and neuroinflammation in MPS-IIIA mice treated with AAV9-IDSspSGSH-IRES-SUMFl
  • Example 5 Treatment with IDSspSGSH-IRES-SUMFl prevents the memory deficits occurring in the late stage of MPS-IIIA pathology
  • the inventors next validated the impact of the ICV AAV9-mediated delivery of IDSspSGSH-IRES- SUMFl on behavioural deficits that manifest in 6 and 9 months MPS-IIIA mice. Based on previous studies, the inventors evaluated exploratory behaviour in the open field. At 6 months, MPS-IIIA male mice show a mild difference in the distance travelled, immobile time and line crossing, in the very first minutes of open field testing, as compared to WT animals ( Figure 8C). This tendency was not evident at 9 months, likely due to the test-retest effects observed also in control animals [27], which did not allow to properly evaluate the rescuing efficacy of IDSspSGSH-IRES- SUMF1 ( Figure 8A-8C).
  • the inventors tested the animals in a memory task that is extensively used to test contextual memory depending on the functional integrity of the medial temporal lobe, namely the fear contextual conditioning test [28]. Using this task, the inventors identified, for the first time in MPS-IIIA mice, an age-dependent long term memory impairment: 6 months-old MPS-IIIA mice show normal freezing time when they are exposed to a mild foot shock during training, as compared to control animals; similarly, exposure to the context (24 hr after training) paired with the mild foot shock is sufficient to elicit freezing as well as in control mice (Figure 6E). This suggests that at this stage MPS-IIIA mice can form emotional contextual memories as well as WT animals.
  • the inventors developed and tested an intra-CSF AAV-mediated gene transfer approach for the MPS- MIA based on intrathecal delivery of AAV9 encoding a modified SGSH expression cassette with enhanced therapeutic potential.
  • Such expression cassette contained an SGSH bearing the signal peptide of IDS, which confer to the engineered enzyme the capability to be secreted at higher efficiency compared to WT enzyme.
  • modification resulted in improved bio-distribution of the enzyme in the CNS of both small (MPS-IIIA mice) and large (WT pigs) animal models.
  • an important additional feature of the SGSH expression cassette used in the present strategy is the insertion of the cDNA codifying for SUMF1, the enzyme responsible for the post- translational activation of sulfatases.
  • Such modification synergistically acts together with enhanced secretion to further improve enzyme CNS bio-distribution upon intra-CSF AAV9- mediated gene delivery.
  • the inventors demonstrated that the modified SGSH expression cassette they developed is therapeutically effective, being able to efficiently rescue CNS and somatic storage pathology and improve memory impairment.
  • Intra-CSF AAV9-mediated gene delivery using WT SGSH has previously been successfully tested in different preclinical animal models using vector doses similar to those used in the present approach (i.e. ⁇ 4-5 xlO 12 GC/Kg) [16].
  • the authors also showed that reducing AAV9 vector dosage in MPS-IIIA mice below 4-5 xlO 12 GC/Kg led to inefficient enzyme biodistribution, thus resulting in only mild neuropathology improvement.
  • Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nature biotechnology. 2009;27(l):59-65.

Abstract

The present invention related to improved gene therapy to treat sulfatase deficiencies, preferably via co-delivery of Sulfatase enzymes and SUMF1 protein.

Description

Therapy of sulfatase deficiencies
TECHNICAL FIELD
The present invention relates to improved gene therapy to treat sulfatase deficiencies, preferably via co-delivery of Sulfatase enzymes and SUMF1 protein.
BACKGROUND
Sulfatases are hydrolases that cleave sulfate esters from a wide range of substrates, including glycosaminoglycans, sulfolipids, and steroid sulfates ("The sulfatase gene family". Parenti, G, Meroni, G, and Ballabio, A.Curr. Opin. Genet. Dev. 1997; 7: 386-391; "Multiple sulfatase deficiency and the nature of the sulfatase family" .Hopwood, J.J and Ballabio, A.) [1]. At least eight inherited metabolic disorders in humans are caused by sulfatase deficiencies, resulting in impaired desulfation of sulfatase natural substrates. In the case of deficiencies of lysosomal sulfatases, sulfated substrates accumulate in the cells and tissues of patients, resulting in Lysosomal Storage Disorders (LSD) with consequences depending on the type and tissue distribution of the stored material. Deficiencies of sulfatases include MLD (metachromatic leukodystrophy), which is characterized by the storage of sulfolipids in the central and peripheral nervous systems, and five different types of Mucopolysaccharidoses (MPSs types II, IMA, MID, IVA and VI), which are due to the accumulation of Glycosaminoglycans (GAGs) in several tissues and organs[2]. Active sulfatases contain a unique FGIy (formylglycine) catalytic residue which derived from a post-translational modification of a cysteine precursor. In a previous study, Ballabio et al. identified the gene encoding the FGE (formylglycine-generating enzyme), named SUMF1 (sulfatase-modifying factor 1) and demonstrated its essential role for sulfatases activity [3]. SUMF1 is both an essential and a limiting factor for sulfatases. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. SUMF1 has been shown to have an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells as well as co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by diseases owing to sulfatase deficiencies or from murine models of the same diseases. Importantly, simultaneous over-expression of SUMF1 and human sulfatase cDNA results in a strikingly synergistic increase in enzyme activity of each sulfatase examined both in cell culture and in in vivo models, indicating that SUMF1 is either essential or a limiting factor for sulfatases[l]. The delivery of a bicistronic construct carrying the wild type sulfamidase and the SUMF cDNA was administered peripherally to mice and the levels of expression measured in muscle, in Fraldi et a I, [1]. Direct administration and activity measurement in the CNS was not attempted.
Moreover, the intraventricular AAV-mediated co-delivery of SGSH and SUMF1 proteins have been shown to rescue the systemic and neurodegenerative problems in MPS-IIIA[4].
Mucopolysaccharidosis type IMA (M PS-11 IA) is one of the most common and severe forms of neurodegenerative lysosomal storage disorders (LSDs) [10, 11]. It is caused by inherited defect of the sulfamidase (SGSH), a soluble lysosomal enzyme belonging to the family of sulfatases [12] and leads to the accumulation of heparan sulfate in cell, particularly within the central nervous system (CNS). Presently, there are no treatments available to treat the CNS in M PS-111 A patients. Gene delivery aimed at correcting defective hydrolytic lysosomal defects represent the most promising replacement strategy for the CNS treatment of M PS-111 A as well as other MPSs with similar causes due to its potential for a one-time treatment [13].
Among viral vectors used for gene transfer, adeno-associated viral (AAV) vectors are most commonly utilized for in vivo gene transfer because they are safe, provide significantly long transgene expression and may be generated with variable serotypes allowing efficient delivery of therapeutic genes to different target tissues [14, 15]. To date, several therapeutic approaches have been designed and developed based on AAV-mediated gene delivery of SGSH using different route of administrations to reach the CNS [4, 5, 16-19]. Although all these approaches have demonstrated potential benefits in preclinical animal models, the effective therapeutic application of these protocols in the clinical management of MPS-IIIA patients is challenging because of the difficulty in achieving widespread distribution of the corrective enzyme in the CNS and in maintaining therapeutic threshold levels of them in targeted cells [20]. Phase I/ll clinical trials based on either AAV9- mediated systemic or AAVrhlO-mediated intra- pa renchymal delivery of SGSH gene are ongoing (clinical trial.gov) and while both are promising, both approaches have drawbacks. The first approach suffers from potential toxicity due to the high doses of systemically delivered AAV9 vectors required for efficacy [21], while the second approach is invasive and preliminary efficacy data did not show evident improvement of CNS pathology likely also due to the inefficient targeting of CNS regions at any significant distance from the injection sites [22].
Therefore, a major medical need in the clinical care of M PS-111 patients is to overcome these limitations and develop safe and minimal invasive gene transfer approaches with improved CNS transduction.
Enhancing the therapeutic potential of sulfamidase, along with developing tools for efficient and safe CNS targeting, may represent a novel area of intervention to improve CNS therapy in MPS- IMA patients. Administration of AAVs by the cerebrospinal fluid (CSF) route of injection provides very limited access to visceral organs while at the same time allowing the circulating virus to access a large CNS surface area, a condition that could reduce cytotoxic effects of whole body exposure and may potentially limit the amount of viral vector required to efficiently transduce CNS cells [23]. Among different AAV serotypes, serotype 9 has been shown to have a broad tropism, including neurons and astrocytes, when delivered either via CSF or through intravenous injection due to its capability to cross the blood-brain barrier (BBB) [24, 25]. The inventors have previously demonstrated that AAV9 outperforms all other AAV serotypes tested in CNS transduction efficiency when administered via CSF even in large animal models [6]. Lysosomal hydrolases, including SGSH can be secreted and taken up by surrounding cells [26]. Such cross-correction capability makes gene replacement protocols potentially more effective since transduced cells (factory cells) may also correct non-transduce cells. In previous work the inventors demonstrated that replacing the signal peptide (sp) of sulfamidase with that belonging to another lysosomal hydrolase that is highly secreted, the iduronate sulfatase (IDS), strongly improved the efficiency of SGSH secretion from multiple cell types both in vivo and in vitro, a property, which enhanced the cross-correction capability of transduced cells upon gene delivery [5].
Therefore, there is still the need for enhancing the therapeutic potential of sulfatase, in particular sulfamidase.
SUMMARY OF THE INVENTION
In the present invention a potentiated SGSH expression cassette was generated by combining enhanced secretion efficiency of SGSH with improved SUMFl-mediated enzyme activation capacity and its therapeutic potential was explored upon intra-CSF AAV9-mediated gene delivery. The present results demonstrate that by using this strategy it is possible to achieve a superior SGSH CNS bio- distribution in a large animal model (wild type pigs) and to fully rescue the CNS phenotype in a mouse model of sulfatase deficiency, as M PS-111 A. In other words, the present inventors have surprisingly obtained increased enzymatic activity and CNS distribution of the sulfamidase gene through intra-cerebral spinal fluid (CSF) administration of a highly secreted version of the sulfamidase gene (eg a sulfamidase modified to include the IDS signal peptide, IDSsp) via AAV-mediated delivery and co-delivery of said modified SGSH and SUMFlproteins.
The inventors approach was to focus on gene transfer approaches to treat the CNS in LSDs with minimal invasiveness and high CNS targeting efficiency.
The inventors found that intra-CSF administration of a modified SGSH was surprisingly effective. The inventors further found that a bicistronic construct of modified SGSH/SUMF1 was superior to either of two single gene vectors (for either modified SGSH or SUMF1, respectively) to enhance SGSH activity, and was surprisingly better than a combination of two single gene vectors for modified SGSH and SUMF1. In cells expressing non-modified SGSH, or expressing modified SGSH, or expressing SUMF1 or expressing both non-modified SGSH and SUMF1, there was a striking improvement in SGSH activity by supplying exogenous modified SGSH together with SUMF1 as a bicistronic construct expressing the two proteins.
The construct IDSsp-SGSH-SUMFl has the highest expression levels in pigs. Further the construct IDSsp-SGSH displays less enzymatic activity than the IDSsp-SGSH-SUMFl construct and the SGSH activity induced by the SGSH-SUMF construct is lower than the one induced by the IDSsp-SGSH-SUMFl construct.
The method of the invention, in comparison with other approaches in the art, is illustrated in the figures.
The vector is structurally different than prior vectors in that it incorporates both the modified SGSH and SUMF1 genes in a single expression cassette under the control of a single promoter. In the present invention, the two genes are co-delivered as one vector, rather than as single gene vectors. Functionally, the bicistronic vector is superior either to modified SGSH alone, or to a combination of single gene vectors for modified SGSH and SUMF1.
The present vector would be used to deliver the modified SGSH and SUMF1 genes to human brain so as to increase SGSH brain distribution and SGSH activity for the treatment of CNS pathology in sulfatase deficiency. While a preferred embodiment of the present invention involves co-delivery of a modified sulfatase, preferably SGSH, and SUMF1 to the brain via an adeno-associated viral vector, it is also anticipated that other delivery mechanisms, including retro viral, adeno viral, herpes simplex viral, cytomegaloviral, could also be used. Methods for formulating pharmaceutical compositions or carriers for the bicistronic constructs or vectors disclosed herein are well known in the art (e.g. U.S. Pat. Nos. 6,054,467, and 5,747,469) incorporated herein by reference). Non-viral delivery vehicles could be used as well, including approaches that utilize liposomes, polylysine carrier complexes, or naked DNA (1992, Proc. Natl. Acad. Sci. USA 89:6099-6103; Zhu et al., Systemic gene expression after intravenous DNA delivery into adult mice, (1993) Science 261:209-211; Yoshimura et al., (1992) Nucleic Acids Research 20: 3233-3240). Methods for combination therapy involving chemotherapy and gene therapy are well known (e.g. U.S. Pat. Nos. 6,054,467; 5,747,469).
The inventors have used the cytomegalovirus promoterto achieve expression of modified SGSH and SUMF1, but other ubiquitous promoters could be used as well, including the Rous Sarcoma Virus promoter, and SV40 promoter, CAG promoter (hybrid of the chicken b-actin promoter and CMV enhancer), as well as glial cell specific promoters, for example GFAP (Glial fibrillar Acid promoter). In some embodiments, the vector could be used in combination with already existing therapies for sulfatase deficiencies, including enzyme replacement therapy and small molecule therapy, wherein small molecules include molecular tweezers such as those described in PCT Publication No: W02010102248. The present invention would also include variants of modified SGSH and SUMF1 (such as mutated or truncated forms of these enzyme activators) that retain the SGSH activity of wild-type SGSH or that retain the sulfatase-modifying activity of SUMF1, eg variants and/or fragments retaining activity, eg functional variants and/or functional fragments thereof. A fundamental aspect of the present invention is the co-delivery of two factors within one vector, a sulfatase gene product and a SUMF1 gene product. Conveniently, co-delivery may be obtained via expression of a bi-cistronic construct encoding the two gene products with an IRES element inserted between the two. Preferably, the sequence encoding the sulfatase gene is placed upstream (eg 5') to the SUMF1 coding sequence, since the coding sequence following the promoter is expressed at a higher rate compared to the second sequence. Hence, the construct is composed of: promoter- sp-Sulfatase- IRES-SUMF1.
Alternatively, the two factors may be expressed from one construct via 2A-peptide-mediated cleavage, eg P2a multi-gene expression system, as described in Wang et al., Scientific Reports volume5, Article number: 16273 (2015). It is anticipated that all types of human sulfatase deficiencies, irrespective of their endogenous sulfatase and SUMF1 status, would be amenable to this approach. Preferred embodiments would be the treatment of M PS-111 A via co-delivery of modified SGSH and SUMF1.
