CN115029360A

CN115029360A - Transgenic expression cassette for treating mucopolysaccharidosis type IIIA

Info

Publication number: CN115029360A
Application number: CN202210600807.3A
Authority: CN
Inventors: 闫梦迪; 郑静; 蒋威; 杜增民; 陈慧; 陈晨; 袁龙辉; 刘强; 侯文秀; 陈新武; 王慧; 赵阳
Original assignee: Shanghai Mianyi Biotechnology Co ltd
Current assignee: Shanghai Mianyi Biotechnology Co ltd
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2022-09-09
Also published as: WO2023231778A1

Abstract

The present disclosure relates to transgenic expression cassettes useful for the treatment of mucopolysaccharidosis type IIIA and uses thereof, optimized nucleic acid molecules encoding glucosamine sulfohydrolase (SGSH), novel combinatorial promoters optimized in design, gene delivery systems and drugs comprising the transgenic expression cassettes. The transgenic expression cassette, the gene delivery system and the drug disclosed by the invention can effectively recover SGSH activity in a patient with mucopolysaccharidosis, reduce the accumulation of glycosaminoglycan and have good treatment effects on peripheral body diseases and central nervous system diseases of mucopolysaccharidosis IIIA.

Description

Transgenic expression cassette for treating mucopolysaccharidosis type IIIA

Technical Field

The present disclosure belongs to the technical field of biomedicine. The present disclosure relates to transgenic expression cassettes useful for the treatment of mucopolysaccharidosis type IIIA and uses thereof, design optimized nucleic acid molecules encoding glucosamine sulfohydrolase (SGSH), design optimized novel combinatorial promoters, gene delivery systems and medicaments comprising the transgenic expression cassettes.

Background

Mucopolysaccharidosis (MPS) is a rare one of lysosomal storage disorders. Mucopolysaccharidosis found can be classified into 7 types including types I, II, III, IV, VI, VII and IX according to causative genes and clinical manifestations, clinically depending on the kind of glycosaminoglycan (GAG) excreted in urine. MPS III is one of the most common subtypes of MPS, with patients having congenital metabolic disorders of Heparan Sulfate (HS). There are four different hydrolases involved in the metabolism of HS. Depending on the lack of hydrolytic enzymes, mucopolysaccharidosis type III can be further classified into A, B, C, D four subtypes.

MPS IIIA, also known as Sanfilippo syndrome, is one of mucopolysaccharidosis, an autosomal recessive genetic disease caused by a gene mutation of glucosamine sulfohydrolase (SGSH), a sulfatase. Currently, MPS IIIA is a relatively common one of mucopolysaccharidosis, accounting for about 30% of the total MPS.

The SGSH gene is located at 17q25.3 and encodes 502 amino acids. The mutation causes the reduction or deletion of SGSH enzyme activity, thereby causing abnormal accumulation of HS, affecting normal functions of cells and tissues, and leading excessive HS to be discharged by urine, thereby causing various diseases.

MPS IIIA typically occurs in infancy, manifested by deterioration of intelligence, behavioral and sleep disturbances, loss of ambulation, and early death. Central Nervous System (CNS) degenerative disorders are the main features of MPS IIIA, but are also manifested by somatic diseases, symptoms including rough face, respiratory impairment, hepatosplenomegaly, skeletal dysplasia, and the like. Patients will usually die within the second or third decade of life, even early puberty, if left untreated.

In clinical applications, therapeutic modalities such as Enzyme Replacement Therapy (ERT) and Hematopoietic Stem Cell Transplantation (HSCT) are currently available for MPS IIIA patients. However, the macromolecular enzymes that are used in ERT do not cross the Blood Brain Barrier (BBB); however, HSCT cannot be applied to all patients due to donor limitations. Therefore, in recent years, a more effective and feasible approach for MPS IIIA patients would be gene therapy.

Gene therapy repair enzyme deficiencies are a disposable and permanent therapeutic regimen, particularly with AAV vector-mediated gene therapy. AAV has high safety, wide host range, low pathogenicity and long-term stable protein expression in various organ tissues. At present, although some gene therapy drugs based on AAV vectors have been applied to the treatment of MPS IIIA, there still exist many disadvantages, such as low expression level of target gene, weak expression level of promoter regulatory gene, low efficiency of delivering target gene to central nervous system, etc., and peripheral body disorders and central nervous system disorders of MPS IIIA cannot be considered effectively at the same time.

Disclosure of Invention

In order to solve the above technical problems, the present inventors constructed a novel transgenic expression cassette.

Accordingly, in a first aspect, the present disclosure provides a transgenic expression cassette comprising: a promoter selected from the group consisting of SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9; and a nucleic acid molecule encoding a glucosamine sulfohydrolase (SGSH) having a nucleotide sequence at least 80% identical, preferably at least 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO. 1.

In a preferred embodiment, the promoter is selected from the group consisting of SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 9. By using the promoter of

SEQ ID NO

3, 6, 7 or 9, SGSH expression can be regulated and controlled to be more efficiently expressed, thereby realizing better treatment effect on MPS IIIA.

In a more preferred embodiment, the promoter is SEQ ID NO 3 or SEQ ID NO 7. By using the promoter of SEQ ID No. 3 or SEQ ID No. 7, the plasma SGSH enzyme activity of MPS IIIA patients can be maintained at physiological and even supraphysiological levels for a longer time, thus achieving a more effective and more stable therapeutic effect.

In a most preferred embodiment, the promoter is SEQ ID NO 7. By using the promoter of SEQ ID No. 7, better SGSH expression can be achieved in MPS IIIA patients brain.

In a preferred embodiment, the nucleic acid molecule encoding a glucosamine sulfohydrolase (SGSH) comprises the nucleotide sequence set forth in SEQ ID NO. 1.

