JP6430980B2 - Therapeutic strategy for treating CNS lesions in mucopolysaccharidosis - Google Patents

Description

  The present invention is a viral vector-mediated gene therapy for crossing the blood brain barrier and treating CNS lesions in mucopolysaccharidosis (MPS), particularly type IIIA MPS, or of modified sulfatases, particularly sulfamidase enzymes. It relates to a therapeutic approach by administration.

  Type IIIA mucopolysaccharidosis (MPS-IIIA) is a genetic disease caused by a deficiency in sulfamidase (SGSH), an enzyme involved in the stepwise degradation of a large macromolecule called heparin sulfate. As a result, undegraded matrix accumulates in the cells and tissues of affected patients, causing cell damage. The central nervous system (CNS) is a major target of injury, and in fact MPS-IIIA patients show severe mental retardation and neuropathological decline that ultimately leads to death (often over 20 years). Clinical symptoms include hyperactivity, aggressive behavior, and sleep disturbances (1).

  The naturally occurring mouse model of MPS-IIIA has been regarded as a pathophysiology and symptom similar to the human condition (2-4). These mice are an ideal model for studying the pathophysiology of this disorder and for testing new treatment protocols.

  Treatment of brain damage is a major goal of all therapeutic approaches to MPS-IIIA. The route to the brain is direct injection of therapeutic molecules directly into the brain. Several different enzyme replacement therapy (ERT) protocols have been tested. In these protocols, recombinant sulfamidase enzyme was administered by direct injection into the brain of MPSIIIA mice. These strategies can delay the appearance of neurodegenerative changes when younger mice are administered sulfamidase (5, 6). Furthermore, a gene therapy protocol based on intracerebral injection of SGSH gene via AAV vector has been successfully developed by the authors of the present invention (7). Although these direct brain targeting approaches have been shown to be clinically effective, they are highly invasive approaches for human therapeutic applicability.

  As each neuron in the brain is perfused by its own blood vessels, another effective minimally invasive route to the brain is intravenous administration of therapeutic molecules (8). However, this very dense network of microvasculature that forms the vascular brain barrier (BBB) is not permeable to all molecules and can interfere with the effective delivery of therapeutic agents (9) . Indeed, intravenous administration of lysosomal enzymes produced a therapeutic effect in many LSD somatic lesions, but little or no effect in CNS lesions due to the impermeability of BBB to macromolecules (10). In MPS-IIIA, intravenous injection of sulfamidase has been shown not to alter lesions or behavioral processes that occur in MPS-IIIA mouse brain when the enzyme is supplied after BBB formation (11 ).

  Importantly, recent studies by Urayama et al. Show that sulfamidase is transported across the BBB in neonatal mice through mannose 6-phosphate receptor-mediated transport, but entry into the adult brain is negligible. A small amount was shown (12).

  In such a context, the actual challenge to the treatment of MPS-IIIA, and generally to all LSDs, including the CNS, develops a CNS systemic treatment strategy that can overcome the major disturbances represented by the BBB That is. An effective strategy for crossing the BBB is protein targeting to the CNS via receptor-mediated transcytosis (13). Well-characterized BBB receptors include low density lipoprotein receptor (LDLR), transferrin receptor (TfR), and insulin-like growth factor receptor (IGF-R). The LDLR family is a group of cell surface receptors that bind apolipoprotein (Apo) complexes (lipid carriers) for internalization into lysosomes 14-16). On the surface of the BBB, LDLR binding to Apo results in transcytosis of the BBB into the lumen, where apolipoprotein is released for uptake by neurons and astrocytes. Recent studies have shown that fusion of Apo's LDLR binding domain to a lysosomal enzyme results in effective delivery of the chimeric enzyme to the CNS (17).

  WO2004108071 refers to a chimeric CNS targeting polypeptide comprising a BBB receptor binding domain, such as an apolipoprotein B binding domain, for therapeutic use in lysosomal storage diseases.

  WO2004064750 is involved in chimeric lysosomal polypeptides (particularly type II lysosomal storage disorder glycogen storage diseases), including secretory signal sequences (ie Vi-antitrypsin and α-1-antitrypsin) and related AAV vectors Reference is made to a nucleic acid encoding lysosomal acid glucosidase GAA).

  WO2005002515 refers to a compound comprising a megalin binding moiety conjugated to a drug of interest for receptor-mediated drug delivery across the blood brain barrier, in particular by transcytosis. Furthermore, this document refers to a method of treating lysosomal storage diseases based on the administration of a composition comprising a megalin binding moiety. Apolipoprotein B and mucopolysaccharidosis IIIA are mentioned.

  WO2009131698 refers to a treatment based on a chimeric NaGlu enzyme characterized by an apolipoprotein B binding domain and specifically directed to mucopolysaccharidosis IIIB.

  Cardone et al. (Hum Mol Gen, 2006, 15 (7): 1225) treats Hunter syndrome (type II lysosomal storage disease mucopolysaccharidosis) in an MPSII mouse model by liver-directed AAV2 / 8-TBG-mediated gene delivery Is described.

  WO2007092563 first delivers an effective amount of a transgene encoding a polypeptide to mammalian liver tissue, and then administers an effective amount of the transgene to the mammalian central nervous system (CNS) Thus, reference is made to methods and compositions for tolerating a mammalian brain against an externally administered acid sphingomyelinase polypeptide. Therapeutic approaches target Niemann-Pick disease and lysosomal storage diseases. Liver-specific promoters and type 8 AAV are mentioned.

  WO2009075815 refers to a method of treating pump disease (lysosomal storage disease) comprising administration of an AAV vector in the context of enzyme replacement therapy. The liver-specific promoter (thyroid hormone binding globulin promoter) and type 8 AAV are mentioned.

  None of the above prior art citations disclose or even suggest the modified sphamidase enzyme of the present invention, and the modified sphamidase enzyme of the present invention may have a therapeutic effect on the treatment of type IIIA MPS. Is not disclosed or even suggested.