The present invention relates to novel viral vectors comprising a construct encoding for a partially modified sulfatase, preferably a sulfamidase, whose signal peptide is substituted with an exogenous signal peptide; preferably the IDS (Iduronate 2-Sulfatase) signal peptide (IDSsp- Sgsh), alternatively, the hAAT signal peptide may be used as described in WO2012085622; the construct is a bicistronic construct further encoding SUMF1. Preferably the bicistronic construct is obtained via insertion of an IRES element between the two coding sequences. Alternatively, the construct is a multigene construct encoding the sulfamidase and SUMF1 as a multi-gene expression system. Preferably the viral vector is an AAV9 or AAV1. Preferably the viral vector is administered by intra-CSF delivery.
The inventors surprisingly found that AAV-mediated intra-cerebral spinal fluid (CSF) administration of the highly secreted version of the SGSH enzyme (IDSspSGSH) and the AAV- mediated intra-cerebral spinal fluid (CSF) administration of the highly secreted version of the SGSH enzyme (IDSspSGSH) and SUMFlprotein in MPS-IIIA mice, yielded a broad biodistribution to the CNS as well as higher enzymatic activity compared to constructs tested previously, both in wild type animals and in animal models of sulfatase deficiencies as MPSIIIA models. Thus, the present inventors obtained a novel therapeutic tool which is an improvement over prior therapeutics, able to increase the enzymatic activity and the CNS distribution of the SGSH enzyme in an MPS-IIIA mouse model.
The advantages of the constructs of the invention are improved biodistribution of therapeutic gene to the CNS.
The construct of the invention can be used at lower doses since it has a lower toxicity.
In the present invention it is shown that intra-CNS (particularly intra-CSF) administration of a sulfatase gene improves sulfatase secretion derived from IDS signal peptide, combined to co expression with SUMF1 gene product which improves activation of the sulfamidase enzyme. The strategy of the present invention is based on the construction of a chimeric sulfatase, in particular a sulfamidase (the sulfatase enzyme which is deficient in MPS-IIIA), optimized with an amino-acid sequence to the N -terminus of the protein which confers to the modified sulfamidase the capability to be highly secreted. In the instant invention, the signal peptide of the human lduronate-2-sulfatase (IDS) gene is fused to the human sulfatase derived amino acid sequence deprived of its signal peptide, as reported in Sorrentino et al. (EMBO Mol Med (2013) 5, 675-690 and in WO2012085622).
Said chimeric sulfamidase is optionally co-expressed with the SUMF 1 (sulfatase-modifying factor 1) protein, which is an essential factor for sulfatase activities.
Sulfatase deficiencies of the present invention include sulfatase deficiencies with a neurological involvement, eg MLD (metachromatic leukodystrophy), MSD, and MPS (mucopolysaccharidosis) II, 111 A, HID, IVA. Sulfatase deficiencies of the present invention may not include MSD.
Said sulfatase deficiencies are due to deficiency of the following factors: MLD (metachromatic leukodystrophy) due to the deficiency of arylsulfatase A (ARSA NC_000022.11,; 607574), galactosamine (N-acetyl)-6-sulfatase (GALNS NC_000016.10, 611458) for MPS IVA and glucosamine (N-acetyl)-6-sulfatase (GNS NC_000012.12; 252940) for MPS HID, iduronate 2- sulfatase (IDS;OMIM ref.300823), for MPS II, MPS IMA (SGSH, 605270).
Therefore, the present invention provides sulfatase and SUMF1 expressed from the same construct, wherein the sulfatase has an exogenous signal peptide with an increased secretion efficiency compared to the native protein.
The present invention in particular provides a nucleotide sequence encoding a modified sulfatase and sulfatase-modifying factor 1 (SUMF1), said modified sulfatase comprising in the N-terminal-C-terminal sequence order: a) an amino acid sequence of an exogenous signal peptide, preferably the signal peptide of the human lduronate-2-sulfatase (IDS) or the hAAT signal peptide, and b) an amino acid sequence of a human sulfatase said human sulfatase being deprived of its signal peptide, wherein said nucleotide sequence optionally comprises a sequence of an IRES element between the sequence encoding for said modified sulfatase and said sulfatase-modifying factor 1 (SUMF1).
The signal peptide of the human lduronate-2-sulfatase (IDS) is preferably characterized by the sequence MPPPRTGRGLLWLGLVLSSVCVALG (SEQ ID NO: 14).
The signal peptide of hAAT is preferably characterized by the sequence MPSSVSWGILLLAGLCCLVPVSLA (SEQ ID NO: 15).
Preferably the sulfatase-modifying factor 1 (SUMF1) consists of SEQ ID No 13 or a functional fragment thereof or a functional variant thereof, more preferably said functional variant has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID No. 13 and functions as a sulfatase- modifying factor. Also preferably, said functional fragment is at least 374 amino acid long a nd functions as a sulfatase-modifying factor.
The expression "functions as a sulfatase-modifying factor" indicates that its function consists in the post-translational activation of all sulfatases in the endoplasmic reticulum. The product of SUM F1 gene oxidises a cysteine residue located within a consensus CxPxR sequence shared by all sulfatases to generate a formylglycine. This cysteine oxidation is absolutely required by sulfatases to exert their enzymatic activity.
Preferably, the human sulfatase is selected from the group consisting of: human N- sulfoglucosamine sulfohydrolase (SGSH), human arylsulfatase A (ARSA), human arylsulfatase B (ARSB), human arylsulfatase D (ARSD human arylsulfatase G (ARSG), human galactosa mine (N- acetyl)-6-sulfate sulfatase (GALNS), human glucosamine (N-acetyl)-6-sulfatase (GNS), human steroid sulfatase (microsomal), isozyme S (STS), human sulfatase 1 (SULF1), human sulfatase 2 (SULF2), human sulfatase modifying factor 2 (SU MF2) or a functional fragment thereof or a functional variant thereof, preferably the human sulfatase is the human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional fragment thereof or a functional variant thereof.
Preferably the human sulfatase is the human sulfamidase (SGSH) or a functional fragment thereof or a functional variant thereof.
Preferably the human sulfatase is not SUM F1.
Human SGSH aminoacid sequence:
MSCPVPACCALLLVLGLCRARPRNALLLLADDGGFESGAYN NSAIATPH LDALARRSLLFRNAFTSVSSCSPS
RASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGI IGKKHVGPETVYPFDFAYTEENGS
VLQ.VGRNITRIKLLVRKFLQ.TQ.DDRPFFLYVAFHDPHRCGHSQ.PQYGTFCEKFGNGESGMGRI PDWTPQA
YDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLN DTLVI FTSDNGIPFPSGRTNL
YWPGTAEPLLVSSPEHPKRWGQ.VSEAYVSLLDLTPTI LDWFSI PYPSYAI FGSKTI HLTGRSLLPALEAEPLWA
TVFGSQSHHEVTMSYPM RSVQHRHFRLVHNLNFKM PFPI DQDFYVSPTFQDLLNRTTAGQPTGWYKDLR
HYYYRARWELYDRSRDPHETQNLATDPRFAQLLEM LRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQ
PLHNEL* (SEQ I D NO 16)
The natural signal peptide is underlined above.
Preferably the modified sulfamidase has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ. I D No. 12 and functions with improved sulfamidase activity (e.g. measured by enzymatic activity assay) and/or secretion (e.g. the modified version of sgsh show an increased secretion efficiency respect to the native version of sulfamidase).
Preferably the nucleotide sequence has at least 75 % ,80%, 85%, 90%, 95%, 99% identity to SEQ. ID No. 4.
The present invention provides a vector comprising the nucleic acid as defined above.
Preferably the vector is a viral vector.
Still preferably the viral vector is a lentiviral vector, an adeno- associated virus vector, an adenoviral vector, a retroviral vector, a polio viral vector, a murine Maloney-based viral vector, an alpha viral vector, a pox viral vector, a herpes viral vector, a vaccinia viral vector, a baculoviral vector, or a parvoviral vector.
Preferably the adeno- associated virus is AAV9, AAV1, AAVSH19, AAV2/7, AAVPHP.B.
The invention also provides the nucleotide sequence as defined above or the vector as defined above in combination with a carrier, preferably the carrier is a lipid or a polypeptide, preferably a liposome, a polylysine carrier complex.
The invention further provides the nucleic acid as defined above or the vector as defined above for medical use, preferably for gene therapy. Preferably, the invention further provides the nucleic acid as defined above or the vector as defined above for use in the treatment of a sulfatase deficiency with a neurological involvement, preferably of a lysosomal sulfatase deficiency.
Preferably the sulfatase deficiency is selected from the group consisting of: metachromatic leukodystrophy (MLD), mucopolysaccharidosis MPS type IMA, mucopolysaccharidosis MPS type MID, mucopolysaccharidosis MPS type IVA, mucopolysaccharidosis MPS type II, preferably the sulfatase deficiency is MPS type IMA.
Preferably, the vector as defined above is administered at a dose of from 0,5 x 1012- 1 x 1013 GC/kg, preferably 1 x 1012 GC/kg to 10 x 1012 GC/kg, more preferably 4.5 xlO12 GC/Kg.
The invention also provides the nucleic acid or the vector for use as defined above in combination with a further therapeutic agent, preferably the further therapeutic agent is selected from the group consisting of: enzyme replacement therapy and small molecule therapy.
Small molecule therapy of particular interest for the present invention is therapy based on molecular tweezers, compounds described for instance in PCT Publication No: W02010102248. Particularly preferred molecular tweezers are of general formulas
Figure imgf000011_0001
Figure imgf000011_0002
pharmaceutically acceptable salt, ester, amide, or prodrug thereof, wherein: XI and X2 are both 0; A alone, or A combined with XI, forms a substituent selected from the group consisting of phosphate, hydrogen phosphate, alkylphosphonate, arylphosphonate, alkylphosphamide, arylphosphamide, sulfate, hydrogen sulfate, alkylcarboxylate, and
Figure imgf000011_0003
. g alone, or B combined with X2, forms a substituent selected from the group consisting of phosphate, hydrogen phosphate, alkylphosphonate, arylphosphonate, alkylphosphamide, arylphosphamide, sulfate, hydrogen sulfate, alkylcarboxylate and
Figure imgf000011_0004
. a nc| each 0f Rl, R2, R3, and R4 is, independently, selected from the group consisting of H, Cl, Br, I, OR, NR2, N02, C02H, and C02R5, wherein R5 is alkyl, aryl or H, or Rl and R2 combine to form an aliphatic or aromatic ring, and/or R3 and R4 combine to form an aliphatic or aromatic ring. Preferred molecular tweezers are (TW1/CLR01), of formula:
Figure imgf000012_0001
(TW4), of formula
Figure imgf000012_0002
(TW5), of formula
Figure imgf000013_0001
The invention also provides a pharmaceutical composition comprising the nucleic acid as defined above or the nucleotide sequence as defined above or the vector as defined above and pharmaceutically acceptable diluents and/or excipients and/or carriers.
Preferably the composition further comprising a therapeutic agent, preferably the therapeutic agent is selected from the group consisting of: enzyme replacement therapy and small molecule therapy.
Preferably the pharmaceutical composition is administered through a route selected from the group consisting of: intra cerebral spinal fluid (CSF), intrathecal, parenteral, intralesional, intraperitoneal, intramuscular, intratumoral, subcutaneous, intraventricular, intra cisterna magna, lumbar, intracranial, intraspinal, intravenous, topical, nasal, oral, ocular, or any combination thereof.
The present invention also provides a nucleotide sequence encoding for a modified sulfatase, said modified sulfatase comprising in the N-terminal-C-terminal sequence order: a) an amino acid sequence of an exogenous signal peptide, preferably the signal peptide of the human lduronate-2-sulfatase (IDS) or the hAAT signal peptide, and b) an amino acid sequence a human sulfatase said human sulfatase being deprived of its signal peptide for medical use, wherein said nucleotide sequence is administered intra-CSF.
Preferably, the human sulfatase is selected from the group consisting of: human N- sulfoglucosamine sulfohydrolase (SGSH), human arylsulfatase A (ARSA), human arylsulfatase B (ARSB), human arylsulfatase D (ARSD), human arylsulfatase E (chondrodysplasia punctata 1) (ARSE), human arylsulfatase F (ARSF), human arylsulfatase G (ARSG), human arylsulfatase family, member H (ARSH), human arylsulfatase family, member I (ARSI), human arylsulfatase family, member J (ARSJ), human arylsulfatase family, member K (ARSK), human galactosamine (N-acetyl)-6-sulfate sulfatase (GALNS), human glucosamine (N-acetyl)-6-sulfatase (GNS), human iduronate 2-sulfatase (IDS), human steroid sulfatase (microsomal), isozyme S (STS), human sulfatase 1 (SULF1), human sulfatase 2 (SULF2), human sulfatase modifying factor 2 (SUMF2), preferably the human sulfatase is the human N-sulfoglucosamine sulfohydrolase (SGSH).
Preferably the human sulfatase is the human sulfamidase. Preferably the modified sulfamidase has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID No. 12.
Preferably the nucleotide sequence has at least 75 % ,80%, 85%, 90%, 95%, 99% identity to SEQ. ID No. 1.
The present invention also provides a vector comprising the above nucleic acid or nucleotide sequence for medical use, wherein said vector is administered intra-CSF. Preferably the vector is a viral vector, preferably the viral vector is a lentiviral vector, an adeno- associated virus vector, an adenoviral vector, a retroviral vector, a polio viral vector, a murine Maloney-based viral vector, an alpha viral vector, a pox viral vector, a herpes viral vector, a vaccinia viral vector, a baculoviral vector, or a parvoviral vector, preferably the adeno- associated virus is AAV9, AAV1, AAVSH19, AAVPHP.B.
Preferably said nucleotide sequence is inserted in a vector, preferably a viral vector, still preferably an adeno-associated vector. Preferably the vector is administered intra-CSF. Preferably the medical use is for the treatment of the treatment of a sulfatase deficiency with neurological impairment, preferably of a lysosomal sulfatase deficiency. Preferably the sulfatase deficiency is selected from the group consisting of: metachromatic leukodystrophy (MLD), mucopolysaccharidosis MPS type II, mucopolysaccharidosis MPS type IMA, mucopolysaccharidosis MPS type MID, mucopolysaccharidosis MPS type IVA, preferably the sulfatase deficiency is MPS type MIA.
Sulfatases (EC 3.1.6. )-are enzymes of the esterase class that catalyze the hydrolysis of sulfate esters. These may be found on a range of substrates, including steroids, carbohydrates and proteins. Sulfate esters may be formed from various alcohols and amines. In the latter case the resultant N-sulfates can also be termed sulfamates.