In a more preferred embodiment, the nucleotide sequence of the nucleic acid molecule encoding a glucosamine sulfohydrolase (SGSH) is set forth in SEQ ID NO 1.

In one embodiment, the transgenic expression cassette further comprises a regulatory element, such as two ITRs at either end; and/or an origin of replication; and/or simian virus 40 intron; and/or a polyadenylation signal.

In one embodiment, the nucleotide sequence of the transgene expression cassette is as set forth in SEQ ID NO: 15. the amino acid sequence of SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20 or SEQ ID NO: 21 is shown in the figure; preferably, the nucleotide sequence of the transgene expression cassette is as set forth in SEQ ID NO: 15. SEQ ID NO: 18. SEQ ID NO: 19 or SEQ ID NO: 21 is shown in the figure; more preferably, the nucleotide sequence of the transgene expression cassette is as set forth in SEQ ID NO: 15 or SEQ ID NO: 19 is shown in the figure; most preferably, the nucleotide sequence of the transgene expression cassette is as set forth in SEQ ID NO: shown at 19.

The transgenic expression cassette of the present disclosure comprises an optimized nucleic acid sequence having improved SGSH expression levels, and one of 6 novel combination-type promoters and CB promoters constructed by artificial design. The transgenic expression cassette disclosed by the invention can effectively recover SGSH activity in patients with mucopolysaccharidosis IIIA, simultaneously reduce the accumulation of GAG, and has good treatment effect on peripheral body diseases and central nervous system diseases of MPS IIIA.

In a second aspect, the present disclosure provides a nucleic acid molecule encoding a glucosamine sulfohydrolase (SGSH) having a nucleotide sequence at least 80% identical, preferably at least 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO. 1.

The optimized nucleic acid molecules of the present disclosure have better SGSH expression levels, thereby enabling better therapeutic effects on mucopolysaccharidosis.

In a third aspect, the present disclosure provides a combined promoter selected from the group consisting of: the MF2 promoter consisting of CMV enhancer, SYN1 enhancer, and chicken β -actin promoter; the MF3 promoter, consisting of the SYN1 enhancer, CMV enhancer, and chicken beta-actin promoter; and the MF5 promoter, which consists of the LXP2.1 enhancer, SYN1 enhancer, and chicken β -actin promoter.

In one embodiment, the nucleotide sequence of the MF2 promoter is shown as SEQ ID NO. 6, the nucleotide sequence of the MF3 promoter is shown as SEQ ID NO. 7, and/or the nucleotide sequence of the MF5 promoter is shown as SEQ ID NO. 9.

The novel combined promoter disclosed by the invention has excellent gene expression regulation and control capability, and can regulate and control SGSH to be expressed more efficiently, so that a better treatment effect on MPS IIIA is realized.

In a preferred embodiment, the combinatorial promoter of the present disclosure is the MF3 promoter. By using MF3 promoter, high expression of SGSH in peripheral system and nervous system can be achieved simultaneously.

In a fourth aspect, the present disclosure provides a gene delivery system comprising: a transgene expression cassette and an AAV capsid protein according to the first aspect.

In one embodiment, the AAV capsid protein is a native AAV capsid protein or an artificially engineered AAV capsid protein; preferably, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, AAVrh10, AAVrh39, AAVrh43, AAV32.33, AAV3B, AAVv66, AAVXL32, AAV. php.b and AAV 2.1.

In a more preferred embodiment, the AAV capsid protein is an AAV9 capsid protein.

In a fifth aspect, the present disclosure provides the use of a transgenic expression cassette according to the first aspect or a gene delivery system according to the fourth aspect in the manufacture of a medicament for the treatment of mucopolysaccharidosis type IIIA.

In a sixth aspect, the present disclosure provides a medicament comprising: a transgene expression cassette according to the first aspect, a nucleic acid molecule according to the second aspect and a gene delivery system according to the fourth aspect, and a excipient.

In one embodiment, the medicament of the present disclosure is for treating mucopolysaccharidosis type IIIA.

The gene delivery system and the drug of the present disclosure can improve the expression of SGSH in the nervous system while maintaining the expression of SGSH in the peripheral system, thereby effectively compromising the treatment of peripheral body disorders and nervous system disorders in MPS IIIA patients.

Accordingly, in a seventh aspect, the present disclosure provides a method of treating mucopolysaccharidosis type IIIA, comprising administering to a subject in need thereof a therapeutically effective amount of a medicament according to the sixth aspect.

In one embodiment, the drug is administered by a systemic route or a local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration, and intralesional administration.

In a preferred embodiment, the drug is administered by a systemic route, such as intravenously.

Drawings

Figure 1A shows a pattern diagram for plasmids comprising hSGSH1, hSGSH2, or hSGSH.

Fig. 1B shows Western Blot results in lysates and supernatants of Huh7 cells.

Fig. 1C shows Western Blot results in HEK293 cell lysates and supernatants.

Figure 1D shows SGSH enzyme activity in lysates of Huh7 and HEK293 cells 48h after vector transfection. P <0.05, p <0.01, p < 0.001.

Fig. 1E shows SGSH enzyme activity in the supernatants of Huh7 and HEK293 cells 48h after vector transfection. P <0.05, p <0.01, p < 0.001.

FIG. 2A shows a schematic diagram of plasmids containing different promoters (CB, MF to MF 5).

FIG. 2B shows Western Blot results in lysates and supernatants of Huh7 cells.

Fig. 2C shows Western Blot results in lysates and supernatants of HEK293 cells.

FIG. 2D shows SGSH enzyme activity in lysates of Huh7 and HEK293 cells 48h after vector transfection. P <0.05, p <0.01, p < 0.001.