WO2004108071 WO2004064750 WO2005002515 WO2009131698 WO2007092563 WO2009075815

Cardone et al. (Hum Mol Gen, 2006, 15 (7): 1225) Naldini, L., Blomer, U., Gage, FH, Trono, D., and Verma, IM (1996a), In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science 272 (5259), 263〜 Page 7 Consiglio A, Quattrini A, Martino S, Bensadoun JC, Dolcetta D, Trojani A, Benaglia G, Marchesini S, Cestari V, Oliverio A, Bordignon C, Naldini, In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice. L. Nat Med. 2001/3; 7 (3): 310-6 Follenzi A, Naldini L, HIV-Based vectors.Preparation and use.Methods Mol Med. 2002; 69: 259-74 Brunetti-Pierri N, Ng P. Progress towards liver and lung-directed gene therapy with helper-dependent adenoviral vectors. Curr Gene Ther. Oct 2009; 9 (5): 329-40 Fraldi et al., HMG2007 Hemsley, K.M. and J.J.Hopwood, Behav Brain Res, 2005 Savas P.S et al., Mol Genet Metab, 2004 Hemsley, K.M., et al., Mol Genet Metab, 2007 Fraldi et al., Hum Mol Gen 2007

  As disclosed in the background art, brain lesions are the most common feature in lysosomal storage disorders. Therefore, the treatment of brain disorders is the main goal of any effective treatment for these disorders.

  A major obstacle for efficiently treating the brain by systemic delivery of therapeutic agents is the blood brain barrier (BBB).

  The authors developed a novel non-invasive therapeutic approach to treat brain lesions in type IIIA mucopolysaccharidosis (MPS-IIIA), a lysosomal storage disorder with severe central nervous system involvement. The strategy involves two amino acid sequences (one of which is the N-terminus of the protein, one that gives the modified sulfamidase the ability to be secreted and effectively targeted to the brain by crossing the blood brain barrier (BBB). Based on the construction of a chimeric sulfamidase (sulfatase enzyme deficient in MPS-IIIA) optimized on the other (to the C-terminus). The modified enzyme is expressed by adeno-associated virus (AAV) serotype 8, which specifically targets the liver and makes the liver a factory organ for therapeutic enzymes.

  Modified sulfamidases can be used effectively both for gene therapy and for enzyme replacement therapy (ERT).

  The modified approach can be used for other lysosomal enzymes that are deficient in other mucopolysaccharidosis with severe CNS involvement.

  Thus, a nucleotide sequence encoding a chimeric sulfatase is an object of the present invention, said chimeric sulfatase being essentially an a) human α-antitrypsin (hAAT) amino acid sequence or a human iduronic acid-2-sulfatase (IDS) amino acid sequence. Signal peptide derived from either, b) human sulfatase-derived amino acid sequence lacking the signal peptide, c) ApoB LDLR binding domain N-terminal to C-terminal sequence.

  In a preferred embodiment, the encoded signal peptide has a sequence belonging to the following group: MPSSVSWGILLLAGLCCLVPVSLA (SEQ ID NO: 2) or MPPPRTGRGLLWLGLVLSSVCVALG (SEQ ID NO: 4 or 6).

In a preferred embodiment, the nucleotide human sulfatase is a human sulfamidase, more preferably essentially the sequence:
EmuesushiPibuiPieishishieierueruerubuierujierushiarueiaruPiaruenueieruerueruerueididijijiefuiesujieiwaienuenuesueiaieitiPieichierudieierueiaruaruesuerueruefuaruenueiefutiesubuiesuesushiesuPiesuarueiesueruerutijieruPikyueichikyuenujiemuwaijierueichikyudibuieichieichiefuenuesuefudikeibuiaruesueruPieruerueruesukyueijibuiarutijiaiaijikeikeieichibuiJipiitibuiwaiPiefudiefueiwaitiiienujiesubuierukyubuijiaruenuaitiaruaikeieruerubuiarukeiefuerukyutikyudidiaruPiefuefueruwaibuieiefueichidiPieichiarushijieichiesukyuPikyuwaijitiefushiikeiefujienujiiesujiemujiaruaiPididaburyutiPikyueiwaidiPierudibuierubuiPiwaiefuBuipienutiPieieiarueidierueieikyuwaititibuijiaruemudikyujibuijierubuierukyuieruarudieijibuieruenuditierubuiaiefutiesudienujiaiPiefupiesujiarutienueruwaidaburyuPijitieiiPieruerubuiesuesuPiieichiPikeiarudaburyujikyubuiesuieiwaibuiesueruerudierutiPitiaierudidaburyuefuesuaiPiwaiPiesuwaieiaiefujiesukeitiaieichierutijiaruesuerueruPieieruieiiPierudaburyueitibuiefujiesukyuesueichieichiibuitiemuesuwaiPiemuaruesubuikyueichiarueichiefuaruerubuieichienueruenuefukeiemuPiefupiaidikyudiefuwaibuiesuPitiefukyudierueruenuarutitieijikyuPitijidaburyuwaikeidieruarueichiwaiwaiwaiarueiarudaburyuieruwaidiaruesuarudiPieichiitikyuenuerueitidiPiaruefueikyuerueruiemueruarudikyuerueikeidaburyukyudaburyuitieichidiPWVCAPDGVLEEKLSPQCQPLHNEL (SEQ ID NO: 8) is a human sulfamide peptidase derived amino acid sequence encoded with a. Such a sequence is encoded by the SEQ ID NO: 7nt sequence. 5 '-3'.

  In a preferred embodiment, the encoded ApoB LDLR binding domain has the sequence: SVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 10).

In a preferred embodiment, the nucleotide sequence has a sequence that essentially belongs to the following group:
SEQUENCES WITH FLAG (The expert shall simply replace the flag sequence with any other suitable spacer sequence):
a) Assembly hAATsp-SGSH-3xFlag cassette (1611) (SEQ ID NO: 11).
5 '-3'
b) Assembly hIDSsp-SGSH-3xflag cassette (1614 bp) (SEQ ID NO: 13).
5 '-3'
c) Assembly hAATsp-SGSH-3xflag-ApoB cassette (1734 bp) (SEQ ID NO: 15).
5 '-3'
d) Assembly hIDSsp-SGSH-3xflag-ApoB cassette (1737 bp) (SEQ ID NO: 17).
5 '-3'
SEQUENCES WITHOUT FLAG:
e) Assembly hAATsp-SGSH cassette (SEQ ID NO: 19).
5 '-3'
f) Assembly hIDSsp-SGSH cassette (SEQ ID NO: 21).
5 '-3'
g) Assembly hAATsp-SGSH-ApoB cassette (SEQ ID NO: 23).
5 '-3'
h) Assembly hIDSsp-SGSH-ApoB cassette (SEQ ID NO: 25).
5 '-3'.