All UniProtKB/Swiss-Prot entries corresponding to class 3.1.6. are:
3.1.6.1 Arylsulfatase
3.1.6.2 Steryl-sulfatase
3.1.6.3 Glycosulfatase
3.1.6.4 N-acetylgalactosamine-6-sulfatase 3.1.6.6 Choline-sulfatase
3.1.6.7 Cellulose-polysulfatase
3.1.6.8 Cerebroside-sulfatase
3.1.6.9 Chondro-4-sulfatase
3.1.6.10 Chondro-6-sulfatase
3.1.6.11 Disulfoglucosamine-6-sulfatase
3.1.6.12 N-acetylgalactosamine-4-sulfatase
3.1.6.13 lduronate-2-sulfatase
3.1.6.14 N-acetylglucosamine-6-sulfatase
3.1.6.15 N-sulfoglucosamine-3-sulfatase
3.1.6.16 Monomethyl-sulfatase
3.1.6.17 D-lactate-2-sulfatase
3.1.6.18 Glucuronate-2-sulfatase
3.1.6.19 (R)-specific secondary-alkylsulfatase
The expression "human sulfatase" refers to a well-defined class of enzymes. Sulfatases are a conserved gene family having the following defined functional representatives in the human genome assembly of April 2003:
ARSA - Homo sapiens arylsulfatase A (ARSA), transcript variant 2, mRNA. (AB448736)
ARSB - Homo sapiens arylsulfatase B (ARSB), transcript variant 1, mRNA. (J05225)
ARSD - Homo sapiens arylsulfatase D (ARSD), mRNA.
ARSG - Homo sapiens arylsulfatase G (ARSG), transcript variant 1, mRNA.
GALNS - Homo sapiens galactosamine (N-acetyl)-6-sulfate sulfatase (GALNS), mRNA.
(D17629.2)
GNS - Homo sapiens glucosamine (N-acetyl)-6-sulfatase (GNS), mRNA. (NM_002076.4)
IDS - Homo sapiens iduronate 2-sulfatase (IDS), transcript variant 1, mRNA. (NG_011900).
SGSH - Homo sapiens N-sulfoglucosamine sulfohydrolase (SGSH), mRNA. (U30894)
SULF1 - Homo sapiens sulfatase 1 (SULF1), transcript variant 4, mRNA.
SULF1 - Homo sapiens sulfatase 1 (SULF1), transcript variant 4, mRNA.
SULF2 - Homo sapiens sulfatase 2 (SULF2), transcript variant 1, mRNA.
SUMF1 - Homo sapiens sulfatase modifying factor 1 (SUMF1), transcript variant 1, mRNA. (AB448737)
SUMF2 - Homo sapiens sulfatase modifying factor 2 (SUMF2), transcript variant 5, mRNA.
The following sulfatases have been shown to be structurally related based on their sequence homology:
• cerebroside-sulfatase
• steroid sulfatase
• arylsulfatase A EC 3.1.6.8 (ASA), a lysosomal enzyme which hydrolyzes cerebroside sulfate;
• arylsulfatase B EC 3.1.6.12 (ASB) which hydrolyzes the sulfate ester group from N- acetylgalactosamine 4-sulfate residues of dermatan sulfate;
• arylsulfatase C (ASD) and E (ASE); steryl-sulfatase EC 3.1.6.2 (STS), a membrane bound enzyme which hydrolyzes 3-beta-hydroxy steroid sulfates;
• iduronate 2-sulfatase EC 3.1.6.13 (IDS), a lysosomal enzyme that hydrolyzes the 2-sulfate groups from iduronic acids in dermatan sulfate and heparan sulfate;
• N-acetylgalactosamine-6-sulfatase EC 3.1.6.4, which hydrolyzes the 6-sulfate groups of the N-acetyl-D-galactosamine of chondroitin sulfate and D-galactose 6-sulfate units of keratan sulfate;
• N-sulfoglucosamine sulfohydrolase EC 3.10.1.1, the lysosomal enzyme that hydrolyses N- sulfo-D-glucosamine into glucosamine and sulfate;
• glucosamine-6-sulfatase EC 3.1.6.14 (G6S), which hydrolyzes the N-acetyl-D-glucosamine 6-sulfate units of heparan sulfate and keratan sulfate;
• N-sulfoglucosamine sulfohydrolase EC 3.10.1.1, the lysosomal enzyme that hydrolyses N- sulfo-D-glucosamine into glucosamine and sulfate;
• sea urchin embryo arylsulfatase EC 3.1.6.1;
• green algae arylsulfatase EC 3.1.6.1, which plays a role in the mineralization of sulfates; and
• arylsulfatase EC 3.1.6.1 from Escherichia coli, Klebsiella aerogenes and Pseudomonas
aeruginosa.
Sulfatase deficiencies of the present invention include sulfatase deficiencies with a neurological involvement, eg MLD (metachromatic leukodystrophy), MSD (Multiple Sulfatase Deficiency), MPS (mucopolysaccharidosis) II, IMA, MID, IVA. Preferably, sulfatase deficiencies of the present invention may not include MSD. Mucopolysaccharidosis type 111 A (MPS-IIIA) is an inherited disease caused by the deficiency of sulfamidase (SGSH), an enzyme involved in the stepwise degradation of large macromolecules called heparan sulfates. As a consequence, undegraded substrates accumulate in the cells and tissues of the affected patients causing cell damage. The central nervous system (CNS) is the predominant target of damage and in fact, MPSIIIA patients show severe mental retardation and neuropathological decline that ultimately leads to death (often< 20 years). Clinical symptoms include hyperactivity, aggressive behaviour and sleep disturbance. A naturally occurring mouse model of MPS-IIIA has been identified with pathophysiology and symptoms that resemble the human condition [11-13]. These mice represent an ideal model to study the physiopathology of this disorder and to test new therapeutic protocols.
Description of the Drawings
Figure 1. Sulfamidase activity in cultured glial cells. Cultured glia cells derived from C57BL/6 mice were transfected with SGSH, AATsp-SGSH, IDSspSGSH constructs. Both cells and 24 h conditioned media were collected and assayed for sulphamidase activity. The secretion efficiency has been indicated as percentage of the SGSH activity in the medium/ activity in the pellet and the medium.
Figure 2. Sulfamidase specific activity in the CNS of pigs injected with AAV9 vectors carrying different SGSH expression cassettes (A) WT pigs at P60 of age were injected via cisterna magna with 4.5X1012 GC/Kg of AAV9 encoding different human SGSH expression cassettes under the CMV promoter: SGSH WT, IDSspSGSH (SGSH bearing the alternative IDS signal peptide), SGSH- IRES-SUMF1 (bicistronic cassette encoding both SGSH and SUMF1 proteins), IDSspSGSH-IRES- SUMF1 (bicistronic cassette encoding both IDSspSGSH and SUMF1 proteins). At one month after injection sulfamidase specific activity (nmol/17h/mg of protein) was measured in the indicated twenty different areas of the CNS (F. cortex and O. cortex mean frontal and occipital cortex respectively) and compared to control WT pigs treated with PBS. N=5 animals for each group. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. One-way ANOVA followed by Tukey's post hoc test. (B) IHC staining with anti-human sulfamidase in main representative CNS areas of injected pigs.
Figure 3. Sulfamidase activity in MPS-IIIA mice injected with AAV9 vectors carrying different SGSH expression cassettes P60 MPS-IIIA mice were intra-CSF injected (via lateral ventricle administration: ICV) with 4.5x1012 GC/Kg of AAV9 encoding under the CMV promoter the following expression cassettes: GFP, SGSH WT, IDSspSGSH, or IDSspSGSH-IRES-SUMFl. The brain and the first region of the spinal cord of treated mice was divided in five slices (A-E) covering the main representative area of the CNS (A: olfactory bulb and prefrontal cortex, B: frontal cortex, lateral septum and basal ganglia regions, C: parietal cortex, hippocampus, striatum, thalamus, D: occipital cortex, pons, hippocampus; E: cerebellum, medulla oblongata, cervical region of spinal cord). One month after injection sulfamidase activity was measured in these areas and expressed as the percentage of the activity found in control GFP-treated WT mice. N = 3 animals per group. Data represent mean ± SEM.
Figure 4. CNS transduction in M PS-111 A mice injected with AAV9 bearing IDSspSGSH-IRES-SUMFl transgene (A) P60 M PS-111 A mice were ICV injected with 4.5x1012 GC/Kg of AAV9 encoding either IDSspSGSH and SUMF1 or GFP. Five different slices covering the main CNS regions (A-E; as described in the figure 3) were collected at both 1-month (ETP) and 7-months (LTP) after injection. Sulfamidase activity was measured in these areas and expressed as percentage of the activity found in age-matched WT untreated mice. N= 6-7 animals for each group. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. VS M PS-111 A GFP-treated. One-way ANOVA followed by Tukey's post hoc test. (B) Anti-human sulfamidase immunostaining in representative brain regions of M PS-111 A mice was shown at both ETP and LTP upon ICV injection. Scale bar: 100 pm. (C) Co- immunofluorescence analysis of hSulfamidase together with either GFAP (astroglial marker) or NeuN (neuronal marker) in the hippocampus of M PS-111 A mice injected with AAV9 encoding GFP or IDSspSGSH-IRES-SUMFl expression cassette. Scale bars: 50 pm.
Figure 5. Sulfamidase protein quantitation in the brain of M PS-111 A mice injected with AAV9 bearing the IDSspSGSH-IRES-SUMFl transgene. Sulfamidase protein was immuno-quantified by ELISA and expressed as ng of SGSH/mg protein in the five CNS slices (A-E; as described in the supplementary figure 1) of the indicated experimental groups of mice at ETP and LTP. Age- matched WT and MPS-IIIA mice ICV injected with AAV9 encoding for GFP were used as control. Scale banlOO pm. Data represent mean ± SEM. N= 5-7 animals for each group. **P<0.01, ***P<0.001, ****P<0.0001 VS MPS-IIIA-GFP. One-way ANOVA followed by Tukey's post hoc test.
Figure 6. Rescue of storage pathology, inflammation and memory impairment in MPS-IIIA mice injected with AAV9 encoding IDSspSGSH-IRES-SUMFl. (A) LAMP1 immunostaining and relative quantification in the cortex and hippocampus of MPS- IMA mice ICV injected with AAV9 encoding IDSspSGSH-IRES-SUMFl collected at LTP. Age- matched WT and M PS-111 A mice ICV injected with AAV9 encoding GFP were used as controls. Scale bar:100 pm. N= 5-7 animals for each group. All data of IHC in mice were analysed with Prism 7 (GraphPad Software). Data represent mean ± SEM. P values were generated by unpaired t test *p<0.05, **p<0.01. (B) Semi quantitative analysis of lysosome vacuolization on ultra-thin parietal cortex section stained with toluidine blue. The analysis was performed by analyzing 100 cells for each experimental group of mice. N=5-7 animals for group. Data represent mean ± SEM; P values were generated by unpaired t test. *p<0.05, **p<0.01. (C) Quantitative analysis of GAG content (pgGAG/pgDNA) in whole brain samples collected at LTP in M PS-111 A mice ICV injected with AAV9 encoding IDSspSGSH- IRES-SUMF1. Age- matched WT and M PS-111 A mice ICV injected with AAV9 encoding GFP were used as controls. N= 4-6 animals per group. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. One-way ANOVA followed by Tukey's post hoc test. (D) Neuroinflammation was evaluated at LTP in M PS-111 A mice injected with AAV9 encoding IDSspSGSH-IRES-SUMFl by immunostaining with anti-GFAP (astrogliosis) in paraffin sections from frontal cortex, parietal cortex and lateral septum. Age-matched WT and M PS-11 IA mice injected with AAV9 encoding GFP were used as controls. Scale bars: 100 pm. Plots represent the quantification of the signal in the indicted area for each treatment group. N= 4-5 animals per group. Data represent mean ± SEM. P values were generated by unpaired t test **P < 0.01; and ***p < 0.001 versus MPS- IIIA GFP-treated. (E) MPS-IIIA mice and relative controls (WT) were tested at 6 and 9 months of age in the fear contextual conditioning test. MPS-IIIA mice show an age-dependent impairment, as evidenced by reduced freezing time, at only 9 months of age. Treatment with SGSH fully rescued the freezing response in 9 months-old MPS-IIIA mice [Age x Group (F2/16= 4.05; p=0.03); Age (Fl/16=25.34, p=0.0001); Test phase (Fl/16=84.5; p<0.0001); Test phase x Group (F2/16=3.85; p=0.04); Test phase x Age (Fl/16=4.3; p=0.053); Age x Test phase x Group (F2/16=3.35; p=0.06)j. * p<0.05, Duncan post hoc analysis.
Figure 7. Liver transduction in MPS-IIIA mice injected with AAV9 bearing IDSspSGSH-IRES- SUMFl transgene. (A-C) Vector genome copy number (expressed as GC/mouse diploid genome; mdg) (A), sulfamidase activity (expressed as the percentage of WT sulfamidase activity) (B) and ELISA immunoquantification of the sulfamidase protein (expressed as ng of SGSH/mg protein) (C) were measured in liver samples from MPS-IIIA mice ICV injected with AAV9 encoding I DSspSGSH-I RES-SUM Fl and age-matched WT and M PS-111 A mice ICV injected with AAV9 encoding GFP at ETP and LTP. Data represent mea n ± SEM . N= 6-8 animals for each group.
*P<0.05, **P<0.01, ***P<0.001. vs M PS- I I IA GFP-treated. One-way ANOVA followed by Tukey's post hoc test. (D) Quantitative analysis of GAG content (pgGAG/pgDNA) in liver samples collected at LTP in M PS-111 A mice ICV injected with AAV9 encoding I DSspSGSH-I RES-SUM Fl. Age- matched WT and M PS-I IIA mice ICV injected with AAV9 encoding GFP were used as controls. N = 6 animals per group. Data represent mean ± SEM; ****P<0.0001
Figure 8. Assessment of exploratory activity in M PS-I I IA mice injected with AAV9 bea ring I DSspSGSH-I RES-SUM Fl transgene. M PS-I IIA mice and relative controls (WT) were tested at 6 and 9 months of age in the open field test. (A- Al l) There were no significant differences between groups at any of the testing age in the total distance travelled (m) [Group (F2/16=1.16; p=0.22); Distance (Fl/16=0.65; p=0.43); Group x Distance x Age (F2/16=0.45; p=0.64)j (A), total number of line crossings [Group (F2/16=1.94; p=0.17); Line crossing (Fl/16=l,77; p=0.20); Group x Line crossing x Age (F2/16=0.24; p=0.78)j (Al) and total immobility time (sec) [Group (F2/16=1.46; p=0.25); I mmobility time (Fl/16=2.31; p=0.14); Group x I mmobility time x Age (F2/16=0.12; p=0.88)j (Al l). (C-CI I) A deeper ana lysis of the results considering 1 min time intervals (T1-T5) evidenced that at 6 months of age M PS-I I IA mice, as compared to WT littermates, showed reduced distance travelled [Time interval (F4/64=17.9; p<0.0001); Time intervals x Group (F4/64=3.16; p=0.004), Age x Time intervals x Group (F4/64=2.6; p=0.01)j (C), increased immobility time [Time intervals (F4/64=2.4; p=0.05); Group x Age x Time interval (F8/64=2.04; p=0.05)j (Cl) and reduced line crossing frequency [Time interva l (F4/64=6.8; p=0.0001); Group x Time intervals (F4/64=2.66; p=0.01)j (CM) mainly present in the very first minute of the task; these behavioral defects, however, were not anymore detectable at 9 months of ageing probably due to a test-retest habituation effect observed in WT animals [distance, age x time interval (F8/64=3.73; p=0.0085); time immobile, age x time intervals (F4/64=1.61; p=0.05), line crossing, age x time intervals (F4/64=2.4; p=0.05)j. Representative track-plots of the trajectory in the open field (B). * p<0.05, Duncan post hoc analysis
Description of some of the Sequences in the Sequence listing
SEQ ID No 1 - IDSspSGSH Insert (1524 bp)
5'-ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTG
GCCCTGGGCCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGT ACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATG
CCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGA
ATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCG
CTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCG
TGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACT
AGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCC
TTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAAC
GGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGG
TGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCC
GCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACT
GGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCA
CTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTAC
GTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCT
TTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGG
G CCACCGTCTTTGG CAG CCAG AGCCACCACG AGGTCACCATGTCCTACCCCATG CG CTCCGTG CAG CAC
CGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCT
CACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTC
CGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGA
ACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAG
TGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGT
G CCAG CCCCTCCACAATG AGCTGTG A-3' .