Fig. 2E shows SGSH enzyme activity in the supernatants of Huh7 and HEK293 cells 48h after vector transfection. P <0.05, p <0.01, p < 0.001.

FIG. 3 shows SGSH enzyme activity in Huh7 cell lysates and supernatants 72h after AAV9-hSGSH1 virus infection. P <0.05, p <0.01, p < 0.001.

Fig. 4A shows a schematic diagram of CRISPR/Cas9 knockout mouse SGSH gene.

Fig. 4B shows comparative photographs of the appearance of 6-month-old MPS IIIA mice (left) and wild-type (WT) mice (right).

Figure 4C shows SGSH enzyme activity in tissues of 6-month-old MPS IIIA and WT mice. P <0.05, p <0.01, p < 0.001.

Figure 4D shows SGSH enzyme activity in plasma of 6-month old MPS IIIA and WT mice. P < 0.01.

Figure 4E shows 6-month-old MPS IIIA and WT mouse brain LAMP1 immunofluorescence staining. Scale bar 50 μm.

Figure 4F shows 6-month-old MPS IIIA and WT mice brain GFAP immunofluorescence staining. Scale bar 50 μm.

Figure 5A shows SGSH enzyme activity in MPS IIIA mouse plasma 28 days after virus injection. P <0.05, p <0.01, p < 0.001.

FIG. 5B shows SGSH enzyme activity in brain, liver, spleen, heart, kidney, lung and muscle tissues of MPS IIIA mice 28 days after virus injection. P <0.05, p <0.01, p < 0.001.

Figure 6 shows GAG content in MPS IIIA mouse tissues, including mouse brain, liver, spleen, heart, kidney, lung and muscle tissues, 28 days after virus injection. P <0.05, p <0.01, p < 0.001.

Figure 7A shows SGSH enzyme activity in MPS IIIA mouse plasma 56 days after virus injection. P < 0.001.

Figure 7B shows SGSH enzyme activity in brain, liver, spleen, heart, kidney, lung and muscle tissues of MPS IIIA mice 56 days after virus injection. P <0.05, p <0.01, p < 0.001.

Figure 8A shows the GAG content in brain, liver, spleen, heart, kidney, lung and muscle tissues of MPS IIIA mice 56 days after virus injection. P <0.05, p <0.01, p < 0.001.

Figure 8B shows HS content in urine of MPS IIIA mice 56 days after virus injection. P <0.05, p <0.01, p < 0.001.

Fig. 9A shows the latency of searching for the escape compartment and the number of errors per search for the middle aged MPS IIIA mice in 16 training sessions for 4 days.

Figure 9B shows the residence time of aged MPS IIIA mice in the target quadrant at day 5 and day 12 testing. P <0.05, p <0.01, p < 0.001.

Figure 9C shows a follow-up plot of the movement trajectory of aged MPS IIIA mice at day 5 and day 12 testing.

Figure 10 shows the improvement of lysosomal storage pathology in the brain of aged MPS IIIA mice 5 months after viral vector treatment. And (3) CBC: cerebral cortex; TH: the thalamus; STR: a striatum; HP: sea horses; CB: the cerebellum. Scale bar 50 μm.

Figure 11 shows the improvement of neuroinflammation in the brain of aged MPS IIIA mice after 5 months treatment with viral vectors. And (3) CBC: the cerebral cortex; TH: the thalamus; STR: a striatum; HP: sea horses; CB: the cerebellum; MY: medulla oblongata. Scale bar 50 μm.

FIG. 12 shows the nucleotide sequence of hSGSH1(SEQ ID NO: 1).

FIG. 13 shows the nucleotide sequence of hSGSH2(SEQ ID NO: 2).

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Abbreviative index

Unless otherwise indicated, the nucleic acid or polynucleotide sequences set forth herein are in single stranded form in the orientation 5 'to 3', left to right. The nucleotides and amino acids provided herein are in the format suggested by the IUPACIUB Biochemical nomenclature Commission, and the single letter code or the three letter code is used for amino acids.

Unless otherwise indicated, "polynucleotide" is synonymous with "nucleic acid" and refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or the like. Polynucleotides may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism that has or is susceptible to a condition that can be prevented or treated by administration of a medicament of the present disclosure, and include both human and non-human animals (e.g., rodents or other mammals).

In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other apes and monkey species; farm animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including poultry, pheasants, and game birds such as chickens, turkeys, and other chickens, ducks, geese, etc.). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

Herein, the term "treatment" includes: (1) inhibiting the condition, disease or disorder, i.e., arresting, reducing, or delaying the progression of the disease or its recurrence or the progression of at least one clinical or subclinical symptom thereof; or (2) ameliorating the disease, i.e., causing regression of at least one of the conditions, diseases or disorders or clinical or subclinical symptoms thereof.

As used herein, the term "therapeutically effective amount" refers to the dosage which produces the therapeutic effect which it is administered to achieve. For example, a therapeutically effective amount of a medicament useful for treating muscular dystrophy may be an amount that is capable of preventing or ameliorating one or more symptoms associated with the muscular dystrophy.

As used herein, the term "amelioration" refers to an improvement in a symptom associated with a disease, and may refer to an improvement in at least one parameter that measures or quantifies the symptom.

Herein, the term "preventing" a condition, disease or disorder includes: preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of a developed condition, disease or disorder in a subject who may be suffering from or susceptible to the condition, disease or disorder but who has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

Herein, the term "topical administration" or "topical route" refers to an administration having a local effect.