  A recombinant plasmid suitable for gene therapy of MPS comprising a nucleotide sequence as disclosed above under the control of a liver-specific promoter is further an object of the present invention, preferably the liver-specific promoter is human thyroid Hormone-globulin (TBG) promoter, more preferably the human thyroid hormone-globulin (TBG) promoter consists essentially of the sequence: 5'- It has ACCTTATCATTTTTTGCTTTGTTCCTCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGTTAATGCTGGGGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATGCTATAAAAATGGAAAGATGTTGCTTTCTGAGAGACTGCAG-3 ′ (SEQ ID NO: 27).

  Those skilled in the art will recognize that the recombinant plasmids of the present invention must be assembled in a viral vector for gene therapy of lysosomal disorders and select a suitable one. Such viral vectors may belong to the group of lentiviral vectors, helper dependent adenoviral vectors or AAV vectors. For example, lentiviral vectors for gene therapy of lysosomal storage disorders are Naldini, L., Blomer, U., Gage, FH, Trono, D., and Verma, IM (1996a), In vivo gene delivery and stable transduction. of nondividing cells by a lentiviral vector. Science 272 (5259), 263-7; Consiglio A, Quattrini A, Martino S, Bensadoun JC, Dolcetta D, Trojani A, Benaglia G, Marchesini S, Cestari V, Oliverio A, Bordignon C, Naldini.In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice, L. Nat Med. March 2001; 7 (3): 310-6; Follenzi A, Naldini L., HIV-based vectors. Preparation and use. Methods Mol Med. 2002; 69: 259-74. As a further example, a helper-dependent adenoviral vector is Brunetti-Pierri N, Ng P. Progress towards liver and lung-directed gene therapy with helper-dependent adenoviral vectors. Curr Gene Ther. October 2009; 9 (5): 329 Described on page ~ 40.

  In a preferred embodiment, the recombinant plasmid is derived from the plasmid vector AAV2.1 and is suitable for AAV viral vectors, preferably AAV serotype 8.

  Then, a viral vector for gene therapy of lysosomal disorders comprising any of the recombinant nucleic acid vectors as disclosed above is a further object of the present invention. Preferably, the lysosomal disorder is MPS, more preferably type IIIA MPS.

  A pharmaceutical composition comprising a viral vector as disclosed above, preferably for systemic administration, is a further object of the present invention.

  a) essentially a signal peptide derived from either the human α-antitrypsin (hAAT) amino acid sequence or the human iduronic acid-2-sulfatase (IDS) amino acid sequence, b) an amino acid sequence derived from human sulfatase lacking the signal peptide A further object of the present invention is a chimeric sulfatase, in which c) the N-terminal to C-terminal sequence of the ApoB LDLR binding domain.

  In a preferred embodiment, the chimeric sulfatase has a signal peptide having a sequence belonging to the following group: (SEQ ID NO: 2) or (SEQ ID NO: 4).

  In a preferred embodiment, the chimeric sulfatase has a human sulfamidase derived sequence, preferably (SEQ ID NO: 8).

  In a preferred embodiment, the chimeric sulfatase essentially comprises an encoded ApoB LDLR binding domain having the sequence of (SEQ ID NO: 10).

In a preferred embodiment, the chimeric sulfatase has a sequence that essentially belongs to the following group:
SEQUENCES WITH FLAG (The expert shall simply replace the flag sequence with any other suitable spacer sequence)
a) hAATsp-SGSH-3xFlag amino acid sequence (* = end) (SEQ ID NO: 12).
**
b) hIDSsp-SGSH-3xflag amino acid sequence (* = terminate) (SEQ ID NO: 14)
**
c) hAATsp-SGSH-3xflag-ApoB amino acid sequence (* = terminate) (SEQ ID NO: 16)
**
d) hIDSsp-SGSH-3xflag-ApoB amino acid sequence (* = terminate) (SEQ ID NO: 18)
**,
SEQUENCES WITHOUT FLAG:
e) hAATsp-SGSH amino acid sequence (* = terminate) (SEQ ID NO: 20)
MPSSVSWGILLLAGLCCLVPVSLARPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQ PLHNEL *
f) hIDSsp-SGSH amino acid sequence (* = terminate) (SEQ ID NO: 22)
MPPPRTGRGLLWLGLVLSSVCVALGRPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHNEL *
g) hAATsp-SGSH-ApoB amino acid sequence (* = terminate) (SEQ ID NO: 24)
**
h) hIDSsp-SGSH-ApoB amino acid sequence (* = terminate) (SEQ ID NO: 26)
**

  A chimeric sulfatase as disclosed above for medical use, preferably for the treatment of MPS, more preferably type IIIA MPS, is another object of the present invention.

  A pharmaceutical composition comprising a chimeric sulfatase as disclosed above and a suitable diluent and / or excipient and / or carrier is another object of the present invention.

  Another object of the present invention is a method for the treatment of MPS lesions comprising the step of administering to a subject a suitable amount of a pharmaceutical composition comprising a viral vector for gene therapy as disclosed above. . Preferably, the MPS lesion is a type IIIA MPS.

  It is another object of the present invention a method for the treatment of MPS lesions comprising the step of administering to a subject a suitable amount of a pharmaceutical composition comprising a chimeric sulfatase as disclosed above. Preferably, the MPS lesion is a type IIIA MPS.

  A major advantage of the present invention is that the chimeric molecules of the present invention produced and secreted by the liver can cross the BBB and thus target all brain areas.

  Compared to the prior art Fraldi et al., HMG2007, which describes AAV2 / 5-mediated gene therapy for MPS-IIIA with respect to gene therapy approaches, the present invention does not allow AAV8 vectors to be administered systemically and directly into the brain. There is no invasiveness.

  For the prior art Hemsley, KM and JJ Hopwood, Behav Brain Res, 2005; Savas, PS et al., Mol Genet Metab, 2004 and Hemsley, KM et al., Mol Genet Metab, 2007 Designed to overcome the need for repeated enzyme injections and pass through the BBB. It is worth pointing out that the high cost of BBB and enzyme production is a very important limitation for the ERT approach.