SEQ ID No 2 - hSGSH Insert (NCBI: U30894) (1509 bp)
5'-ATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCT
CTGCCGGGCGCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAG GCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTG GACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGT CAG CAGCTG CTCTCCCAGCCGCGCCAG CCTCCTCACTGG CCTG CCCCAG C ATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCC TTCG ACAAG GTG CG G AG CCTG CCG CTG CTG CTCAG CCAAG CTGGTGTGCG
CACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGT
TTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGG AACAT CACT AG AATT AAGCTG CT CGT CCGG AAATT CCTG CAG ACT CAGG A
TGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTG
GGCACTCCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGA
GAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCC
ACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAG
CCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTT
GGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACT
GGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCA
ACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAG
CACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGA
CCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACG
CCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCG
GCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCA
CCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACT
TCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAG
GACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGC
TGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGG
CGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAAC
CTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCA
GCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCG
ACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAAT
GAGCTGTGA-3'
SEQ ID No 3 - AATspSGSH Insert (1521bp)
ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTG
GCTcgtccccggaacgcactgctgctcctcgcggatgacggaggctttgagagtggcgcgtacaacaacagcgccatcgccaccccg cacctggacgccttggcccgccgcagcctcctctttcgcaatgccttcacctcggtcagcagctgctctcccagccgcgccagcctcctca ctggcctgccccagcatcagaatgggatgtacgggctgcaccaggacgtgcaccacttcaactccttcgacaa
ggtgcggagcctgccgctgctgctcagccaagctggtgtgcgcacaggcatcatcgggaagaagcacgtggggccggagaccgtgta cccgtttgactttgcgtacacggaggagaatggctccgtcctccaggtggggcggaacatcactagaattaagctgctcgtccggaaat tcctgcaga ctcagga tga ccggcctttcttcctcta cgtcgccttcca cga ccccca ccgctgtgggca ctcccagccccagta cgga a ccttctgtgagaagtttggcaacggagagagcggcatgggtcgtatcccagact gga ccccccaggccta cga ccca ctgga cgtgctggtgcctta cttcgtcccca a ca ccccggcagcccgagccga cctggccgctcag tacaccaccgtcggccgcatggaccaaggagttggactggtgctccaggagctgcgtgacgccggtgtcctgaacgacacactggtga tcttcacgtccgacaacgggatccccttccccagcggcaggaccaacctgtactggccgggcactgctgaacccttactggtgtcatccc cggagca ccca a a a cgctggggcca agtcagcgaggccta cgtgagcctccta
gacctcacgcccaccatcttggattggttctcgatcccgtaccccagctacgccatctttggctcgaagaccatccacctcactggccggt ccctcctgccggcgctggaggccgagcccctctgggccaccgtctttggcagccagagccaccacgaggtcaccatgtcctaccccatg cgctccgtgcagcaccggcacttccgcctcgtgcacaacctcaacttcaagatgccctttcccatcgaccaggacttctacgtctcaccc a ccttccagga cctcctga a ccgca cca cagctggtcagccca cgggctg
gtacaaggacctccgtcattactactaccgggcgcgctgggagctctacgaccggagccgggacccccacgagacccagaacctggc caccgacccgcgctttgctcagcttctggagatgcttcgggaccagctggccaagtggcagtgggagacccacgacccctgggtgtgcg cccccgacggcgtcctggaggagaagctctctccccagtgccagcccctccacaatgagctgtga
SEQ ID No 4 - IDSspSGSH -IRES-SUMF1 Insert (3283 bp):
5
ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTGG
CCCTGGGCCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTAC
AACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCC
TT CACCT CGGT CAGCAG CTGCTCTCCCAG CCG CG CCAG CCTCCTCACTGG CCTG CCCCAG CATC AG A AT
GGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCT
GCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGT
GTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTA
GAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTT
CCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACG
GAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGT
GCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCG
CATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTG
GTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCAC
TGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACG
TGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTT
TGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGG
CCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACC
GGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTC ACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCC
GTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAA
CCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGT
GGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTG
CCAGCCCCTCCACAATGAGCTGTGATCTAGAAATTCCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTG
GCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTT
TGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCT
CGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGAC
AAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGC
CAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGAT
AGTTGTG G AAAG AGTC AAATG G CTCTCCTC AAG CGTATTC AACAAG GG G CTGAAGGATGCCCAGAAG
GTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTA
AAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATTGATC
AACTAGTCCTGCAGGTTTAAACGAATTCGCCCTTCGTGATCAATGGCTGCGCCCGCACTAGGGCTGGT
GTGTGGACGTTGCCCTGAGCTGGGTCTCGTCCTCTTGCTGCTGCTGCTCTCGCTGCTGTGTGGAGCGGC
AGGGAGCCAGGAGGCCGGGACCGGTGCGGGCGCGGGGTCCCTTGCGGGTTCTTGCGGCTGCGGCAC
GCCCCAGCGGCCTGGCGCCCATGGCAGTTCGGCAGCCGCTCACCGATACTCGCGGGAGGCTAACGCTC
CGGGCCCCGTACCCGGAGAGCGGCAACTCGCGCACTCAAAGATGGTCCCCATCCCTGCTGGAGTATTT
ACAATGGGCACAGATGATCCTCAGATAAAGCAGGATGGGGAAGCACCTGCGAGGAGAGTTACTATTG
ATGCCTTTTACATGGATGCCTATGAAGTCAGTAATACTGAATTTGAGAAGTTTGTGAACTCAACTGGCT
ATTTGACAGAGGCTGAGAAGTTTGGCGACTCCTTTGTCTTTGAAGGCATGTTGAGTGAGCAAGTGAAG
ACCAATATTCAACAGGCAGTTGCAGCTGCTCCCTGGTGGTTACCTGTGAAAGGCGCTAACTGGAGACA
CCCAGAAGGGCCTGACTCTACTATTCTGCACAGGCCGGATCATCCAGTTCTCCATGTGTCCTGGAATGA
TGCGGTTGCCTACTGCACTTGGGCAGGGAAGCGGCTGCCCACGGAAGCTGAGTGGGAATACAGCTGT
CGAGGAGGCCTGCATAATAGACTTTTCCCCTGGGGCAACAAACTGCAGCCCAAAGGCCAGCATTATGC
CAACATTTGGCAGGGCGAGTTTCCGGTGACCAACACTGGTGAGGATGGCTTCCAAGGAACTGCGCCTG
TTGATGCCTTCCCTCCCAATGGTTATGGCTTATACAACATAGTGGGGAACGCATGGGAATGGACTTCAG
ACTGGTGGACTGTTCATCATTCTGTTGAAGAAACGCTTAACCCAAAAGGTCCCCCTTCTGGGAAAGACC
GAGTGAAGAAAGGTGGATCCTACATGTGCCATAGGTCTTATTGTTACAGGTATCGCTGTGCTGCTCGG
AGCCAGAACACACCTGATAGCTCTGCTTCGAATCTGGGATTCCGCTGTGCAGCCGACCGCCTGCCCACT
ATGGACTGA SEQ ID No 5 - pAAV2.1-CMV-SGSH construct (5749 bp)
agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgga attgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggctgcgcgctcgctcgctcact gaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcct
cagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctactta tctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagc ccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatg
ggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaa atggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga gtttgttttggca cca a a a tea a cggga ctttcca a a a tgtcgta a ca
actccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctg cagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgag acagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgca tgagctgccccgtgcccgcctgctgcgcgctgctgctagtcctggggctctgc
cgggcgcgtccccggaacgcactgctgctcctcgcggatgacggaggctttgagagtggcgcgtacaacaacagcgccatcgccaccc cgcacctggacgccttggcccgccgcagcctcctctttcgcaatgccttcacctcggtcagcagctgctctcccagccgcgccagcctcct cactggcctgccccagcatcagaatgggatgtacgggctgcaccaggacgtgcaccacttcaactccttcgacaaggtgcggagcctg ccgctgctgctcagccaagctggtgtgcgcacaggcatcatcgggaagaagca
cgtggggccggagaccgtgtacccgtttgactttgcgtacacggaggagaatggctccgtcctccaggtggggcggaacatcactaga attaagctgctcgtccggaaattcctgcagactcaggatgaccggcctttcttcctctacgtcgccttccacgacccccaccgctgtgggc actcccagccccagtacggaaccttctgtgagaagtttggcaacggagagagcggcatgggtcgtatcccagactggaccccccaggc eta ega ccca ctgga cgtgctggtgcctta cttcgtcccca a ca ccccggcag
cccgagccgacctggccgctcagtacaccaccgtcggccgcatggaccaaggagttggactggtgctccaggagctgcgtgacgccgg tgtcctgaacgacacactggtgatcttcacgtccgacaacgggatccccttccccagcggcaggaccaacctgtactggccgggcactg ctgaacccttactggtgtcatccccggagcacccaaaacgctggggccaagtcagcgaggcct
acgtgagcctcctagacctcacgcccaccatcttggattggttctcgatcccgtaccccagctacgccatctttggctcgaagaccatcca cctcactggccggtccctcctgccggcgctggaggccgagcccctctgggccaccgtctttggcagccagagccaccacgaggtcacca tgtcctaccccatgcgctccgtgcagcaccggcacttccgcctcgtgcacaacctcaacttcaagatgccctttcccatcgaccaggactt eta cgtctca ccca ccttccagga cctcctga a ccgca ccacagctggt
cagcccacgggctggtacaaggacctccgtcattactactaccgggcgcgctgggagctctacgaccggagccgggacccccacgag acccagaacctggccaccgacccgcgctttgctcagcttctggagatgcttcgggaccagctggccaagtggcagtgggagacccacg acccctgggtgtgcgcccccgacggcgtcctggaggagaagctctctccccagtgccagcccctccacaatgagctgtgaatcgattct agtagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctc
ccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgt cattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaa gggcgaattcccgattaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctag tgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccggg
cgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactgg ccgtcgtttta ca a cgtcgtga ctggga a a a ccctggcgtta ccca a etta a tcgccttgcagca ca tccccctttcgccagctggcgta atagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaa gcgcggcgggtgtggtggtta cgcgcagcgtga ccgcta ca cttgccagcgccct
agcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccccgatagacggttttt cgccctttgacgctggagttcacgttcctcaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggatttttccgatttcggcctattggttaaaaaatg
agctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttcggggaaatgtgcgcggaa cccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagg aagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtg aaagtaaaagatgctgaagatcagttgggtgcacgagtgggttac
atcgaactggatctcaatagtggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctat gtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcac cagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaa etta cttctga ca a egateggagga ccga aggageta a ccgcttttttgca
caacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgat gcctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgg aggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggt ctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtag ttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggta actgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataa tctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttt tttctgcgcgta a tctgctgcttgcaa a ca a a a a a a cca ccgcta cca
gcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcctt ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttc gtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct
acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgcggttttgctcaca tgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttg
agtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag
SEQ ID No 6 - pAAV2.1-CMV-SGSH-IRES-SUMFl construct (7508 bp)
agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgga attgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggctgcgcgctcgctcgctcact gaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcct
cagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctactta tctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagc ccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatg
ggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaa atggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga gtttgttttggca cca a a a tea a cggga ctttcca a a a tgtcgta a ca
actccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctg cagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgag acagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgca tgcccccgccccgcaccggccgcggcctgctgtggctgggcctggtgctgagc agcgtgtgcgtggccctgggccgtccccggaacgcactgctgctcctcgcggatgacggaggctttgagagtggcgcgtacaacaaca gcgccatcgccaccccgcacctggacgccttggcccgccgcagcctcctctttcgcaatgccttcacctcggtcagcagctgctctccca gccgcgccagcctcctcactggcctgccccagcatcagaatgggatgtacgggctgcaccaggacgtgcaccacttcaactccttcgac aaggtgcggagcctgccgctgctgctcagccaagctggtgtgcgcacaggcat
catcgggaagaagcacgtggggccggagaccgtgtacccgtttgactttgcgtacacggaggagaatggctccgtcctccaggtgggg cggaacatcactagaattaagctgctcgtccggaaattcctgcagactcaggatgaccggcctttcttcctctacgtcgccttccacgac ccccaccgctgtgggcactcccaaccccagtacggaaccttctgtgagaagtttggcaacggagagagcggcatgggtcgtatcccag a ctgga ccccccaggccta cga ccca ctgga cgtgctggtgcctta cttcgtcc
ccaacaccccggcagcccgagccgacctggccgctcagtacaccaccgtcggccgcatggaccaaggagttggactggtgctccagg agctgcgtga cgccggtgtcctga a cga ca ca ctggtga tcttca cgtccga ca a cggga tccccttccccagcggcagga cca a cctg tactggccgggcactgctgaacccttactggtgtcatccccggagcacccaaaacgctggggccaagtcagcgaggcctacgtgagcc tcctagacctcacgcccaccatcttggattggttctcgatcccgtaccccagctac
gccatctttggctcgaagaccatccacctcactggccggtccctcctgccggcgctggaggccgagcccctctgggccaccgtctttggc agccagagccaccacgaggtcaccatgtcctaccccatgcgctccgtgcagcaccggcacttccgcctcgtgcacaacctcaacttcaa gatgccctttcccatcgaccaggacttctacgtctcacccaccttccaggacctcctgaaccgcaccacagctggtcagcccacgggctg gta ca agga cctccgtca tta eta eta ccgggcgcgctgggagctcta cga
ccggagccgggacccccacgagacccagaacctggccaccgacccgcgctttgctcagcttctggagatgcttcgggaccagctggcc aagtggcagtgggagacccacgacccctgggtgtgcgcccccgacggcgtcctggaggagaagctctctccccagtgccagcccctac acaatgagctgtgatctagaaattccgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgc gtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggc
ccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaa ggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacagg tgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtgg aaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgccc
agaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccc cccgaaccacggggacgtggttttcctttgaaaaacacgatgataattgatcaactagtcctgcaggtttaaacgaattcgcccttcgtg atcaatggctgcgcccgcactagggctggtgtgtggacgttgccctgagctgggtctcgtcctcttgctgctgctgctctcgctgctgtgtg gagcggcagggagccaggaggccgggaccggtgcgggcgcggggtccc
ttgcgggttcttgcggctgcggcacgccccagcggcctggcgcccatggcagttcggcagccgctcaccgatactcgcgggaggctaac gctccgggccccgtacccggagagcggcaactcgcgcactcaaagatggtccccatccctgctggagtatttacaatgggcacagatg atcctcagataaagcaggatggggaagcacctgcgaggagagttactattgatgccttttacatggatgcctatgaagtcagtaatact gaatttgagaagtttgtgaactcaactggctatttgacagaggctgagaagttt
ggcgactcctttgtctttgaaggcatgttgagtgagcaagtgaagaccaatattcaacaggcagttgcagctgctccctggtggttacct gtgaaaggcgctaactggagacacccagaagggcctgactctactattctgcacaggccggatcatccagttctccatgtgtcctggaa tgatgcggttgcctactgcacttgggcagggaagcggctgcccacggaagctgagtgggaatacagctgtcgaggaggcctgcataat agacttttcccctggggcaacaaactgcagcccaaaggccagcattatgccaa
catttggcagggcgagtttccggtgaccaacactggtgaggatggcttccaaggaactgcgcctgttgatgccttccctcccaatggtta tggcttatacaacatagtggggaacgcatgggaatggacttcagactggtggactgttcatcattctgttgaagaaacgcttaacccaa aaggtcccccttctgggaaagaccgagtgaagaaaggtggatcctacatgtgccataggtcttattgttacaggtatcgctgtgctgctc ggagccagaacacacctgatagctctgcttcgaatctgggattccgctgtg
cagccgaccgcctgcccactatggactgaactagtagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccc cgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcat tctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagg gcgaattcccgattaggatcttcctagagcatggctacgtagataagtagca
tggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcg accaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggcc gtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaat agcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg
gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccct agcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccccgatagacggttttt cgccctttgacgctggagttcacgttcctcaatagtggactcttgttc
caaactggaacaacactcaaccctatctcggtctattcttttgatttataagggatttttccgatttcggcctattggttaaaaaatgagct gatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttcggggaaatgtgcgcggaacccc tatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaag agtatgagtattcaacatttccgtgtcgcccttattcccttttttgc
ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacat cgaactggatctcaatagtggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgt ggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcacc agtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcac aacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg cctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgga ggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggttt
attgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagtt atctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaac tgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatc tcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcaga
ccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttcta gtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttac
cggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc tacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggccttttta
cggttcctggccttttgctgcggttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgag ctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag
SEQ ID No 7 - pAAV2.1-CMV-IDSspSGSH construct (5752 bp)
agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgga attgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggctgcgcgctcgctcgctcact gaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcct
cagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctactta tctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagc ccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatg
ggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaa atggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga gtttgttttggca cca a a a tea a cggga ctttcca a a a tgtcgta a ca actccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctg cagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgag acagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgca tgcccccgccccgcaccggccgcggcctgctgtggctgggcctggtgctgagc
agcgtgtgcgtggccctgggccgtccccggaacgcactgctgctcctcgcggatgacggaggctttgagagtggcgcgtacaacaaca gcgccatcgccaccccgcacctggacgccttggcccgccgcagcctcctctttcgcaatgccttcacctcggtcagcagctgctctccca gccgcgccagcctcctcactggcctgccccagcatcagaatgggatgtacgggctgcaccaggacgtgcaccacttcaactccttcgac aaggtgcggagcctgccgctgctgctcagccaagctggtgtgcgcacaggcat
catcgggaagaagcacgtggggccggagaccgtgtacccgtttgactttgcgtacacggaggagaatggctccgtcctccaggtgggg cggaacatcactagaattaagctgctcgtccggaaattcctgcagactcaggatgaccggcctttcttcctctacgtcgccttccacgac ccccaccgctgtgggcactcccaaccccagtacggaaccttctgtgagaagtttggcaacggagagagcggcatgggtcgtatcccag a ctgga ccccccaggccta cga ccca ctgga cgtgctggtgcctta cttcgtcc
ccaacaccccggcagcccgagccgacctggccgctcagtacaccaccgtcggccgcatggaccaaggagttggactggtgctccagg agctgcgtga cgccggtgtcctga a cga ca ca ctggtga tcttca cgtccga ca a cggga tccccttccccagcggcagga cca a cctg tactggccgggcactgctgaacccttactggtgtcatccccggagcacccaaaacgctggggccaagtcagcgaggcctacgtgagcc tcctagacctcacgcccaccatcttggattggttctcgatcccgtaccccagctac
gccatctttggctcgaagaccatccacctcactggccggtccctcctgccggcgctggaggccgagcccctctgggccaccgtctttggc agccagagccaccacgaggtcaccatgtcctaccccatgcgctccgtgcagcaccggcacttccgcctcgtgcacaacctcaacttcaa gatgccctttcccatcgaccaggacttctacgtctcacccaccttccaggacctcctgaaccgcaccacagctggtcagcccacgggctg gta ca agga cctccgtca tta eta eta ccgggcgcgctgggagctcta cga
ccggagccgggacccccacgagacccagaacctggccaccgacccgcgctttgctcagcttctggagatgcttcgggaccagctggcc aagtggcagtgggagacccacgacccctgggtgtgcgcccccgacggcgtcctggaggagaagctctctccccagtgccagcccctac acaatgagctgtgaagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaa ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcatt
gtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctg gggactcgagttaagggcgaattcccgattaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaacta caaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgc ccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacc
taattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgcc agctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcg gcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcc cttcctttctcgcca cgttcgccggctttccccgtca agctcta a a tcggg
ggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgcc ccgatagacggtttttcgccctttgacgctggagttcacgttcctcaatagtggactcttgttccaaactggaacaacactcaaccctatc tcggtctattcttttgatttataagggatttttccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaatttt aacaaaatattaacgtttataatttcaggtggcatctttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaa tatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgc ccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtg cacgagtgggttacatcgaactggatctcaatagtggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactt t
taaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgactt ggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgat aacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcg ccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtg
acaccacgatgcctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaata gactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggc aactatggatgaacgaaatagacagatcgctgagataggtgcctcactgatt
aagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttct tgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctac caactctttttccgaaggtaactggcttcagcagagcgcagatacc
aaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtta ccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctg aacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcg
gaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgcggtt ttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga acgaccgagcgcagcgagtcagtgagcgaggaagcggaag
SEQ ID No 8 - pAAV2.1-CMV-IDSspSGSH-IRES-SUMFl construct (7517) agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgga attgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggctgcgcgctcgctcgctcact gaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcct
cagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctactta tctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagc ccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatg
ggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaa atggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga gtttgttttggca cca a a a tea a cggga ctttcca a a a tgtcgta a ca
actccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctg cagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgag acagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgca tgcccccgccccgcaccggccgcggcctgctgtggctgggcctggtgctgagc
agcgtgtgcgtggccctgggccgtccccggaacgcactgctgctcctcgcggatgacggaggctttgagagtggcgcgtacaacaaca gcgccatcgccaccccgcacctggacgccttggcccgccgcagcctcctctttcgcaatgccttcacctcggtcagcagctgctctccca gccgcgccagcctcctcactggcctgccccagcatcagaatgggatgtacgggctgcaccaggacgtgcaccacttcaactccttcgac aaggtgcggagcctgccgctgctgctcagccaagctggtgtgcgcacaggcat
catcgggaagaagcacgtggggccggagaccgtgtacccgtttgactttgcgtacacggaggagaatggctccgtcctccaggtgggg cggaacatcactagaattaagctgctcgtccggaaattcctgcagactcaggatgaccggcctttcttcctctacgtcgccttccacgac ccccaccgctgtgggcactcccaaccccagtacggaaccttctgtgagaagtttggcaacggagagagcggcatgggtcgtatcccag a ctgga ccccccaggccta ega ccca ctgga cgtgctggtgcctta cttcgtcc
ccaacaccccggcagcccgagccgacctggccgctcagtacaccaccgtcggccgcatggaccaaggagttggactggtgctccagg agctgcgtgacgccggtgtcctgaacgacacactggtgatcttcacgtccgacaacgggatccccttccccagcggcaggaccaacctg tactggccgggcactgctgaacccttactggtgtcatccccggagcacccaaaacgctggggccaagtcagcgaggcctacgtgagcc tcctagacctcacgcccaccatcttggattggttctcgatcccgtaccccagctac
gccatctttggctcgaagaccatccacctcactggccggtccctcctgccggcgctggaggccgagcccctctgggccaccgtctttggc agccagagccaccacgaggtcaccatgtcctaccccatgcgctccgtgcagcaccggcacttccgcctcgtgcacaacctcaacttcaa gatgccctttcccatcgaccaggacttctacgtctcacccaccttccaggacctcctgaaccgcaccacagctggtcagcccacgggctg gta ca agga cctccgtca tta eta eta ccgggcgcgctgggagctctacga
ccggagccgggacccccacgagacccagaacctggccaccgacccgcgctttgctcagcttctggagatgcttcgggaccagctggcc aagtggcagtgggagacccacgacccctgggtgtgcgcccccgacggcgtcctggaggagaagctctctccccagtgccagcccctac acaatgagctgtgatctagaaattccgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgc gtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggc
ccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaa ggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacagg tgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtgg aaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgccc
agaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccc cccgaaccacggggacgtggttttcctttgaaaaacacgatgataattgatcaactagtcctgcaggtttaaacgaattcgcccttcgtg atcaatggctgcgcccgcactagggctggtgtgtggacgttgccctgagctgggtctcgtcctcttgctgctgctgctctcgctgctgtgtg gagcggcagggagccaggaggccgggaccggtgcgggcgcggggtccc
ttgcgggttcttgcggctgcggcacgccccagcggcctggcgcccatggcagttcggcagccgctcaccgatactcgcgggaggctaac gctccgggccccgtacccggagagcggcaactcgcgcactcaaagatggtccccatccctgctggagtatttacaatgggcacagatg atcctcagataaagcaggatggggaagcacctgcgaggagagttactattgatgccttttacatggatgcctatgaagtcagtaatact gaatttgagaagtttgtgaactcaactggctatttgacagaggctgagaagttt
ggcgactcctttgtctttgaaggcatgttgagtgagcaagtgaagaccaatattcaacaggcagttgcagctgctccctggtggttacct gtgaaaggcgctaactggagacacccagaagggcctgactctactattctgcacaggccggatcatccagttctccatgtgtcctggaa tgatgcggttgcctactgcacttgggcagggaagcggctgcccacggaagctgagtgggaatacagctgtcgaggaggcctgcataat agacttttcccctggggcaacaaactgcagcccaaaggccagcattatgccaa
catttggcagggcgagtttccggtgaccaacactggtgaggatggcttccaaggaactgcgcctgttgatgccttccctcccaatggtta tggcttatacaacatagtggggaacgcatgggaatggacttcagactggtggactgttcatcattctgttgaagaaacgcttaacccaa aaggtcccccttctgggaaagaccgagtgaagaaaggtggatcctacatgtgccataggtcttattgttacaggtatcgctgtgctgctc ggagccagaacacacctgatagctctgcttcgaatctgggattccgctgtg
cagccgaccgcctgcccactatggactgaactagtagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccc cgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcat tctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagg gcgaattcccgattaggatcttcctagagcatggctacgtagataagtagca tggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcg accaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggcc gtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaat agcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg
gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccct agcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccccgatagacggttttt cgccctttgacgctggagttcacgttcctcaatagtggactcttgttc
caaactggaacaacactcaaccctatctcggtctattcttttgatttataagggatttttccgatttcggcctattggttaaaaaatgagct gatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttcggggaaatgtgcgcggaacccc tatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaag agtatgagtattcaacatttccgtgtcgcccttattcccttttttgc
ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacat cgaactggatctcaatagtggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgt ggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcacc agtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca
gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcac aacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg cctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgga ggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggttt
attgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagtt atctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaac tgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatc tcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcaga
ccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttcta gtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttac
cggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc tacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggccttttta
cggttcctggccttttgctgcggttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgag ctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag
SEQ ID No 9 - pAAV2.1-CMV-GFP construct (5504 bp)
agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgga attgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggctgcgcgctcgctcgctcact gaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggc caactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgc ccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggta aatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggacttt ccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattga cgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtc atcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccc attgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg cggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctgcagaagttggtcgtgaggcactgg gcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctga taggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgccatggtgagcaagggcgaggagctg ttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgat gcca ccta cggca agctga ccctga agttca tctgca cca ccggca agctgcccgtgccctggccca ccctcgtga cca ccctga ccta cggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggag cgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgag ctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatg gccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccacta ccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccc caacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaataa gcttggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacg ctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagt tgtggcccgttgtcaggca a cgtggcgtggtgtgca ctgtgtttgctga cgca a ccccca ctggttggggca ttgcca cca cctgtcagc tcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg ttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcggga cgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcga gatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactg tcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagg gggaggattgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccgattaggatcttcctagagcatggctacg tagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctca ctgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaac eta a ttca ctggccgtcgtttta ca a cgtcgtga ctggga a a a ccctggcgtta ccca a etta a tcgccttgcagca ca tccccctttcgc cagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagc ggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttc ccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctc gaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccccgatagacggtttttcgccctttgacgctggagttcacg ttcctcaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggatttttccgatttcg gcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttc ggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgct tcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgct cacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaatagtggta agatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgac gccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacgg atggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggagg accgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccatac caaacgacgagcgtgacaccacgatgcctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcc cggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgat aaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacac gacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagac caagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacc aaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcg taatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaa ctggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgccta catacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttacc ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgcggttttgctcaca tgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccga gcgcagcgagtcagtgagcgaggaagcggaag