As used herein, the term "vector" refers to a macromolecule or series of macromolecules encapsulating a polynucleotide that facilitates delivery of the polynucleotide into a target cell in vitro or in vivo. Classes of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotide to be delivered is sometimes referred to as a "transgene," including, but not limited to, the coding sequence for certain proteins or synthetic polypeptides that can enhance, inhibit, attenuate, protect, trigger or prevent certain biological and physiological properties, or the coding sequence of interest in vaccine development (e.g., a polynucleotide that expresses a protein, polypeptide or peptide suitable for eliciting an immune response in a mammal), the coding sequence for an RNAi component (e.g., shRNA, siRNA, antisense oligonucleotide), or an optional marker.

As used herein, the terms "transduction," "transfection," "transformation" and "transforming" refer to the process of delivering an exogenous polynucleotide into a host cell, transcription and translation to produce a polypeptide product, including the introduction of the exogenous polynucleotide into the host cell using a recombinant virus.

As used herein, the term "gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene delivery, including targeting, binding, uptake, transport, replicon integration, and expression.

As used herein, the term "gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce the RNA or protein product of the gene.

As used herein, the term "infection" refers to the process by which a virus or viral particle comprising a polynucleotide component delivers a polynucleotide into a cell and produces its RNA and protein products, and may also refer to the process of replication of the virus in a host cell.

The terms "expression cassette", "transgene cassette" and "transgenic expression cassette" are used interchangeably herein and refer to a polynucleotide fragment encoding a particular protein, polypeptide or RNAi element, which can be cloned into a plasmid vector.

As used herein, the term "codon optimized" refers to a polynucleotide sequence that is modified from its native form. Such modifications result in differences in one or more base pairs, with or without changes in their corresponding amino acid sequences, which may enhance or inhibit expression of the gene and/or cellular response to the modified polynucleotide sequence.

The term "adeno-associated virus (AAV)" includes native AAV (AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV), and other artificially engineered AAV known or later discovered or invented. Such as Bernard N.FIELDS et al, VIROLOGY, Vol.2, Chapter 69 (fourth edition, Lippincott-Raven Publishers and e.g. Gao et al, J.Virol (2004)78: 6381-. The genomic sequences and ITR sequences, Rep and Cap proteins of different serotypes of AAV are known in the art. These sequences can be found in the literature or in public databases, such as genbank (r) libraries, e.g., genbank (r) accession nos. NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, JO1901, J02275, XO1457, AF 2861, AHO09962, AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY 631966; the summary of which is incorporated herein in its entirety. Such as Srivistava et al, J.Virol (1983)45: 555; chiorini et al, J.Virol (1998)71: 6823; chiorini et al, J.Virol (1999)73: 1309; Bantel-Schaal et al, J.Virol (1999)73: 939; xiao et al, J.Virol (1999)73: 3994; muramatsu et al, Virology (1996)221: 208; choudhury SR et al, 2016, 24(7): 1247-57; hsu HL. et al, Nat Commun 11,3279 (2020); international patent publications WO 00/28061, WO 99/61601, WO 98/11244, WO 2021050970a1, US 2019/036676 a1, US patent No. 6,156.303.

In this context, the term "inverted terminal repeat" encompasses any AAV viral terminal repeat or synthetic sequence that constitutes a hairpin structure and mediates replication, packaging and integration of the virus as a cis structure. The ITRs herein include, but are not limited to, terminal repeats of AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In addition, the AAV terminal repeats need not be native, so long as they can be used for replication, packaging, and integration of AAV.

In this context, the term "targeted" refers to the preferential entry of the virus into some cell or tissue, followed by further expression of the viral genome or sequence carried by the recombinant transgene in the cell.

In one embodiment, the promoter in the transgenic expression cassette of the present disclosure is selected from the group consisting of: CB promoter, MF promoter (MHC enhancer and SYN1 promoter), MF1 promoter (MHC enhancer, SYN1 enhancer and SYN1 promoter), MF2 promoter (CMV enhancer, SYN1 enhancer and chicken β -actin promoter), MF3 promoter (SYN1 enhancer, CMV enhancer and chicken β -actin promoter), MF4 promoter (LXP2.1 enhancer, SYN1 enhancer and chicken SYN1 promoter), and MF5 promoter (LXP2.1 enhancer, SYN1 enhancer and chicken β -actin promoter).

In a preferred embodiment, the promoter in the transgenic expression cassette of the present disclosure is a CB promoter or MF3 promoter. By using the CB promoter or MF3 promoter, plasma SGSH enzyme activity in MPS IIIA patients can be maintained at physiological and even supraphysiological levels for longer periods of time, thus achieving more effective and stable therapeutic effect.

In a more preferred embodiment, the promoter in the transgenic expression cassette of the present disclosure is the MF3 promoter. Compared to the CB promoter, better SGSH expression in MPS IIIA patient brain can be achieved using MF3 promoter.

In one embodiment, the transgenic expression cassettes or gene delivery systems of the present disclosure are formulated for pharmaceutical administration to humans or other mammals.

The medicine disclosed by the invention can stably express SGSH protein in MPS IIIA patients, and recover central nervous system and body diseases caused by enzyme deficiency, thereby realizing a good treatment effect on mucopolysaccharidosis IIIA.

The present disclosure is described in further detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1: optimization and screening of hSGSH sequence

Firstly, a single-stranded vector framework pAAV2.1-CB-SV40-MCS which is stored in a laboratory at present is used for selecting a proper enzyme cutting site and inserting a cDNA original sequence of hSGSH to construct and obtain pAAV2.1-CB-SV40-hSGSH, wherein the vector structure is shown in figure 1A.