FIG. 6 shows unmodified SGSH: preliminary in vivo test 1 (neonatal treatment). Analysis of GFP signal in the liver of newborn MPSIIIA mice injected with AAV2 / 8-TBG-GFP. Neonatal MPSIIIA was injected with the AAV2 / 8-TBG-SGSH vector (expressing unmodified sulfamidase). As a control, newborn MPSIIIA and heterozygous (phenotypically normal) mice were injected with the AAV2 / 8-TBG-GFP vector. Liver sections from MPS-IIIA injected mice were analyzed for GFP staining at different time points after injection (1, 2, 3, 5 and 10 weeks after injection). The GFP signal was very strong at an early time point. However, a significant decrease in GFP signal was observed at a later time point after injection. FIG. 6 shows unmodified SGSH: preliminary in vivo test 1 (neonatal treatment). SGSH activity in liver and serum of newborn injected mice. Sulfamidase activity was measured in serum (A) and liver (B) of MPSIIIA mice injected with AAV2 / 8-TBG-SGSH and control mice (MPS-IIIA and heterozygous mice injected with AAV2 / 8-TBG-GFP). ). SGSH activity in the plasma of AAV2 / 8-TBG-SGSH treated MPS-IIIA mice increases during the first 2 weeks after neonatal treatment and then reaches the level measured in control GFP injected MPS-IIIA mice Decreased over time. FIG. 6 shows unmodified SGSH: preliminary in vivo test 1 (neonatal treatment). SGSH activity in liver and serum of newborn injected mice. Sulfamidase activity was measured in serum (A) and liver (B) of MPSIIIA mice injected with AAV2 / 8-TBG-SGSH and control mice (MPS-IIIA and heterozygous mice injected with AAV2 / 8-TBG-GFP). ). Analysis of liver SGSH activity showed a trend similar to that detected in plasma with higher levels of activity detected within the first week after injection. FIG. 6 shows unmodified SGSH: preliminary in vivo test 2 (adult treatment). Analysis of GFP signal in the liver of adult MPSIIIA mice injected with AAV2 / 8-TBG-GFP. 1.5 month old MPSIIIA was injected with the AAV2 / 8-TBG-SGSH vector (expressing unmodified sulfamidase). As a control, 1.5 month old MPSIIIA heterozygous (phenotypically normal) mice were injected with the AAV2 / 8-TBG-GFP vector. Liver sections from MPS-IIIA injected mice were analyzed for GFP staining 1 and 5 weeks after injection. High and stable GFP expression was observed. FIG. 6 shows unmodified SGSH: preliminary in vivo test 2 (adult treatment). SGSH activity in serum and liver of injected adult mice. Sulfamidase activity was measured in liver (A) and serum (B) of MPSIIIA mice injected with AAV2 / 8-TBG-SGSH and control mice (MPS-IIIA and heterozygous mice injected with AAV2 / 8-TBG-GFP). ). A strong increase in SGSH activity was observed in the liver of MPSIIIA mice injected with AAV2 / 8-TBG-SGSH, compared to the low enzyme activity detected in animals injected with GFP vector. Furthermore, this activity remained stable for 5 weeks after injection (last time point analyzed). FIG. 6 shows unmodified SGSH: preliminary in vivo test 2 (adult treatment). SGSH activity in serum and liver of adult injected mice. Sulfamidase activity was measured in liver (A) and serum (B) of MPSIIIA mice injected with AAV2 / 8-TBG-SGSH and control mice (MPS-IIIA and heterozygous mice injected with AAV2 / 8-TBG-GFP). ). (B) Similarly, the analysis of SGSH activity in the serum of MPS-IIIA mice treated with AAV2 / 8-TBG-SGSH was very high and stable throughout the post-injection time period analyzed. It is a figure which shows a chimeric sulfamidase construct. The sulfamidase signal peptide (SP) was replaced with either human α-antitrypsin (hAAT) or iduronic acid-2-sulfatase (IDS). The construct was designed as “partially engineered sulfamidase protein” (IDSsp-SGSHflag and hAATsp-SGSHflag). To construct the final chimeric sulfamidase protein, the ApoB LDLR binding domain (ApoB-BD) was fused at the C-terminus of the Flag tag and the resulting “final genetically engineered construct” (IDSsp- SGSHflag-ApoB and hAATsp-SGSHflag-ApoB) were obtained. The ApoB sequence (114 bp) was amplified by PCR from human blood cDNA using forward and reverse oligonucleotides with 5 'BglII sites. A backbone plasmid containing the SP-SGSH sequence was prepared by inserting a BglII site in front of the stop codon of the Flag tag. All resulting chimeric sulfamidase sequences (IDSsp-SGSHflag, hAATsp-SGSHflag, IDSsp-SGSHflag-ApoB and hAATsp-SGSHflag-ApoB) were inserted into the mammalian expression plasmid under the CMV promoter. It is a figure which shows receptor mediated transport. BBB passage through receptor-mediated transcytosis. Apolipoprotein B low-density lipoprotein receptor (LDLR) binding domain (ApoB LDLR-BD) binds to sulfamidase, abundant on BBB epithelial cells, by binding to LDL receptors in brain cells Give the ability to reach. This mechanism can replace inefficient, mannose-6-phosphate receptor (M6PR) -mediated transport of sulfamidases throughout the BBB. It is a figure which shows an in vitro test. SGSH activity in pellets and media of transfected MPS-IIIA MEF cells. MEF cells from MPS-IIIA mice were transfected with the genetically engineered constructs either partially or ultimately. Sulfamidase activity was measured in the medium (light grey) and pellet (dark grey) of transfected cells. It is a figure which shows an in vitro test. The corresponding secretion efficiency (activity in the medium / total activity) was also evaluated. It is a figure which shows an in vitro test. Western blot analysis of all engineered sulfamidase proteins. MEF cells from MPS-IIIA mice were transfected with either a partial or final engineered construct or with a control SGSH unmodified construct. (A) Blot analysis with anti-flag antibody showing correct expression of all chimeric proteins. As a control for transfection efficiency, cells were co-transfected with a plasmid containing the same concentration of an irrelevant protein, flag-tagged syntaxin 7. (B) Pulse and follow-up experiments were performed in the transfected cells to assess the turnover rate of the chimeric protein. (C) Cos-7 cells were transfected with either a partially or ultimately engineered construct, or a control SGSH unmodified construct. Lysosomal location was observed in all transfected cells by immunostaining with anti-SGSH antibody. It is a figure which shows an in vivo test. Preliminary in vivo results in MPSIIIA mice finally injected with genetically modified sulfamidase. The authors obtained preliminary but extremely promising results in MPS-IIIA mice injected with one of the final sulfamidase constructs: hAATsp-SGSHflag-ApoB. Adult MPS-IIIA mice were systemically injected with AAV2 / 8-TBG-hAATsp-SGSHflag-ApoB. A group of MPS-IIIA was also injected with AAV2 / 8-TBG-SGSH (containing unmodified sulfamidase) as a control. Mice were sacrificed one month after injection. We observed higher liver sulfamidase activity and a very strong increase in sulfamidase secretion in mice injected with chimeric sulfamidase compared to control mice. In addition, we detected a significant increase in SGSH activity in the brain of mice injected with chimeric sulfamidase compared to measuring SGSH activity in the brain of mice injected with unmodified sulfamidase. did. It is a figure which shows the map of AAV2.1 plasmid. Map of pAAV2.1 plasmid used to generate AAV2.8 viral vector. This plasmid contains the GFP gene under the control of the liver-specific promoter TBG. The GFP sequence was replaced with a cDNA encoding a chimeric sulfamidase cassette by using NotI and HindIII restriction sites. The resulting plasmid was transfected with pAd helper, pAAV rep-cap plasmid in 293 cells to generate the AAV2.8 viral vector (see methods).