SEQ ID No 10 - pAAV2.1-CMV-AATspSGSH entire plasmid (5749 bp)
agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgga attgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggctgcgcgctcgctcgctcact gaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcct
cagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctactta tctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagc ccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtaca tcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatg ggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaa tgtcgtaaca
actccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctg cagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgag acagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgcA
TGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTG
GTCCCTGTCTCCCTGGCTcgtccccggaacgcactgctgctcctcgcggatgacggaggctttgagagtggcgcgtacaacaac agcgccatcgccaccccgcacctggacgccttggcccgccgcagcctcctctttcgcaatgccttcacctcggtcagcagctgctctccc agccgcgccagcctcctcactggcctgccccagcatcagaatgggatgtacgggctgcaccaggacgtgcaccacttcaactccttcga caaggtgcggagcctgccgctgctgctcagccaagctggtgtgcgcacaggcatcat
cgggaagaagcacgtggggccggagaccgtgtacccgtttgactttgcgtacacggaggagaatggctccgtcctccaggtggggcgg aacatcactagaattaagctgctcgtccggaaattcctgcagactcaggatgaccggcctttcttcctctacgtcgccttccacgaccccc accgctgtgggcactcccagccccagtacggaaccttctgtgagaagtttggcaacggagagagcggcatgggtcgtatcccagactg gaccccccaggcctacgacccactggacgtgctggtgccttacttcgtccccaacaccccggcagcccgagccgacctggccgctcagt acaccaccgtcggccgcatggaccaaggagttggactggtgctccaggagctgcgtgacgccggtgtcctgaacgacacactggtgat cttca cgtccga ca a cggga tccccttccccagcggcagga cca a cctgta ctggccgggca ctgctga a ccctta ctggtgtca tcccc ggagcacccaaaacgctggggccaagtcagcgaggcctacgtgagcctcctagacctcacgcccaccatcttggattggttctcgatcc cgtaccccagctacgccatctttggctcgaagaccatccacctcactggccggtccctcctgccggcgctggaggccgagcccctctggg ccaccgtctttggcagccagagccaccacgaggtcaccatgtcctaccccatgcgctccgtgcagcaccggcacttccgcctcgtgcac aacctcaacttcaagatgccctttcccatcgaccaggacttctacgtctcacccaccttccaggacctcctgaaccgcaccacagctggt cagcccacgggctggtacaaggacctccgtcattactactaccgggcgcgctgggagctctacgaccggagccgggacccccacgag acccagaacctggccaccgacccgcgctttgctcagcttctggagatgcttcgggaccagctggccaagtggcagtgggagacccacg acccctgggtgtgcgcccccgacggcgtcctggaggagaagctctctccccagtgccagcccctccacaatgagctgtgaagatctgcc tcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcct aataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagga ttgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccgattaggatcttcctagagcatggctacgtagataa gtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggc cgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattca ctggccgtcgtttta ca a cgtcgtga ctggga a a a ccctggcgtta ccca a ctta a tcgccttgcagca ca tccccctttcgccagctggc gtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcatt aagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttccttt ctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgacccca aaaaacttgattagggtgatggttcacgtagtgggccatcgccccgatagacggtttttcgccctttgacgctggagttcacgttcctcaa tagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggatttttccgatttcggcctattg gttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttcggggaaa tgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataat attgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccag aaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaatagtggtaagatcct tgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccggg caagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcat gacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaag gagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacga cgagcgtgacaccacgatgcctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaac aattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctg gagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggg agtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt actcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatc ccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctg ctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggctt cagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagc gtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcg cacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgc tcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgcggttttgctcacatgttct ttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgca gcgagtcagtgagcgaggaagcggaag
SEQ ID No 11 - I nsert SGSH -I RES-SUM F1 (3268 bp)
ATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCTCTGCCGGGCGCGTCCCCG
GAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATC
GCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGC
AGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCT
GCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAG
CTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTT
TGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTC
GTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACC
GCTGTGGGCACTCCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCAT
GGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCC
CAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGA
GTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTC
CGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTAC
TGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGAC
CTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCA
TCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCA
GCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCG
TGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGA
CCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCG GGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCG
CGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCC
CTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATG
AGCTGTGATCTAGAAATTCCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGA
ATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCC
CGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAA
GGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGC
GACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTAT
AAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGT
CAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGG
GATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCC
CCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATTGATCAACTAGTCCTGCAGGTT
TAAACGAATTCGCCCTTCGTGATCAATGGCTGCGCCCGCACTAGGGCTGGTGTGTGGACGTTGCCCTG
AGCTGGGTCTCGTCCTCTTGCTGCTGCTGCTCTCGCTGCTGTGTGGAGCGGCAGGGAGCCAGGAGGCC
GGGACCGGTGCGGGCGCGGGGTCCCTTGCGGGTTCTTGCGGCTGCGGCACGCCCCAGCGGCCTGGCG
CCCATGGCAGTTCGGCAGCCGCTCACCGATACTCGCGGGAGGCTAACGCTCCGGGCCCCGTACCCGGA
GAGCGGCAACTCGCGCACTCAAAGATGGTCCCCATCCCTGCTGGAGTATTTACAATGGGCACAGATGA
TCCTCAGATAAAGCAGGATGGGGAAGCACCTGCGAGGAGAGTTACTATTGATGCCTTTTACATGGATG
CCTATGAAGTCAGTAATACTGAATTTGAGAAGTTTGTGAACTCAACTGGCTATTTGACAGAGGCTGAG
AAGTTTGGCGACTCCTTTGTCTTTGAAGGCATGTTGAGTGAGCAAGTGAAGACCAATATTCAACAGGC
AGTTGCAGCTGCTCCCTGGTGGTTACCTGTGAAAGGCGCTAACTGGAGACACCCAGAAGGGCCTGACT
CTACTATTCTGCACAGGCCGGATCATCCAGTTCTCCATGTGTCCTGGAATGATGCGGTTGCCTACTGCA
CTTGGGCAGGGAAGCGGCTGCCCACGGAAGCTGAGTGGGAATACAGCTGTCGAGGAGGCCTGCATA
ATAGACTTTTCCCCTGGGGCAACAAACTGCAGCCCAAAGGCCAGCATTATGCCAACATTTGGCAGGGC
GAGTTTCCGGTGACCAACACTGGTGAGGATGGCTTCCAAGGAACTGCGCCTGTTGATGCCTTCCCTCCC
AATGGTTATGGCTTATACAACATAGTGGGGAACGCATGGGAATGGACTTCAGACTGGTGGACTGTTCA
TCATTCTGTTGAAGAAACGCTTAACCCAAAAGGTCCCCCTTCTGGGAAAGACCGAGTGAAGAAAGGTG
GATCCTACATGTGCCATAGGTCTTATTGTTACAGGTATCGCTGTGCTGCTCGGAGCCAGAACACACCTG
ATAGCTCTGCTTCGAATCTGGGATTCCGCTGTGCAGCCGACCGCCTGCCCACTATGGACTGA
Aminoacidic sequences:
I DSspSGSH SEQ ID No 12 M PPPRTGRGLLWLGLVLSSVCVALGRPRNALLLLADDGGFESGAYN NSAIATPH LDALARRSLLFRNAFTSV
SSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYT
EENGSVLQVGRNITRI KLLVRKFLQTQDDRPFFLYVAFHDPH RCGHSQPQYGTFCEKFGNGESGMGRI PD
WTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRM DQGVGLVLQELRDAGVLNDTLVIFTSDNGI PFP
SGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTI LDWFSI PYPSYAI FGSKTIHLTGRSLLPALE
AEPLWATVFGSQSHH EVTMSYPMRSVQH RHFRLVHN LNFKM PFPI DQDFYVSPTFQDLLNRTTAGQPTG
WYKDLRHYYYRARWELYDRSRDPHETQN LATDPRFAQLLEM LRDQLAKWQWETHDPWVCAPDGVLEE
KLSPQCQPLHNEL*
SUM F1 SEQ ID No 13
MAAPALGLVCGRCPELGLVLLLLLLSLLCGAAGSQEAGTGAGAGSLAGSCGCGTPQRPGAHGSSAAAHRYS
REANAPGPVPGERQLAHSKMVPIPAGVFTMGTDDPQIKQDGEAPARRVTI DAFYMDAYEVSNTEFEKFV
NSTGYLTEAEKFGDSFVFEGM LSEQVKTN IQQAVAAAPWWLPVKGANWRHPEGPDSTI LHRPDHPVLHV
SWN DAVAYCTWAGKRLPTEAEWEYSCRGGLHN RLFPWGNKLQPKGQHYAN IWQGEFPVTNTGEDGFQ
GTAPVDAFPPNGYGLYN IVGNAWEWTSDWWTVHHSVEETLN PKGPPSGKDRVKKGGSYMCHRSYCYRY
RCAARSQNTPDSSASNLGFRCAADRLPTM D*
DETAILED DESCRIPTION OF THE INVENTION MATERIAL AND METHODS
Generation of modified Sulfamidase cassettes and AAV vectors
SGSH and SGSH-IRES-SUMF1 cassettes
The generation of pAAV2.1-CMV-SGSH and pAAV2.1-CMV-SGSH-I RES-SUM Fl for the AAV9 vector preparations has been described in the paper of Fraldi et al.2003 [4].
IDSspSGSH cassette
The assem bly of the I DSspSGSH construct (I DSsp: human iduronate-2-sulfatase signal peptide) has been described in the paper of Sorrentino et al. 2013 [5]. To generate the pAAV2.1-CMV- I DSspSGSH for the AAV9 preparation, the inventors amplified the I DSspSGSH from the p3xFLAG- CMV-I DSspSGSH by using the following oligos:
Forward Notl IDSnew: TTTGCGGCCGCATGCCCCCGCC (SEQ I D NO: 17)
Reverse Bglll SGSH : G AAG AT CTT C ACAG CT C ATTGTG (SEQ I D NO: 18)
The PCR product was digested with Notl/Bglll restriction enzymes and cloned into the pAAV2.1- CMV-GFP by replacing the GFP protein. IDSspSGSH-IRES-SUMFl cassette
The IDSspSGSH cassette to use for the production of the IDSspSGSH-IRES-SUMFl cassette was amplified from the p3xFLAG-CMV-IDSspSGSH by the following oligos:
Forward Notl IDSnew: TTTGCGGCCGCATGCCCCCGCC (SEQ ID NO: 19)
Reverse Xbal SGSH: TG CTCT AG AT C ACAG CT CATTGTG (SEQ ID NO: 20)
The PCR product was digested with Notl/Xbal restriction enzymes and cloned into the pAAV2.1- CMV-SGSH-IRES-SUMF1, already developed in Fraldi el al paper [4], by replacing the SGSH protein.
The resulting plasmids were used to generate the correspondent AAV serotype 9 (AAV2/8) viral vectors according to the protocols established by AAV Vector Core of TIGEM institute.
Transfection and Secretion in cells
Primary cultured Glia cells were transfected using LipofectamineTM 2000 (Invitrogen) according to manufacturer's protocols. 5 hours after transfection the medium was replaced with DMEM 10% FBS. One day after transfection the inventors induced the secretion replacing the DMEM 10% FBS with a DMEM 0.5% FBS. Two days after transfection the conditioned media and the pellets were collected for the enzymatic assays.
Pig Study
Animal model, rAAV administration and tissue collection
The study was conducted in accordance with the provisions of European Economic Community Council Directive 86/609 adopted by the Italian Government (DL 27/01/1992 No. 116) under the local approval of the Ethical Committee of the University of Bologna and under the approval of Italian Ministry of Health. All animal studies have been approved by the authors. The animals enrolled were WT large White x Duroc hybrids, and the sex ratio between females and castrated males was ~1:1. These animals were transferred to inventors' facility on the day of weaning (28th day after birth) and housed in multiple stalls with infrared heating lamps. They were strictly monitored in order to rule out any pathology that may have affected the entire experiment.
rAAV administration and CNS samples collection was carried out according to the Sorrentino el al. paper [6]. In detail, the dorsal area of the neck was trimmed and surgically prepared, and the puncture of the cisterna magna was performed. One ml of CSF was collected before the injection in order to analyze it as a preinjection physiological standard. The dose of 4,5 x 1012 GC/Kg of viral vector in a volume range from 1.5 to 2.8 ml was injected slowly to avoid a sudden increase in intracranial pressure. Piglets were then placed in Trendelenburg position for 2 minutes in order to help the injected compound to spread toward the more rostral parts of the CNS. Animals were then monitored until complete recovery.
Mouse study
Animals, rAAV administration and tissue collection
Homozygous mutant (sgsh-/-; phenotypically M PS-111 A affected) and WT C57BL/6 mice were used [7], [4], [9]. After being collected, the brain samples were harvested and stored frozen until use. To collect brain, mice were perfused with PBS (pH 7.4), and to prepare brain samples for Sulphamidase assays standard capillary depletion protocols were used according to the Fraldi et al. protocols as published [4]. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Cardarelli Hospital in Naples and authorized by the Italian Ministry of Health. In detail, M PS-111 A mice at 2 months of age were anesthetized with ketamine/medetomidine. The AAV vectors were injected with a dosage of 5.4X1012 GC/Kg bilaterally into the lateral ventricles.
AAV administration
Pigs. All of the activities performed on the day of the injection have been thoroughly described in Sorrentino et al. [6]. For the intra cisterna magna (ICM) injection the dorsal area of the neck was trimmed and surgically prepared, and the puncture of the cisterna magna was performed as previously described [8]. In brief, animal received an i.m. bolus of tiletamine-zolazepam (5 mg/kg) 10 minutes before induction; general anaesthesia was achieved using sevoflurane with an induction mask. After orotracheal intubation and stabilization, venous access for fluid therapy was achieved from an auricular vein. Blood samples (6 ml) were collected through the femoral artery.
The dose of 4.5 x 1012 GC/Kg of viral vector in the volume range from 1.5 to 2.8 ml was injected slowly to avoid a sudden increase in intracranial pressure. Piglets were then placed in Trendelenburg position for 2 minutes in order to help the injected compound to spread toward the more rostral parts of the CNS. Animals were then monitored until complete recovery. During the following days, all animals were strictly monitored in order to rule out any possible side effect of the procedure and to evaluate any changes in behaviour and consequentially in welfare.
Mice. Mice were anesthetized by were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed on a stereotaxic instrument with a motorized stereotaxic injector. A midline incision was made to expose the bregma. A hole in the skull was made by a drill (anteroposterior +2.18 mm, mediolateral 0,6 mm, dorsoventral -1.7 mm). rAAV9 vectors 5.4 x 1012 GC/Kg were injected in a volume of 10 ul into the lateral ventricles at a rate of 1 mI/min. After allowing the needle to remain in place for 5 min, the needle was slowly raised at a rate of 0.1 cm/min.