Meanwhile, the original human SGSH gene is subjected to codon optimization so as to screen out a target gene sequence with better expression. Cloning the human SGSH gene optimized by different codons into a single-chain rAAV carrier framework, and driving the human SGSH gene to express through a CB promoter. Wherein the CB promoter is a combined promoter comprising a Cytomegalovirus (CMV) enhancer and a Chicken beta-actin promoter. The cDNA sequence of hSGSH is searched by NCBI database, the length is 1509bp, EcoRI and BglII enzyme cutting sites (the enzyme cutting sites of FseI and Bstbi are added under the other condition) are respectively added at the 5 'end and the 3' end of the sequence, and the codon optimization is carried out on the hSGSH sequence under the premise of protecting the enzyme cutting sites. The two SGSH sequences obtained by different optimization modes are named as hSGSH1(SEQ ID NO:1) and hSGSH2(SEQ ID NO:2) respectively, and the original sequence of the SGSH cDNA which is not subjected to codon optimization is numbered as hSGSH (the carrier structure is shown in figure 1A). After artificial gene synthesis is carried out on hSGSH1, hSGSH2 and hSGSH, fragments with double cohesive ends are obtained through double enzyme digestion reaction, wherein hSGSH1 is subjected to double enzyme digestion through EcoRI and BglII restriction endonucleases, hSGSH2 and hSGSH are subjected to double enzyme digestion through FseI and BstbI restriction endonucleases, then the synthesized gene fragments are connected to pAAV2.1-CB-SV40-MCS vectors through T4 DNA ligase, pAAV2.1-CB-SV40-hSG 1, pAAV2.1-CB-SV40-hSGSH2 and pAAV2.1-CB-SV40-hSGSH vectors are obtained respectively, and the sequencing results are compared to show that the vectors are successfully constructed.

In order to verify that the constructed vector can successfully express SGSH protein, Huh7 cells and HEK293 cells with good cell growth state are used for plating in the research, the number of cells in each hole of a six-hole plate is 1E6, after cells grow adherently 12h, pAAV2.1-CB-SV40-hSGSH1, pAAV2.1-CB-SV40-hSGSH2 and pAAV2.1-CB-SV40-hSGSH plasmids are transferred into the cells by using PEI transfection reagents, each hole is transfected with 5 mu g of plasmids, and 2 times of volume of PEI reagents are added according to the proportion. Culturing for 48h after transfection, collecting cells and supernatant, extracting proteins in the cells by using non-denatured cell tissue lysate, quantifying the proteins by using a BCA kit, and carrying out Western blot analysis and SGSH enzyme activity determination.

Western blot results show that compared with blank control, the SGSH protein expression levels in cell lysates and cell supernatants of 3 vector transfection groups (pAAV2.1-CB-SV40-hSGSH1, pAAV2.1-CB-SV40-hSGSH2 and pAAV2.1-CB-SV40-hSGSH) are remarkably improved (FIG. 1B and FIG. 1C), wherein the SGSH protein expression levels in Huh7 cells transfected with pAAV2.1-CB-SV40-hSGSH1 vectors and supernatants are obviously better than those of pAAV2.1-CB-SV40-hSGSH2 and pAAV2.1-CB-SV40-hSGSH vectors (FIG. 1B); in HEK293 cells and supernatants, the difference in SGSH protein expression was not significant for the 3 vector-transfected groups (fig. 1C).

Furthermore, the enzyme activity of SGSH in Huh7 and HEK293 cells and supernatants after vector transfection was detected, the results thereof are identical to the Western blot results, and the SGSH enzyme activity of 3 vector transfection groups was significantly improved compared with that of the blank control group (fig. 1D and fig. 1E). In Huh7 cells, the SGSH enzyme activity in cell lysates of the transfection vectors paav2.1-CB-SV 40-hsgssh 1 was 66.05 ± 7.5nmol/17h/mg, higher than in cell lysates transfected with paav2.1-CB-SV40-hSGSH2 and paav2.1-CB-SV 40-hsgssh vectors, (SGSH enzyme activities 23.82 ± 4.46 nmol/17h/mg and 57.44 ± 2.75nmol/17h/mg, respectively), and significantly higher than in the blank control group of untransfected plasmids 0.74 ± 0.07 nmol/17h/mg (fig. 1D, # p <0.05, # p <0.01, # p < 0.001). Further analysis showed that in Huh7 cells, SGSH enzyme activity in the transfection vector paav2.1-CB-SV 40-hsgssh 1 cell lysate and cell supernatant was elevated to 88.96 and 52.58 times that of the untransfected control group, and that in the transfection vector paav2.1-CB-SV 40-hsgssh enzyme activity was inferior to that in Huh7 cell lysate and cell supernatant to 77.36 and 42.43 times that of the untransfected control group, while SGSH enzyme activity in the transfection vector paav2.1-CB-SV40-hSGSH2 was elevated to 32.08 and 13.25 times that of the control group; in HEK293 cells, SGSH enzyme activity in the transfection vector pAAV2.1-CB-SV40-hSGSH1 cell lysates and cell supernatants was elevated to 49.18 and 5.40 times higher than that of the untransfected control group, 47.77 and 4.59 times higher than that of the transfection vector pAAV2.1-CB-SV40-hSGSH2 and 46.86 and 4.16 times higher than that of the transfection vector pAAV2.1-CB-SV40-hSGSH, respectively. (figure 1D and figure 1E, # p <0.05, # p <0.01, # p < 0.001). In general, the result of enzyme activity measurement is identical with that of Western Blot, and the expression of pAAV2.1-CB-SV40-hSGSH1 vector in Huh7 cells and supernatant is obviously better than that of the other two vectors.

As described above, the 3 pAAV2.1-CB-SV40-hSGSH vectors constructed in this study successfully expressed hSGSH at the cellular level and were efficiently secreted extracellularly. In contrast, the first optimized hSGSH1 sequence can express a large amount of SGSH proteins with high activity, and the effect is optimal among the three. Therefore, in subsequent studies, optimized hSGSH1 sequences were used in the expression cassettes.