Methods Construction of chimeric SGSH cassettes, recombinant nucleic acid vectors and viral vectors
Another signal peptide was generated by ligation of sequences from two fragments, human SGSH cDNA (fragment I) and a signal peptide sequence (fragment II). Fragment I was amplified from the hSGSH expression plasmid and started at the 3 'end of the hSGSH signal peptide sequence (corresponding to the nucleotide in position 61 on the SGSH sequence), extended to a unique XBaI site, and contained all SGSH cDNA (Oligo used: SGSHFOR 5'-CGT CCC CGG AAC GCA CTG CTG CTC CT-3 '(SEQ ID NO: 28) and SGSHREV 5'-GCG GCC TCT AGA TGA CAG CTC ATT GTG GAG GGG CTG-3' (SEQ ID NO: 29 )). Fragment II was unique for each expression cassette. For hAATsp-SGSH-cFlag, fragment II is composed of two specific oligonucleotide sequences (hAATspFOR 5'-GGC CGC ATG CCG TCT TCT containing the human α1-antitrypsin signal peptide sequence [human a1-antitrypsin cDNA: 72 bp]. GTC TCG TGG GGC ATC CTC CTG CTG GCA GGC CTG TGC TGC CTG GTC CCT GTC TCC CTG GCT 3 '(SEQ ID NO: 30) and hAATspREV 5'-AGC CAG GGA GAC AGG GAC CAG GCA GCA CAG GCC TGC CAG CAG GAG GAT GCC CCACGA GAC AGA AGA CGG CAT GC-3 ′ (SEQ ID NO: 31)) was synthesized by annealing.

A fragment encoding such a signal peptide is
5′-ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCT-3 ′ (SEQ ID NO: 1).

With respect to the IDSsp-SGSH-cFlag expression cassette, fragment II contains two specific oligonucleotide sequences (IDSspFOR 5'- containing the human iduronic acid sulfatase signal peptide sequence [Homosapien suizuronic acid 2-sulfatase (IDS) cDNA: 75 bp). GGC CGC ATG CCC CCG CCC CGC ACC GGC CGC GGC CTG CTG TGG CTG GGC CTG GTG CTG AGC AGC GTG TGC GTG GCC CTG GGC-3 '(SEQ ID NO: 32) and IDSspREV 5'-GCC CAG GGC CAC GCA CAC GCT GCT CAG CAC CAG GCC CAG CCA CAG CAG GCC GCG GCC GGT GCG GGG CGG GGG CAT GC-3 '(SEQ ID NO: 33) was synthesized by annealing.
5'-ATGCCGCCACCCCGGACCGGCCGAGGCCTTCTCTGGCTGGGTCTGGTTCTGAGCTCCGTCTGCGTCGCCCTCGGA-3 '(SEQ ID NO: 3) or optimized sequence
5′-ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTGGCCCTGGGC-3 ′ (SEQ ID NO: 5). The above two sequences differ only in terms of codon usage and encode the same signal peptide aa. Sequence (SEQ ID NO: 4 or 6). The oligonucleotide sequence of fragment II has a 5 ′ NotI site and a 3 ′ blunt end site. Forward and reverse oligonucleotide sequences were incubated at 100 ° C. for 3 minutes. After cooling to RT, we added PNK to the oligo for 30 minutes at 37 ° C. Fragment I (5′NotI-3 ′ blunt) and fragment II (5 ′ blunt-3′XBa) were ligated with the p3xFlag-CMV14 vector plasmid (5′Not-3′XBa). DH5α competent cells were transformed with the resulting ligation mixture.

  To obtain a complete SGSH chimera construct, human ApoB amino acid sequence 3371-3409 (114 bp: 5'TCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGACAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGT-3 ' ATG CAC TGC AGT-3 '(SEQ ID NO: 34) and ApoBDREV 5'-AGA TCT ACT ACC CTC CAC AAA TTT GTT GC-3' (SEQ ID NO: 35)), hAATsp-SGSH-cFlag or IDSsp-SGSH- It was cloned into the BglII site at the 5 ′ end of any 3 × Flag tag of cFlag.

  Different expression cassettes containing either partial chimeric constructs (hAATsp-SGSH-cFlag and hIDSsp-SGSH-cFlag) or full chimeric constructs (hAATsp-SGSH-cFlag-ApoB and hIDSsp-SGSH-cFlag-ApoB) , Subcloned into pAAV2.1-TBG-GFP between NotI (5 ′) and HindIII (3 ′) (GFP sequence replaced with expression cassette). The resulting plasmid (FIG. 10) was used to generate recombinant AAV serotype 8 (AAV2 / 8) (19). Three AAV vectors in 293 cells, pAd helper, pAAV rep-cap (a package plasmid containing the AAV2 rep gene fused with the cap gene of AAV serotype 8), pAAV Cis (this plasmid is a chimeric sulfamidase) It was generated using transient transfection of the protein expressing pAAV2.1-TGB vector). As previously described (19), the recombinant AAV2 / 8 viral vector was purified by two rounds of CsCl. Vector titers expressed as genomic copies (GC / ml) were evaluated by real-time PCR (GeneAmp 7000 Applied Biosystem). AAV vectors were generated by the TIGEM AAV Vector Core Facility (http://www.tigem.it/core-facilities/adeno-associated-virus-aav-vector-core).

Transfection and intracellular secretion
Hela and MPSIIIA MEF cells were maintained in DMEM (normal culture medium) supplemented with 10% FBS and penicillin / streptomycin. Subconfluent cells were transfected using Lipofectamine ™ 2000 (Invitrogen) according to the manufacturer's protocol. One day after transfection, the medium was replaced with DMEM 0.5% FBS. Two days after transfection, we collected conditioned media and pellets for enzyme assay and Western blot analysis.