Tissue collection
Pigs. Animals were euthanized 1 month after injection with a single bolus (0.3 ml/kg) of Tanax and total body perfusion with Dulbecco's phosphate-buffered saline was started. After median sternotomy, the right atrium was opened and the left ventricle was infused with 500 ml of warm Dulbecco's phosphate-buffered saline (+38 °C) and 1,000 ml of cold Dulbecco's phosphate- buffered saline (+4 °C); blood ejected from the right atrium was drained using a surgical aspirator. As far as CNS samples, collected tissues were the whole brain and cervical region of spinal cord. Dissection was performed using the technique previously described [6]. Twelve coronal sections (0.5 cm) were cut covering the main regions of the brain (right and left hemispheres) and cervical region of the spinal cord. Sections were then frozen in liquid nitrogen for biochemical analysis or fixed in 4% (w/v) paraformaldehyde in PBS for OCT embedding. Mice. Mice were euthanized at 1, 6 and 7 months by injection with ketamine and xylazine before blood and CSF collection. CSF was collected by glass capillary inserted into the cisterna magna. For tissue collection, mice were intraca rdially perfused with PBS (pH 7,4) and brains were removed, divided into halves and fixed for further analysis. The right half was sliced in five slices (A-E) and frozen in liquid nitrogen, the left half was sliced in two coronally anterior and posterior parts fixed in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin. For Tol blue staining analysis a coronally slice derived from left half of brain was fixed in 4% paraformaldehyde, 25% glutaraldehyde in phosphate buffer for plastic embedding. Additionally, the left lateral lobe of the liver from the mice were fixed in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin, and the right medial lobe of the liver of the mice was frozen in liquid nitrogen.
SGSH activity Twenty main regions covering the entire CNS of injected pigs were dissected and homogenized with Tissue Lyser using 10 volumes of H20 mQ (700 mI). In mice 5 different slices covering the entire brain and liver samples were homogenized separately with Tissue Lyser using 8 volumes of H20 mQ. The SGSH activity was assayed by a 4-methylumbelliferone-derived fluorogenic substrate (4- MU; Moscerdam Substrates), following established protocols [5].
Immunoassay of SGSH
Immunoquantification of SGSH was performed in brain and liver samples of mice by using an Enzyme-Linked Immunosorbent Assay (ELISA) technology. 100 pL of anti-SGSH antibody R3074 from SHIRE (1 pg/mL) diluted in 0.05 M carbonate-bicarbonate buffer (pH 9.6, Sigma-Aldrich Japan, Tokyo, Japan) was used to coat multi-well plates at 37°C for lh. Afterward samples or standards were incubated at 37 °C for 1 h and then with horseradish peroxidase (HRP)- conjugated rabbit anti-HNS antibody R1315 (1:4000) at 37 °C for 1 h. The plate was then incubated with the HRP substrate, TMB peroxidase substrate (Bio-Rad, Hercules, California, USA #1721066) for 30 min at 37°C. This enzyme-substrate reaction was stopped using a stop buffer (2N of H2S04) and the absorbance of each well was measured at the absorbance wavelength of 450 nm (Lml) with a reference wavelength of 655 nm (Lm2) using a microplate reader (GloMax®-Multi+ Detection System, Promega, Madison, Wisconsin, USA). The concentrations of HNS in samples were calculated using the HNS calibration curve in the same plate.
Evaluation of AAV vector genome copy number in the CNS
Genomic DNA was extracted from mouse brain samples using a DNeasy Blood and Tissue Extraction kit (Qjagen, Valencia, CA). DNA concentration was determined by using a Nanodrop. Real-time PCR was performed on 100 ng of genomic DNA using a LightCycler SYBR green I system (Roche, Almere, The Netherlands). Amplification was run on a LightCycler 96 device (Roche) with standard cycles. The primers forward SGSH (5' CATCCACTTTGCCTTTCTCTCCA 3', SEQ ID No.: 21) and reverse SGSH (5' T CAAAG CCTCCGT CAT CCG C 3', SEQ ID No.: 22) were used. A standard curve was generated, using the corresponding AAV vector plasmid pAAV2.1CMV-IDSspSGSH- IRES-SUMF1.
GAG quantification
Brain samples and liver samples were lysed in water by Tissue Lyser equipment. The lysates were then digested with proteinase K and extracts were clarified by centrifugation and filtration. GAG levels in brain extracts were determined using Blyscan sulfated glycosaminoglycan kit (Biocolor, Carrickfergus, UK) with chondroitin 4-sulfate as the standard.
Immunohistochemistry
Immunohistochemical experiments were performed in 5micron paraffin sections with BondRX Stainer following the standard protocol. The primary antibodies used were mouse anti-Human SGSH (Shire 2C7) 1:25 for immunofluorescence and 1:500 for IHC, rabbit anti-LAMPl (ab24170 Abeam), rabbit anti-GFAP (ab7260Abcam), rabbit anti-NeuN (abl77487). 5-micron paraffin- embedded tissue sections were incubated overnight at 4°C with rabbit anti-GFAP (Z0334; Dako Cytomation). The detection system including secondary antibodies used for immunohistochemistry was Bond Polymer refine kit (Leica, DS9800) for the visualization of SGSH and LAMP1 signals. The secondary antibody used for the detection of GFAP and IBAI signal was biotinylated universal antibody of Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).
Bright-field sections were stained with 3.3-diaminobenzidine (Sigma-Aldrich) and counterstained with haematoxylin. The SGSH and LAMP1 stained slides were scanned with Aperio ScanScope AT2 scanner. The whole digital slides were viewed and analyzed by ImageScope. The positive pixel count algorithm was selected and adjusted to cover each individual positive staining of LAMP1 for analysis. The data was presented as positivity which was obtained from the following formula: Positivity (%)
= positive area (pixels) / Total analysed area (pixels) x 100%.
The GFAP and IBAI stained slides were scanned with Hamamatsu Nanozoomer 2.0-RS scanner and viewed with NDP.view2. Total number of GFAP and IBAI positive signals were counted using the cell-counter program (ImageJ software) with a fixed threshold.
For immunofluorescence, secondary antibodies Donkey anti mouse IgG-Alexa Fluor 488 (R37114 Thermo Fisher) and Goat anti rabbit IgG-Alexa Fluor 568 (A-11011 Thermo Fisher) were used for the visualization. Stained slides were read and representative photos were taken with Nikon fluorescent microscope.
Toluidine blue staining
Fixed samples of specific brain regions were post-fixed in 1% osmium tetroxide, dehydrated and embedded in resin. One-micron sections were stained with 1% toluidine blue and examined by light microscopy. Ultra-thin sections from the selected region of cortex were cut and stained with 0,5% uranyl acetate.
Behavioral tests
Behavioral tests were carried out in a behavioral testing room maintained under constant light, temperature, and humidity. The mice were tested during daylight hours (between 9am and 6pm). Before testing, animals were habituated to the testing room for at least 30 min. The same groups of animals were tested at 6 and 9 months of age. The inventors performed the open field which in previous studies they have found to be impaired in adult M PS-11 IA mice. Additionally, the inventors tested them in the contextual fear conditioning task, which allows to evaluate animal ability to learn and remember a Pavlovian association between a mild food-shock and a specific context.
Open field task: Open field task was performed as previously described [5]. Mice were placed in the middle of a Plexiglas arena with a masonite base (43 x 32 x 40 cm). Animals were left free to explore the device for 10 min. The distance travelled (m), the immobility time (s) and line crossing, were recorded using a video camera (Panasonic WV-BP330) hanging over the arena that was connected to a video-tracking system (Any-Maze, Stoelting, USA).
Contextual fear conditioning: Each mouse was trained in a conditioning chamber (30 cm x 24 cm x 21 cm; Ugo Basile) that had a removable grid floor and waste pan. The grid floor contained 36 stainless steel rods (3-mm diameter) spaced 8 mm center to center. When placed in the chamber, the grid floor contacted a circuit board through which scrambled shock was delivered. The shock intensity was 0.5 mA with a duration of 2 sec and it was presented for three times and was associated to a context. After 24 hours after training, mice were tested without foot- shock but with the same context. Freezing behavior was defined as complete lack of movement, except for respiration and scored with a video-tracking system (ANY-MAZE, Stoelting, USA). Statistical analysis
Data of Behavioural Tests were expressed as mean ± S.E.M. Two-way ANOVA for repeated measures with the factor group as independent factor and trials or test phase as repeated measures was performed to assess significance among multiple experimental groups and at different time points, followed by Duncan post-hoc test when appropriate. A p<0.05 was considered as statistically significant. Forthe enzymatic activity, ELISA, vector copy number and GAG analysis the data were expressed as mean ± S.E.M. One-way comparisons were performed to calculate the significance among the experimental groups, followed by Tukey post-hoc. A p<0.05 was considered as statistically significant.
Data of LAMP1, toluidine blue and GFAP staining quantification were expressed as mean ± S.E.M. P values were generated by unpaired t test. *p<0.05, **p<0.01, ***p<0.001.
EXAMPLES
Example 1: IN VITRO study - Testing of IDSspSGSH cassette in glia cells
To test the functionality of the IDSspSGSH modified sulfamidase (SEQ ID No. 1) and the capability to be highly secreted from the brain cells, the inventors transfected primary cultured glia derived from C57BL/6 mice with a vector expressing the IDSspSGSH cDNA of SEQ. ID No. 1 (pAAV2.1-CMV-IDSspSGSH, SEQ. ID No.7) and compared it to two other constructs: one bearing the unmodified version of SGSH of SEQ SEQ ID No. 2 (pAAV2.1-CMV-SGSH, SEQ ID No. 5) and a construct carrying the human a-antitrypsin (hAATAATspSGSH cDNA of SEQ ID No. 3 (pAAV2.1- CMV- AATspSGSH, SEQ ID No. 10), obtained by the replacement of the endogenous signal peptide with the alpha antitrypsin signal peptide (AATsp). The secretion experiment was started one day after transfection, by adding the conditioned media to the primary transfected glia cells. 24h after secretion, the enzymatic activity in both the pellet and the medium of glia cells was evaluated, demonstrating that the IDS sp replacement strongly increased the secretion rate of ISDsp-modified sulfamidase compared to wild-type sulfamidase (Fig.l). The secretion efficiency has been indicated as percentage of the SGSH activity in the medium/ activity in the pellet and the medium.
The IDSspSGSH construct showed increased enzymatic activity in the medium associated with improved secretion efficiency in glia cells transfected with the IDSspSGSH compared to cells transfected with the other constructs, demonstrating the capability of the IDSspSGSH protein to be functional and highly secreted in the highly relevant in vitro model of primary glia cells.
Example 2: Usage of a SGSH expression cassette with enhanced enzyme secretion and activation improved enzyme bio-distribution in the CNS of pigs upon intra-CSF AAV9- mediated gene delivery
In order to improve the bio-distribution potential of the SGSH enzyme, the inventors generated a bi-cistronic SGSH expression cassette containing a chimeric SGSH bearing the iduronate sulfatase signal peptide (IDSsp) and the gene encoding SUMF1 (IDSspSGSH-IRES-SUMFl). The SGSH CNS bio- distribution triggered by IDSspSGSH-IRES-SUMFl expression was then evaluated upon intra-CSF AAV-mediated delivery in wild type pigs (Sus Scrofa white model). Thirty days after birth (Postnatal - P30) WT pigs were intra CSF injected via the cisterna magna with 4.5xl012 GC/Kg of AAV9 encoding IDSspSGSH-IRES-SUMFl under the CMV promoter. As control, pigs were injected with AAV9 encoding only either WT SGSH (AAV9-SGSH) or IDSsp-modified SGSH (AAV9-IDSspSGSH) or bicistronic cassette containing SUMF1 together with WT SGSH (AAV9- SGSH-IRES-SUMF1). One month after injection, twenty CNS regions covering the main representative areas of the brain and the spinal cord were collected and assayed for both SGSH enzymatic activity and protein levels. The expression of highly secreted forms of SGSH (IDSspSGSH and IDSspSGSH-IRES-SUMFl cassettes) resulted in a remarkable and significant increase of the specific enzymatic activity over the entire CNS as compared to control (pigs injected with unmodified SGSH constructs: SGSH and SGSH-IRES-SUMF1) (Figure 2A). Interestingly, the enhancing effect of SUMF1 was higher when SUMF1 was co-expressed with IDSsp-modified SGSH (IDSspSGSH-IRES-SUMFl) rather than with unmodified SGSH (SGSH-IRES- SUMF1) (Figure 2A). Immunostaining analysis against exogenous human sulfamidase revealed the strongest SGSH expression in multiple brain areas of pigs injected with IDSspSGSH-IRES- SUMFl, which indicated that the increased enzymatic activity led to a functional change in protein levels (Figure 2B). The present results showed that combining improved secretion and enhanced SUMFl-mediated enzyme activation gave rise to a synergistic effect on CNS bio distribution of SGSH in a large animal model.
Example 3: AAV9-mediated intra-CSF delivery of IDSspSGSH-IRES-SUMFl resulted in a strong and sustained SGSH activity in the brain of Sgsh -/- mice
To evaluate the potential for therapeutic effectiveness of intra-CSF AAV9-mediated delivery of IDSspSGSH-IRES-SUMFl the inventors used the B6Cg-Sgsh(mps3a/PstJ) mouse (Sgsh-/-), a spontaneous MPS- IMA mouse model largely used in preclinical studies of this disease [9]. Initially, the inventors performed a short-term comparative study in a small cohort of Sgsh-/- mice (hereinafter referred to as MPS-IIIA mice) to evaluate the efficacy of the IDSspSGSH-IRES- SUMFl expression cassette on a background of disease-associated enzymatic activity (MPS-IIIA levels in this model show 3-5% of normal SGSH activity). Five MPS-IIIA mice, age post-natal day 60 (P60), were intraventricularly (ICV) injected with 4.5 xlO12 GC/Kg of AAV9 vectors encoding WT SGSH, IDSspSGSH or IDSspSGSH-IRES-SUMFl. One month after injection, the inventors found a significant increase in SGSH activity in brain sections from the IDSspSGSH-injected MPS- 111 A mice and a further significant increase in enzymatic activity upon the administration of AAV9-IDSspSGSH-IRES-SUMFl (until to 50% of WT levels) (Figure 3). These results confirmed the data obtained in pigs showing enhanced therapeutic potential of the IDSspSGSH-IRES- SUMF1 expression cassette.
The inventors then performed an efficacy study in a larger cohort of M PS-111 A mice in which AAV9 encoding IDSspSGSH-IRES-SUMFl was delivered by ICV. Following one month (early time point; ETP) or seven months (late time point; LTP) after injection the enzyme biodistribution and the pathological phenotype were analysed. Consistently with previous results (Figure 3) a significant increase of SGSH activity in the brain of injected animals with peaks of 35% of WT levels was observed at ETP (Figure 4A). Importantly, such increased activity was sustained at LTP (25% of WT levels) (Figure 4A). Increased SGSH activity correlated strongly with the increase in SGSH protein levels evaluated by both ELISA (Figure 5) and IHC analyses in different brain regions (Figure 4B). Co-immunolabeling analysis of cell-specific transduction in the brains of MPS-IIIA mice ICV-injected with AAV9-encoding IDSspSGSH-IRES-SUMFl showed that SGSH signal co-localized with both NeuN (neuronal) and GFAP (astroglial) markers in several brain regions with NeuN co-localization being prevalent over GFAP-colocalization (Figure 4C).