Example 2 construction of expression cassettes containing novel combinatorial promoters and their expression in cells

In the present study, the selected hSGSH1 sequence was used, and 6 different novel combination promoters (MF, MF1, MF2, MF3, MF4, MF5) were redesigned, so as to obtain a novel combination promoter capable of maintaining the expression of SGSH in the peripheral system and simultaneously improving the expression of SGSH in the nervous system.

The pattern diagram of the expression cassette of the specific vector is shown in FIG. 2A, wherein the MF promoter is composed of an MHC enhancer and a SYN1 promoter, the MF1 promoter is composed of an MHC enhancer, a SYN1 enhancer and a SYN1 promoter, the MF2 promoter is composed of a CMV enhancer, a SYN1 enhancer and a chicken beta-actin promoter, the MF3 promoter is composed of a SYN1 enhancer, a CMV enhancer and a chicken beta-actin promoter, the MF4 promoter is composed of an LXP2.1 enhancer, a SYN1 enhancer and a SYN1 promoter, and the MF5 promoter is composed of an LXP2.1 enhancer, a SYN1 enhancer and a chicken beta-actin promoter.

The promoters from MF to MF5 are respectively used for replacing the CB promoter in the vector pAAV2.1-CB-SV40-hSGSH1 to obtain 6 new vectors (figure 2A), and the sequencing results are compared to show that the vector construction is successful. The 6 new vectors and the constructed pAAV2.1-CB-SV40-hSGSH1 vector are respectively transfected into cells to verify the expression of the vectors in vitro. Huh7 cells and HEK293 cells with good growth state are cultured in a twelve-well plate, the inoculation amount of each well is 2.5E5 cells, 2.5 mu g of plasmids are used for transfection through a PEI transfection reagent after 12h of adherent growth of the cells, the cells and supernatant are collected after 48h of culture, protein in the cells is extracted by using non-denatured cell tissue lysate, protein quantification is carried out by using a BCA kit, and Western Blot analysis and SGSH enzyme activity determination are carried out.

Western Blot results show that in Huh7 cells, the expression levels of SGSH protein in cell lysates and cell supernatants of transfection vectors pAAV2.1-CB-SV40-hSGSH1, pAAV2.1-MF2-SV40-hSGSH1 and pAAV2.1-MF5-SV40-hSGSH1 are obviously improved compared with blank controls; the expression level of SGSH protein in cell lysates and cell supernatants of transfection vectors pAAV2.1-MF-SV40-hSGSH1, pAAV2.1-MF3-SV40-hSGSH1 and pAAV2.1-MF4-SV40-hSGSH1 is also improved to a certain extent compared with that of a blank control, but the expression level is slightly lower than that of the 3 vectors; in contrast, the expression level was not significantly increased in the cell lysates and cell supernatants of the transfection vectors pAAV2.1-MF1-SV40-hSGSH1 and pAAV2.1-MF4-SV40-hSGSH1 (FIG. 2B).

Similarly, in the HEK293 cell lysate and supernatant, the SGSH protein expression levels of pAAV2.1-CB-SV40-hSGSH1, pAAV2.1-MF2-SV40-hSGSH1, pAAV2.1-MF3-SV40-hSGSH1, pAAV2.1-MF5-SV40-hSGSH1 vectors were significantly better than those of pAAV2.1-MF-SV40-hSGSH1, pAAV2.1-MF1-SV40-hSGSH1, pAAV2.1-MF4-SV40-hSGSH1 vectors (FIG. 2C).

Further, for the expression of SGSH, quantitative analysis was performed using SGSH enzyme activity detection assay. The results show that the SGSH enzyme activity in both the lysate and supernatant of transfected vehicle was significantly improved compared to the control blank without transfected vehicle. Notably, the transfection vectors paav2.1-CB-SV40-hSGSH1 had SGSH enzyme activity 86.97 ± 0.85nmol/17h/mg and 46.31 ± 0.4nmol/17h/mg in Huh7 and HEK293 cell lysates, significantly higher than 0.69 ± 0.03nmol/17h/mg and 1.1 ± 0.04nmol/17h/mg of the untransfected controls (fig. 2D, < p <0.01, > p <0.001), increased to 125.54 and 42.01 times of the untransfected controls, and increased to 44.75 and 8.15 times of the SGSH enzyme activity in Huh7 and HEK293 cell supernatants, respectively (fig. 2E, < p <0.05, < p < 0.01).

In conclusion, the SGSH enzyme activity expressed by the transfection vector on the cellular level and the Western blot expression result thereof are mutually proved, and shows that the vectors of pAAV2.1-MF-SV40-hSGSH1, pAAV2.1-MF1-SV40-hSGSH1, pAAV2.1-MF2-SV40-hSGSH1, pAAV2.1-MF3-SV40-hSGSH1, pAAV2.1-MF4-SV40-hSGSH1 and pAAV2.1-MF5-SV40-hSGSH1 and the constructed pAAV2.1-CB-SV40-hSGSH1 successfully express hSGSH at cellular level and are effectively secreted out of cells, wherein the newly constructed pAAV2.1-MF2-SV40-hSGSH1, pAAV2.1-MF3-SV40-hSGSH1 and pAAV2.1-MF5-SV40-hSGSH1 vectors and the constructed pAAV2.1-CB-SV40-hSGSH1 vectors have certain advantages in the expression of two cell levels.

Example 3 packaging and validation of AAV9-hSGSH1 viruses

Furthermore, in the research, 7 vectors which are constructed and can successfully express SGSH are respectively packaged into virus capsids of AAV9 by a three-plasmid cotransfection method, and are produced by CsCl density gradient centrifugation purification, and AAV9-CB-hSGSH1, AAV9-MF-hSGSH1, AAV9-MF1-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 virus vectors are prepared. These virus titers were finally determined by qPCR to range from 8.7E 12-2.2E 13 vg/mL.