WB analysis
3 × Flag Lysis buffer 1 × (50 mM Tris-HCl, pH 8, 200 mM NaCl, 1% Triton X100, 1 mM EDTA, 50 mM HEPES) was added to the cell pellet. Lysates were obtained by incubating cell pellets with lysis buffer for 1 hour in ice. Protein concentration was determined using the Bio-Rad (Bio-Rad, Hercules, CA, USA) colorimetric assay. Conditioned medium was concentrated in vivaspin500 (Sartorius) by centrifugation of the medium at 13,000 rpm for 7 minutes. Flagged sulfamidase protein was revealed by Western blot analysis using anti-FLAG M2 monoclonal peroxidase-conjugated antibody (A8592 Sigma-Aldrich) diluted 1: 1000 in 5% milk.

Immunofluorescent cells were washed 3 times in cold PBS and fixed in 4% paraformaldehyde (PFA) for 15 minutes. Fixed cells were washed 4 times in cold PBS, permeabilized with blocking solution (0.1% saponin and 10% FBS in PBS) for 30 minutes, and appropriate primary antibody, rabbit anti-h-sulfamidase (1: 300 , Sigma). After four washes in PBS, we incubated the cells with the secondary antibody anti-rabbit Alexa fluor-488 conjugate (1: 1000). Cells were then washed 4 times in cold PBS and mounted in Vectashield mounting medium.

To determine the degradation rate of the sulfamidase enzyme, MPSIIIA MEFs transfected with different chimeric constructs were treated with 30 μCi for 30 minutes in methionine: cysteine-free medium (Sigma) supplemented with 1% fetal bovine serum. / 10 ⁶ cells [35S] methionine: cysteine labeled (EasyTag ™ EXPRE35S35S Protein Labeling Mix, [3S]; PerkinElmer). After extensive washing, the cells were maintained in the presence of 5% fetal calf serum and supplemented with methionine and cysteine. Cells were harvested at different time points and lysed using 3 × flag Lysis buffer. Lysates were clarified by centrifugation and supernatants were immunoprecipitated by using an agarose-conjugated antibody against flag (anti-flag M2 affinity gel, A2220 Sigma-Aldrich). After extensive washing with lysis buffer, immunoprecipitates were subjected to SDS-PAGE. The dried gel was exposed to a PhosphorImager screen and quantified with the PhosphorImager system.

Animals Homozygous mutants (MPS-IIIA, − / −) and heterozygous (phenotypically normal, +/−) C57BL / 6 mice were used. Thus, the term “normal mouse” is used to represent the mouse phenotype. The experiment was conducted according to the guidelines of the Animal Care and Use Committee of Caldarelli Hospital in Naples, approved by the Italian Ministry of Health.

Systemic injection and tissue recovery Neonatal MPS-IIIA and normal mice from 0 to 1 day after birth were cold-anaesthetized. The vector was delivered into the systemic route via the temporal vein (2 × 10 ¹¹ particles in 100 μl). Adult MPSIIIA mice (1 month) were injected via the tail vein (2 × 10 ¹¹ particles in 100 μl). Animal serum was collected at different time points after injection for enzyme assays. To evaluate transduction with liver and brain traits, animals were sacrificed at different time points. Some of these were perfused / fixed with 4% (w / v) paraformaldehyde in PBS and then the liver was removed for GFP staining. The remaining mice were sacrificed and the liver and brain were removed and SGSH activity was measured.

SGSH activity assay
SGSH activity was measured by the following protocol described in Fraldi et al., Hum Mol Gen 2007.

GFP analysis Liver tissue was subjected to a sucrose gradient (10% -30%) and incubated with O / N in 30% sucrose at 4 ° C. Finally, the tissue was embedded in OCT embedded matrix (KaItek) and snap frozen in a bath of dry ice and ethanol. Tissue frozen sections were cut to a thickness of 10 μm, washed with PBS for 10 minutes, mounted in Vectashield mounting medium and processed for GFP analysis.

Results The purpose of this project was to develop a minimally invasive systemic gene therapy strategy based on intravenous injection of AAV serotype 8. This serotype exhibits a high liver tropism (18-20) and (i) a chimeric modified sulfamidase that is highly secreted from the liver and thus optimized to reach high levels of circulating enzymes in the bloodstream. It can be used to deliver genetically modified genes that encode. Sulfamidase is less secreted than other sulfatase enzymes such as iduronic acid-2-sulfatase (IDS). (ii) The sulfamidase signal peptide was replaced with either IDS or human α-antitrypsin (AAT), a highly secreted enzyme, to efficiently pass through the BBB. The chimeric sulfamidase was further genetically engineered with the specific brain targeting protein domain, the (LDLR) binding domain of Apolipoprotein B (ApoB LDLR-BD).

In vivo results in MPSIIIA mice The effect of the new treatment strongly depends on the ability of the liver to be susceptible to transgene transduction, so that sulfamidase is effectively secreted into the bloodstream and then BBB Pass through and transduce the brain by means of its brain target sequence. Thus, serum levels of therapeutic enzymes can represent an important factor that determines the effect of treatment. Previous studies have not performed the analysis of SGSH liver transduction and systemic levels when exogenous SGSH is delivered systemically to MPS-IIIA mice. The inventors have therefore decided to study this issue to obtain useful preliminary data for designing effective treatment strategies.

Delivery of therapeutic enzymes to neonatal mice is a useful tool to prevent lesions in MPS-IIIA mice. The inventors then decided to test whether systemic injection via AAV2 / 8 into neonatal MPSIIIA could be a feasible approach to develop our new therapeutic strategy. did. To this end, the inventors have undertaken sulfamid under the control of a liver-specific promoter (thyroid hormone-globulin, TBG) to specifically target the liver and make it a factory organ of the therapeutic enzyme. AAV2 / 8 containing the dase coding sequence was injected into MPS-IIIA neonatal mice. Mice were injected with 1 × 10 ¹¹ viral particles via the temporal vein. Experimental group of 3 mice, control mice treated with AAV2 / 8-TBG-GFP (heterozygous mice, these mice show normal phenotype), MPS-IIIA mice treated with AAV2 / 8-TBG-GFP And MPS-IIIA mice treated with AAV2 / 8-TBG-SGSH.

  To test the efficiency of injection, we analyzed GFP fluorescence in the liver of GFP injected mice (normal and MPS-IIIA mice). The GFP signal was present at either early or late time points after injection, but at the late post injection time point, a significant decrease in GFP signal was observed in the analyzed mouse liver (FIG. 1).