Example 4: Rescue of storage pathology, lysosomal enlargement and neuroinflammation in MPS-IIIA mice treated with AAV9-IDSspSGSH-IRES-SUMFl
Abnormal lysosomal storage (including GAGs) is associated with lysosomal compartment enlargement and neuroinflammation, both hallmarks of MPS-IIIA neuropathology [4, 9]. Immunostaining for lysosomal-associated membrane protein-1 (LAMP-1) on brain sections of MPS-IIIA mice injected with AAV9-IDSspSGSH-IRES-SUMFl showed a significant reduction in the lysosomal compartment enlargement compared to control MPS-IIIA mice (Figure 6A). Such improvement was associated to a significant reduction in the abnormal vacuolization as shown by toluidine blue staining analysis (Figure 6B). Consistently, also GAGs levels were significantly reduced following IDSspSGSH-IRES-SUMFl treatment (Figure 6C). Next, to evaluate the inflammation levels in treated MPS-IIIA mice brain sections were stained forglial fibrillary acidic protein (GFAP), a marker of astrocytes. As expected, excessive astrocyte levels were seen in MPS- IMA control mice (Figure 6D). Remarkably, the brain of M PS-111 A mice treated with IDSspSGSH- IRES-SUMF1 showed a strong reduction of GFAP signal as compared to control affected mice indicating a striking reduction of inflammatory processes (Figure 6D).
Along with the severe manifestations MPS-IIIA also shows mild somatic pathology characterized by GAG storage in several somatic tissues, particularly in the liver which leads to increased mass (hepatomegaly). Being that the CSF is continuously absorbed and returned to the blood stream, AAV9 vectors may travels from the CSF to the blood stream and eventually reach the somatic tissues. The presence of such leakage was supported by the fact that following ICV AAV9- mediated delivery of IDSspSGSH-IRES-SUMFl gene transfer the livers of treated mice were efficiently transduced as shown by significant increased levels of vector genomes (Figure 7A) and a subsequent significant increase in SGSH enzymatic activity (Figure 7B). This increase was associated with higher levels of SGSH protein as well (Figure 7C). Importantly, quantitative measurement of GAGs in the liver of IDSspSGSH-IRES-SUMFl treated MPS-IIIA mice showed an almost complete normalization of GAG content (Figure 7D). Together these results demonstrated the effectiveness of AAV9-mediated delivery of the enhanced version of SGSH with improved secretion and activation in rescuing both CNS and somatic pathology in MPS-IIIA mice.
Example 5: Treatment with IDSspSGSH-IRES-SUMFl prevents the memory deficits occurring in the late stage of MPS-IIIA pathology
The inventors next validated the impact of the ICV AAV9-mediated delivery of IDSspSGSH-IRES- SUMFl on behavioural deficits that manifest in 6 and 9 months MPS-IIIA mice. Based on previous studies, the inventors evaluated exploratory behaviour in the open field. At 6 months, MPS-IIIA male mice show a mild difference in the distance travelled, immobile time and line crossing, in the very first minutes of open field testing, as compared to WT animals (Figure 8C). This tendency was not evident at 9 months, likely due to the test-retest effects observed also in control animals [27], which did not allow to properly evaluate the rescuing efficacy of IDSspSGSH-IRES- SUMF1 (Figure 8A-8C). Therefore, the inventors tested the animals in a memory task that is extensively used to test contextual memory depending on the functional integrity of the medial temporal lobe, namely the fear contextual conditioning test [28]. Using this task, the inventors identified, for the first time in MPS-IIIA mice, an age-dependent long term memory impairment: 6 months-old MPS-IIIA mice show normal freezing time when they are exposed to a mild foot shock during training, as compared to control animals; similarly, exposure to the context (24 hr after training) paired with the mild foot shock is sufficient to elicit freezing as well as in control mice (Figure 6E). This suggests that at this stage MPS-IIIA mice can form emotional contextual memories as well as WT animals. However, when the same test, is repeated 3 months later, although there are no differences in basal freezing during training, on the testing day MPS-IIIA mice have impaired freezing response (Figure 6E). Interestingly, IDSspSGSH-IRES-SUMFl fully rescues the memory deficit in 9 months old MPS-IIIA mice (Figure 6E). These behavioural effects in MPS-IIIA mice (both treated and untreated) cannot be explained as due to reduced immobility time (a possible correlate of reduced freezing time), as in the open field test both MPS-IIIA groups show similar levels of immobility time at this testing age. Considering the specificity of this test for memory deficits occurring in animal models of neurodegenerative diseases, these results have a high relevance for the validation of the neuronal dysfunctions correction in MPS-IIIA mice treated with IDSspSGSH-IRES-SUMFl.
In the present invention, the inventors developed and tested an intra-CSF AAV-mediated gene transfer approach for the MPS- MIA based on intrathecal delivery of AAV9 encoding a modified SGSH expression cassette with enhanced therapeutic potential. Such expression cassette contained an SGSH bearing the signal peptide of IDS, which confer to the engineered enzyme the capability to be secreted at higher efficiency compared to WT enzyme. Such modification resulted in improved bio-distribution of the enzyme in the CNS of both small (MPS-IIIA mice) and large (WT pigs) animal models. Very recently Chen et colleagues reported that intra-CSF delivery of AAV4 encoding a highly secreted variant of SGSH mutated in the M6P binding site was therapeutically superior to the intra-CSF AAV4-mediated delivery system based on the use WT enzyme [19]. Both the present and Chen et al. studies support the concept that enhancing SGSH secretion improves distribution of enzyme in the CNS. Of note, while in the approach reported by Chen et al., the mutation of M6P binding site makes SGSH uptake M6P- independent, in the present approach the M6PR-mediated system, which underlie the uptake in disease-relevant cellular targets such as neurons and astrocytes, is preserved [5]. Moreover, an important additional feature of the SGSH expression cassette used in the present strategy is the insertion of the cDNA codifying for SUMF1, the enzyme responsible for the post- translational activation of sulfatases. Such modification synergistically acts together with enhanced secretion to further improve enzyme CNS bio-distribution upon intra-CSF AAV9- mediated gene delivery. Importantly, the inventors demonstrated that the modified SGSH expression cassette they developed is therapeutically effective, being able to efficiently rescue CNS and somatic storage pathology and improve memory impairment. It is remarkable that the performance of M PS-111 A mice treated IDSspSGSH-IRES- SUMF1 in the long-term memory task was undistinguishable from that of WT animals, thus showing that together to enhancing the biodistribution of the therapeutic enzyme, IDSspSGSH- IRES-SUMF1 treatment also allows to obtain maximal improvement in functional tests.
Intra-CSF AAV9-mediated gene delivery using WT SGSH has previously been successfully tested in different preclinical animal models using vector doses similar to those used in the present approach (i.e.~ 4-5 xlO12 GC/Kg) [16]. In that study the authors also showed that reducing AAV9 vector dosage in MPS-IIIA mice below 4-5 xlO12 GC/Kg led to inefficient enzyme biodistribution, thus resulting in only mild neuropathology improvement. Here, the inventors demonstrated that intra-CSF delivery of AAV9 bearing the modified SGSH expression cassette at doses of 4.5 xl012 GC/Kg led to a more efficient CNS enzyme biodistribution compared to WT SGSH both in small and large animal models. This makes the present approach more attractive for clinical purpose, since makes potentially possible to achieve and maintain therapeutic threshold levels of the enzyme throughout the CNS at reduced and, therefore, safer AAV9 vector dosage.
Overall the results presented here pave the way for developing gene delivery replacement protocols with enhanced therapeutic potential for the treatment of MPS-IIIA patients and also provide a proof of principle for potential use of this strategy for the treatment of other forms of LSDs, caused by sulfatase deficiency.
REFERENCES
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Claims

1. A nucleotide sequence encoding a modified sulfatase and sulfatase-modifying factor 1 (SUMF1), said modified sulfatase comprising in the N-terminal-C-terminal sequence order: a) an amino acid sequence of an exogenous signal peptide, preferably the signal peptide of the human lduronate-2-sulfatase (IDS) or the hAAT signal peptide, and b) an amino acid sequence of a human sulfatase said human sulfatase being deprived of its signal peptide, wherein said nucleotide sequence optionally comprises a sequence of an IRES element between the sequence encoding for said modified sulfatase and said sulfatase-modifying factor 1 (SUMF1).
2. The nucleotide sequence according to claim 1 wherein the sulfatase-modifying factor 1 (SUMF1) consists of SEQ ID No 13 or a functional fragment thereof or a functional variant thereof.
3. The nucleotide sequence according to claim 1 or 2 wherein the sulfatase-modifying factor 1 (SUMF1) has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ. ID No. 13 and functions as a sulfatase-modifying factor.
4. The nucleotide sequence according to any one of previous claim wherein the human sulfatase is selected from the group consisting of: human N-sulfoglucosamine sulfohydrolase (SGSH), human arylsulfatase A (ARSA), human arylsulfatase B (ARSB), human arylsulfatase D (ARSD human arylsulfatase G (ARSG), human galactosamine (N-acetyl)-6-sulfate sulfatase (GALNS), human glucosamine (N-acetyl)-6-sulfatase (GNS), human steroid sulfatase (microsomal), isozyme S (STS), human sulfatase 1 (SULF1), human sulfatase 2 (SULF2), human sulfatase modifying factor 2 (SUMF2), preferably the human sulfatase is the human N-sulfoglucosamine sulfohydrolase (SGSH).
5. The nucleotide sequence according to any one of previous claim wherein the modified sulfamidase has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID No. 12.
6. The nucleotide sequence according to any of previous claims having at least 75 %, 80%, 85%, 90%, 95%, 99% identity to SEQ ID No. 4.
7. A vector comprising the nucleic acid according to any one of claim 1 to 6.
8. The vector according to claim 7, wherein the vector is a viral vector.
9. The vector according to claim 8, wherein the viral vector is a lentiviral vector, an adeno- associated virus vector, an adenoviral vector, a retroviral vector, a polio viral vector, a murine Maloney-based viral vector, an alpha viral vector, a pox viral vector, a herpes viral vector, a vaccinia viral vector, a baculoviral vector, or a parvoviral vector.
10. The vector according to claim 9 wherein the adeno- associated virus is AAV9, AAV1, AAVSH19, AAV2/7, AAVPHP.B.
11. The nucleotide sequence according to any one of claim 1 to 6 or the vector according to claim 7 to 10 in combination with a carrier, preferably the carrier is a lipid or a polypeptide, preferably a liposome, a polylysine carrier complex.
12. The nucleotide sequence according to any one of claim 1 to 6 and 11 or the vector according to any one of claim 7 to 11 for medical use, preferably for gene therapy.
13. The nucleotide sequence or the vector for use according to claim 12 for use in the treatment of a sulfatase deficiency with a neurological involvement, preferably of a lysosomal sulfatase deficiency.
14. The nucleotide sequence or the vector for use according to claim 13 wherein the sulfatase deficiency is selected from the group consisting of: metachromatic leukodystrophy (MLD), mucopolysaccharidosis MPS type IMA, mucopolysaccharidosis MPS type HID, mucopolysaccharidosis MPS type IVA, mucopolysaccharidosis MPS type II, preferably the sulfatase deficiency is MPS type IMA.
15. The vector for use according to any one of claim 12 to 14 administered at a dose of from 0,5 x 1012- 1 x 1013 GC/kg, preferably 1 x 1012 GC/kg to 10 x 1012 GC/kg, more preferably 4.5 xlO12 GC/Kg.
16. The nucleotide sequence or the vector for use according to claim 12 to 15 in combination with a further therapeutic agent, preferably the further therapeutic agent is selected from the group consisting of: enzyme replacement therapy and small molecule therapy.
17. A pharmaceutical composition comprising the nucleotide sequence according to any one of claim 1 to 6 and 11 or the vector according to any one of claim 7 to 11 and pharmaceutically acceptable diluents and/or excipients and/or carriers.
18. The pharmaceutical composition according to claim 17 further comprising a therapeutic agent, preferably the therapeutic agent is selected from the group consisting of: enzyme replacement therapy and small molecule therapy.
19. The pharmaceutical composition according to claim 17 or 18 being administered through a route selected from the group consisting of: intra cerebral spinal fluid (CSF), intrathecal, parenteral, intralesional, intraperitoneal, intramuscular, intratumoral, subcutaneous, intraventricular, intra cisterna magna, lumbar, intracranial, intraspinal, intravenous, topical, nasal, oral, ocular, or any combination thereof.
20. A nucleotide sequence encoding for a modified sulfatase, said modified sulfatase comprising in the N-terminal-C-terminal sequence order: a) an amino acid sequence of an exogenous signal peptide, preferably the signal peptide of the human lduronate-2-sulfatase (IDS) or the hAAT signal peptide, and b) an amino acid sequence a human sulfatase said human sulfatase being deprived of its signal peptide for medical use, said nucleotide sequence being administered intra-CSF.
21. The nucleotide sequence according to claim 20 wherein the human sulfatase is selected from the group consisting of: human N-sulfoglucosamine sulfohydrolase (SGSH), human arylsulfatase A (ARSA), human arylsulfatase B (ARSB), human arylsulfatase D (ARSD), human arylsulfatase E (chondrodysplasia punctata 1) (ARSE), human arylsulfatase F (ARSF), human arylsulfatase G (ARSG), human arylsulfatase family, member H (ARSH), human arylsulfatase family, member I (ARSI), human arylsulfatase family, member J (ARSJ), human arylsulfatase family, member K (ARSK), human galactosamine (N-acetyl)-6-sulfate sulfatase (GALNS), human glucosamine (N-acetyl)-6-sulfatase (GNS), human iduronate 2-sulfatase (IDS), human steroid sulfatase (microsomal), isozyme S (STS), human sulfatase 1 (SULF1), human sulfatase 2 (SULF2), human sulfatase modifying factor 2 (SUMF2), preferably the human sulfatase is the human N- sulfoglucosamine sulfohydrolase (SGSH).
22. The nucleotide sequence according to claim 20 or 21 wherein the modified sulfamidase has at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID No. 12.
23. The nucleotide sequence according to any of claim 22 to 22 having at least 75 % ,80%, 85%, 90%, 95%, 99% identity to SEQ ID No. 1.
24. A vector comprising the nucleotide sequence according to any one of claim 20 to 23 for medical use wherein said vector is administered intra-CSF.
25. The vector for use according to claim 24, wherein the vector is a viral vector, preferably the viral vector is a lentiviral vector, an adeno- associated virus vector, an adenoviral vector, a retroviral vector, a polio viral vector, a murine Maloney-based viral vector, an alpha viral vector, a pox viral vector, a herpes viral vector, a vaccinia viral vector, a baculoviral vector, or a parvoviral vector, preferably the adeno- associated virus is AAV9, AAV1, AAVSH19, AAV2/7, AAVPHP.B.
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WO2023240220A1 (en) * 2022-06-09 2023-12-14 The University Of North Carolina At Chapel Hill Aav-sgsh vectors for treatment of mucopolysaccharidosis iiia

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