In order to verify that these viral vectors can produce functional SGSH, the 7 AAV9-hSGSH1 viral vectors with different promoters and the AAV9-CB-GFP viral vectors expressing Green Fluorescent Protein (GFP) that are already in the laboratory were used to infect Huh7 cells (MOI ═ 1E6) with good growth status in 12-well plates, and AAV9-CB-GFP was used as a control group to observe the success of infection procedure. Cells were harvested 72h after infection and expression of functional SGSH was determined by measuring the enzymatic activity of SGSH.

The experimental results showed that the SGSH enzyme activity levels of Huh7 cells after infection with the 7 AAV9-hSGSH1 viruses were 1.45-3.30 times higher than the control group, significantly higher than those of the AAV9-CB-GFP control group (fig. 3, # p <0.01, # p < 0.001). Wherein, the SGSH activity in the cell lysate infected by AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 viruses is remarkably increased to 3.06, 3.30, 3.00 and 2.42 times of the control group respectively (FIG. 3, p <0.001), the cell lysate infected by AAV9-MF-hSGSH1, AAV9-MF1-hSGSH1 and AAV9-MF3-hSGSH1 viruses is increased to 1.50, 1.47 and 1.93 times of the control group respectively (FIG. 3, p <0.01, p < 0.001). Similarly, in the supernatant of Huh7 cells, the SGSH enzyme activity in the supernatants infected with AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1, and AAV9-MF5-hSGSH1 viruses increased to 1.74, 1.80, 2.15, 1.57, and 2.11 times that of the control group (fig. 3, <0.05, and <0.001), and the SGSH activity in the supernatants infected with AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 viruses also increased to 1.39 and 1.10 times that of the control group.

In conclusion, the AAV9-hSGSH1 virus packaged by 7 vectors can still effectively express SGSH in Huh7 cells and is secreted to the outside of cells, and the in vitro effectiveness of the AAV9-hSGSH1 virus is successfully verified. Wherein, the in vitro expression of the SGSH mediated by AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 virus vectors is superior to that of AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 virus vectors. In addition, it is remarkable that the AAV9-MF3-hSGSH1 virus vector mediates SGSH expression in cells to a slightly lower degree than 4 vectors such as AAV9-CB-hSGSH1, but AAV9-MF3-hSGSH1 virus mediates SGSH expression in Huh7 supernatant higher than other 6 virus vectors, and shows better secretion ability.

Example 4 MPS IIIA mouse model building and phenotypic validation

According to the study of Lau et al (DOI:10.1007/s10545-017-0044-4), the MPS IIIA model mouse constructed and used in the study is an SGSH-KO gene knockout mouse, and through the CRISPR/Case9 technology, about 1kb of fragment on the exon 2 of the SGSH gene of a C57BL/6N mouse is knocked out by using 2 pre-designed sgRNAs, so that DNA double-strand break and non-homologous recombination are caused, and the SGSH gene knockout is realized (FIG. 4A), and the knockout process is entrusted to Beijing Bai Oersai biology technology limited company to carry out. The study obtained homozygous F1 mouse after genotype identification, propagated and developed the experiment. The MPS IIIA model mouse can truly reflect most symptoms of MPS IIIA human patients, the phenotype of the MPS IIIA model mouse is preliminarily verified by the research, and the conditions of plasma and main cathepsin activity, other disease injuries including intracerebral lysosomal membrane protein expression and astrocytosis are specifically analyzed, and the result shows that the mouse model is successfully constructed.

This study was divided into 4 groups of MPS IIIA male and female mice, 6 months of age, respectively, and Wild Type (WT) male and female mice of the same strain and age served as controls (n ═ 3). First, MPS IIIA mice were obese in size compared to WT mice analyzed by appearance and clinical observation; abnormal hair condition, manifested as disorganized loss of luster; rough face with typical mucopolysaccharide facial appearance characteristics; the dorsal bones are deformed to bulge and become humpback (fig. 4B). And MPS IIIA mice were hyperactive with individual accompanying aggressive behavior. In addition, the MPS IIIA mice were found to have enlarged liver and spleen during the dissection; most of them are accompanied by a severe swelling of the bladder, which has a certain degree of dysuria, and some individuals show symptoms of hematuria.

SGSH enzyme activity assay was performed by bleeding mice orbital and isolating peripheral plasma, showing that the SGSH enzyme activity in MPS IIIA male and female mouse plasma was 0.03 + -0.04 nmol/17h/mL and 0.1 + -0.07 nmol/17h/mL, respectively (FIG. 4D,. sup.p <0.01), with almost no detectable SGSH enzyme activity in MPS IIIA mouse plasma, 0.1% -0.2% of WT control mice alone, significantly lower than WT mice of the same strain and age. Furthermore, there was no significant difference in SGSH activity in plasma of female and male MPS IIIA mice.

Similar results were also shown for the determination of SGSH enzyme activity in major tissue organs of mice. MPS IIIA mice had much lower enzyme activity in heart, kidney and muscle tissues than WT levels (fig. 4C, # p <0.05, # p <0.01, # p <0.001) and were well below the limit of detection; for spleen and liver tissues, little enzymatic activity was detected in MPS IIIA male mice, MPS IIIA female mice had slightly higher enzymatic activity in spleen and liver than MPS IIIA male mice, but there was no significant difference between them, and SGSH activity in liver and spleen was significantly lower than WT mice, both male and female MPS IIIA mice (fig. 4C, <0.01, < 0.001); in the brain, MPS IIIA male and female mice SGSH enzyme activities were 0.05 ± 0.01nmol/17h/mg and 0.04 ± 0.01nmol/17h/mg, respectively, significantly lower than 0.34 ± 0.10nmol/17h/mg and 0.35 ± 0.03nmol/17h/mg in WT control mice (fig. 4C, # p <0.05, # p < 0.01).