  MPS-IIIA mice injected with AAV2 / 8-TBG-SGSH were tested for SGSH activity in plasma and liver at different time points after injection (5, 8, 10, 14 days and 3, 4, 5 and 10 weeks). Checked for. SGSH activity in plasma of AAV2 / 8-TBG-SGSH treated MPS-IIIA mice increased for the first 2 weeks after neonatal treatment and then reached a level measured in control GFP injected MPS-IIIA mice It decreased with time (FIG. 2A). Analysis of liver SGSH activity showed a trend similar to that observed in plasma detected at higher activity levels within the first week after injection (FIG. 2B).

  This preliminary study in neonatal mice shows that the liver is effectively transduced by AAV2 / 8-mediated neonatal delivery of sulfamidase, but one week after injection, the enzyme is low in both liver and serum. Presents (equivalent to control GFP-injected MPS-IIIA mice), indicating that this approach would be unsuitable for treating the brain.

  In order to assess whether the proliferation of liver cells in the post-treatment period causes dilution of the vector in the liver after injection into the neonate, we have determined that the liver has completed its growth. A new study based on systemic (tail vein injection) AAV2 / 8-mediated delivery of SGSH in adult mice (1.5 months of age) was conducted.

  In this study, we also used three mouse experimental groups, normal mice treated with AAV2 / 8-TBG-GFP, MPS-IIIA and AAV2 / 8-TBG- treated with AAV2 / 8-TBG-GFP. MPS-IIIA mice treated with SGSH were provided. Analysis of GFP expression at different time points after treatment (1 week and 5 weeks after injection) clearly showed high and stable expression of the transgene in the liver of adult treated mice (FIG. 3).

  MPSIIIA treated mice were also checked for SGSH activity in liver and serum at different time points after treatment (1 week, 2, 3, 4, 5 weeks). We observed a strong increase in SGSH activity in the liver of MPSIIIA mice injected with AAV2 / 8-TBG-SGSH, compared to the low enzymatic activity in animals injected with GFP vector. Remained stable until 5 weeks after injection (last time point analyzed) (FIG. 4A). Also, the analysis of SGSH activity in the serum of treated mice was very high and stable until the analyzed post-injection period (FIG. 4B). Importantly, this treatment did not produce detectable sulfamidase activity in the brain of AAV2 / 8 injected MPS-IIIA mice (not shown).

  In conclusion, these preliminary studies show that (i) the liver is highly transduced by AAV2 / 8-mediated systemic injection, and (ii) the decrease in SGSH activity in neonatal-treated mice is due to dilution of the vector in the liver. And allow the inventors to consider adult mice to be a good model for testing systemic treatment with AAV2 / 8 containing chimeric sulfamidase, (iii) It shows that secreted (unmodified) sulfamidase did not produce detectable enzyme activity in the brain. The latter is an expected result and further justifies the rationale behind our project objectives.

Construction and validation of chimeric sulfamidase proteins In order to increase sulfamidase secretion from the liver and thus the amount of enzyme in the bloodstream available to specifically target the brain, we have The sulfamidase was genetically engineered by replacing the unique signal peptide (SP) of the sulfamidase with another. Two signal peptides, iduronic acid-2-sulfatase (IDS) signal peptide and human α-antitrypsin (AAT) signal peptide were tested (FIG. 5). The rationale behind the use of these two signal peptides is that IDS is a liposomal enzyme that has been shown to be secreted at high levels from the liver, while AAT is a highly secreted enzyme That is. The ultimate goal of our project is to generate a modified sulfamidase that can cross the BBB and target the CNS via receptor-mediated transcytosis (Figure 6). . For this reason, before starting an experiment aimed at evaluating the therapeutic effect of replacing the SP signal in SGSH, the present inventors reduced the low density of apolipoprotein B, a specific brain-targeting protein domain. The modified SGSH was further engineered with a lipoprotein (Low Density Lipoprotein) receptor (LDLR) binding domain (ApoB LDLR-BD. This binding domain of ApoB is an abundant LDL receptor on the endothelial cells of the BBB. By binding to the body, it will allow sulfamidase to reach brain cells (Figure 6) .The two finally engineered sulfamidase constructs are C-terminal. Contains ApoB LDLR-BD, and contains either IDS or hAAT signal peptides (IDSsp-SGSHflag-ApoB and hAATsp-SGSHflag-ApoB) at the N-terminus (FIG. 5).

  To assess the function of the chimeric sulfamidase protein, we transfected MPSIIIA MEF cells with either partially or final genetically modified sulfamidase protein, The results were compared with those obtained from transfection with unmodified sulfamidase. Surprisingly, we compared SGSH activity in cells transfected with the final chimeric construct in pellets and in conditioned media compared to the activity measured in cells transfected with other constructs. Higher, suggesting that the finally engineered sulfamidase was efficiently secreted (FIG. 7A). In fact, these results were associated with a higher secretory effect of the finally engineered sulfamidase enzyme compared to sulfamidase that was not engineered (FIG. 7B). However, this secretory effect was similar to that measured after transfection with a partial chimeric sulfamidase (containing only the alternative signal peptide) (FIG. 7B). Clearly, the inventors have found that sulfamidase modifications, particularly those present in the finally engineered sulfamidase, are not engineered to a chimeric protein. It was observed to give higher stability compared to famidase (FIGS. 8A and B). Thus, the inventors have shown that increases in sulfamidase protein levels in the medium of cells transfected with genetically engineered sulfamidase proteins are associated with increased secretion efficiency and genetically engineered sulfamidase. It was concluded that it was due to both increased stability.

  Furthermore, immunostaining with anti-SGSH antibody showed lysosome-like localization for both partially and ultimately engineered constructs (FIG. 8C).

  In conclusion, these results show that (i) chimeric sulfamidase enzymes containing alternative signal peptides are functional and active, and (ii) they are compared to unmodified sulfamidases. More stable, (iii) they are secreted more efficiently compared to sulfamidase enzymes that have not been genetically modified, (iv) ultimately genetically modified The introduction of ApoB LDLR-BD to produce a modified sulfamidase resulted in either a functional or increased secretory effect observed in cells transfected with partially engineered sulfamidase Also showed no effect. Furthermore, the final genetically engineered construct appears to be more stable compared to the partially genetically engineered construct.

In vivo results in MPSIIIA mice that were finally injected with genetically modified sulfamidase We underwent final genetically modified sulfamidase (hAATsp-SGSHflag under the liver-specific promoter TBG. AAV2 / 8 vector containing one of -ApoB) was generated. We have obtained very promising but very promising results with MPS-IIIA injected with this viral vector. Adult MPS-IIIA mice were systemically injected with AAV2 / 8-TBG-hAATsp-SGSHflag-ApoB. A group of MPS-IIIA was also injected with AAV2 / 8-TBG-SGSH (containing unmodified sulfamidase) as a control. Mice were sacrificed one month after injection. We observed higher liver sulfamidase activity and a very strong increase in sulfamidase secretion in mice injected with chimeric sulfamidase compared to control mice (FIG. 9). Furthermore, we detected a significant increase in SGSH activity in the brain of mice injected with chimeric sulfamidase (FIG. 9).

Utilization of other vectors We have completed the generation of AAV2 / 8 vectors containing all genetically modified sulfamidase proteins (partial and final). In particular, the inventors have here noted that in addition to AAV2 / 8-TBG-hAATsp-SGSHflag-ApoB, here AAV2 / 8-TBG-hIDSsp-SGSHflag-ApoB, AAV2 / 8-TBG-hAATsp-SGSHflag and AAV2 / 8 -TBG-hIDSsp-SGSHflag was generated.

  These vectors can be used to perform large-scale in vivo studies by the following procedure. To test the clinical effect of the chimeric sulfamidase enzyme, MPS-IIIA mice (1 month old) are injected (by tail vein route) with an AAV2 / 8 vector containing a genetically engineered construct . The results are useful to evaluate (i) the efficiency of CNS transduction and (ii) the rescue of CNS lesions in treated mice.

[References]

Claims (17)

  A pharmaceutical composition for medical use, derived from either a) human α-antitrypsin (hAAT) amino acid sequence or human iduronic acid-2-sulfatase (IDS) amino acid sequence in N-terminal to C-terminal sequence order A pharmaceutical composition comprising a chimeric sulfatase consisting essentially of a signal peptide, b) an amino acid sequence derived from human sulfatase lacking that signal peptide, and c) an ApoB LDLR binding domain.   2. The pharmaceutical composition for medical use according to claim 1, wherein the signal peptide has a sequence belonging to the group of SEQ ID NO: 2 or SEQ ID NO: 4.   The pharmaceutical composition for medical use according to claim 1 or 2, wherein the human sulfatase is human sulfamidase.   4. The pharmaceutical composition for medical use according to claim 3, wherein the human sulfamidase-derived amino acid sequence essentially has the sequence of SEQ ID NO: 8.   5. The pharmaceutical composition for medical use according to any one of claims 1 to 4, wherein the encoded ApoB LDLR binding domain has the sequence of SEQ ID NO: 10. Said chimeric sulfatase is essentially a group of
a) hAATsp-SGSH-3 × flag amino acid sequence (SEQ ID NO: 12),
b) hIDSsp-SGSH-3 × flag amino acid sequence (SEQ ID NO: 14),
c) hAATsp-SGSH-3 × flag-ApoB amino acid sequence (SEQ ID NO: 16),
d) hIDSsp-SGSH-3 × flag-ApoB amino acid sequence (SEQ ID NO: 18),
e) hAATsp-SGSH amino acid sequence (SEQ ID NO: 20),
f) hIDSsp-SGSH amino acid sequence (SEQ ID NO: 22),
g) hAATsp-SGSH-ApoB amino acid sequence (SEQ ID NO: 24),
h) hIDSsp-SGSH-ApoB amino acid sequence (SEQ ID NO: 26),
6. A pharmaceutical composition for medical use according to any one of claims 1 to 5, having a sequence belonging to   7. A pharmaceutical composition for medical use according to any one of claims 1 to 6, for the treatment of mucopolysaccharidosis.   8. The pharmaceutical composition for medical use according to claim 7, wherein the mucopolysaccharidosis is type IIIA mucopolysaccharidosis.   A pharmaceutical composition comprising a chimeric sulfatase and a suitable diluent and / or excipient and / or carrier, wherein the chimeric sulfatase is a) human α-antitrypsin (hAAT) in order of N-terminal to C-terminal sequence ) A signal peptide derived from either the amino acid sequence or the human iduronic acid-2-sulfatase (IDS) amino acid sequence, b) a human sulfatase-derived amino acid sequence lacking that signal peptide, c) consisting essentially of the ApoB LDLR binding domain, Pharmaceutical composition.   10. The pharmaceutical composition according to claim 9, wherein the signal peptide has a sequence belonging to the group of SEQ ID NO: 2 or SEQ ID NO: 4.   The pharmaceutical composition according to claim 9 or 10, wherein the human sulfatase is human sulfamidase.   12. The pharmaceutical composition according to claim 11, wherein the human sulfamidase-derived amino acid sequence has the sequence of SEQ ID NO: 8.   13. The pharmaceutical composition according to any one of claims 9 to 12, wherein the encoded ApoB LDLR binding domain essentially has the sequence of SEQ ID NO: 10. Said chimeric sulfatase is essentially a group of
a) hAATsp-SGSH-3 × flag amino acid sequence (SEQ ID NO: 12),
b) hIDSsp-SGSH-3 × flag amino acid sequence (SEQ ID NO: 14),
c) hAATsp-SGSH-3 × flag-ApoB amino acid sequence (SEQ ID NO: 16),
d) hIDSsp-SGSH-3 × flag-ApoB amino acid sequence (SEQ ID NO: 18),
e) hAATsp-SGSH amino acid sequence (SEQ ID NO: 20),
f) hIDSsp-SGSH amino acid sequence (SEQ ID NO: 22),
g) hAATsp-SGSH-ApoB amino acid sequence (SEQ ID NO: 24),
h) hIDSsp-SGSH-ApoB amino acid sequence (SEQ ID NO: 26),
14. The pharmaceutical composition according to any one of claims 9 to 13, having a sequence belonging to.   15. A pharmaceutical composition according to any one of claims 9 to 14 for the treatment of mucopolysaccharidosis lesions.   16. The pharmaceutical composition according to claim 15, wherein the mucopolysaccharidosis lesion is type IIIA mucopolysaccharidosis.   A viral vector for medical use comprising a recombinant plasmid, the recombinant plasmid comprising a nucleotide sequence under the control of a liver-specific promoter, the nucleotide sequence encoding a chimeric sulfatase, The chimeric sulfatase is in the order of N-terminal-C-terminal sequence, a) a signal peptide derived from either a human α-antitrypsin (hAAT) amino acid sequence or a human iduronic acid-2-sulfatase (IDS) amino acid sequence, b) A viral vector consisting essentially of an amino acid sequence derived from human sulfatase lacking a signal peptide, c) ApoB LDLR binding domain.