Further, to assess neuroinflammatory conditions in the central nervous system of MPS IIIA mice, the present study used LAMP-1 and GFAP antibodies, respectively, for immunofluorescent staining of 6-month old MPS IIIA and WT male mouse brain sections, indicating changes in lysosomal storage and astrocyte activation, respectively, in MPS IIIA disease. The results show that positive signal for LAMP I in MPS IIIA mice brain is significantly more pronounced in the periventricular striatal sites than in WT mice (fig. 4E); in the hippocampal region, however, a significantly greater positive GFAP signal was observed in MPS IIIA mice than in WT mice (fig. 4F), indicating a strong manifestation of MPS IIIA mice in lysosomal storage pathology and neuroinflammation.

In conclusion, MPS IIIA mice have phenotypically apparent somatic disorders, including some growth and development and body area manifestations, disorders of tissues such as bone and hair, urinary and hepatobiliary phenotypes, etc. Characteristically, the SGSH activity in the tissue and organ of MPS IIIA mice was very low, and even below the normal level of 0.1% SGSH activity in plasma, indicating a loss of SGSH activity. Pathophysiological features of aberrantly activated neuroinflammation and lysosomal storage of astrocytes were shown in MPS IIIA mouse central nervous system. In summary, the MPS IIIA mouse model with SGSH gene knockout used in this study was successfully established, and this model mouse could reflect to a large extent most of the conditions of MPS IIIA human patients, and could be used for subsequent in vivo studies.

Example 5 treatment of MPS IIIA mice with AAV9-hSGSH1 Virus

To compare the therapeutic effect of these 7 AAV9-hSGSH1 viruses in MPS IIIA model mice, a dose of 5E13 vg/kg (number of viral genomes/kg body weight) of the virus was injected into 2-month-old MPS IIIA male mice by tail vein injection. Because the reproductive capacity of the model mouse is unstable and limited by the breeding quantity, 7 AAV9-hSGSH1 viruses packaged and purified are divided into two parts for screening. In the first part of research, AAV9-CB-hSGSH1, AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 virus vectors are used for carrying out short-term treatment on MPS IIIA mice, and AAV9-CB-hSGSH1 with optimal treatment effect is obtained through screening. Next, the second study used AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 viral vectors to perform short-term treatment on MPS IIIA mice in order to screen out therapeutic vectors that can simultaneously correct somatic and neurological disorders in MPS IIIA patients.

AAV9-CB/-MF/-MF1-hSGSH1 Virus treatment MPS IIIA mice

First, in order to investigate the therapeutic profile of the viral vector in MPS IIIA model mice, 2-month-old male MPSIIIA model mice were administered by tail vein injection at a dose of 5E13 vg/kg in this study. In the first study 3 treatment groups were set up, AAV9-CB-hSGSH1(n ═ 5), AAV9-MF-hSGSH1(n ═ 5), AAV9-MF1-hSGSH1(n ═ 5), and untreated MPS IIIA model mice of the same age (n ═ 5) and WT mice of the same age and strain (n ═ 5) were set as controls. Wherein the untreated MPS IIIA model mouse is an MPS IIIA model mouse administered with the same volume of PBS. Blood was collected from the orbital 3, 7, 14, 21, and 28 days after virus injection and plasma was isolated for SGSH enzyme activity assay. Mice were sacrificed 28 days after the administration, and various tissues and organs of the mice were taken for analysis and examination.

The results of SGSH enzyme activity in plasma showed that SGSH expression peaked 7 days after virus injection, and slightly decreased after 7 days, but remained at a relatively stable level. At 28 days post-injection, SGSH enzyme activity was significantly increased in blood circulation and approached WT levels in AAV9-CB-hSGSH1 treated group compared to AAV9-MF-hSGSH1 treated group (fig. 5A,. xp < 0.01). However, the peripheral plasma activity of mice in the AAV9-MF1-hSGSH 1-treated group was not greatly increased compared with that in the untreated model group, and was always at a lower level.

SGSH enzyme activity results in tissues show that AAV9-CB-hSGSH1, AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 treated groups have increased SGSH enzyme activity in vivo compared to untreated model mice, showing significant differences (FIG. 5B, p <0.05, p <0.01, p < 0.001. AAV9-CB-hSGSH1, AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 treated groups have significantly increased SGSH enzyme activity in liver compared to untreated model mice (FIG. 5B, p <0.05, p <0.001), achieving supraphysiological level expression, wherein AAV9-CB-hSGSH1 treated groups having better therapeutic effects have increased SGSH enzyme activity in liver compared to mice treated with HSSH 24-HSSH groups (SGSH 24-HSSH 8653 and HSSH 1504.24 treated mice, and AAV 5928 times as compared to untreated model mice (AAV 8628-HSSH 2-HSSH 6327 and AAV 1-HSSH 8427, p < 0.001). In the brain, AAV9-CB-hSGSH1 (FIG. 5B, p <0.01), AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 (FIG. 5B, p <0.05) treated groups were slightly elevated compared to untreated model mice. In addition, the SGSH activity level of the AAV9-CB-hSGSH1 treated group is 1.80 times that of the untreated model mice (figure 5B, p <0.01), 23.31 percent of WT mice, and is higher than that of the AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 treated groups, but none of the groups returns to the WT level. Furthermore, SGSH enzyme activity was increased in the treatment group of 3 AAV viruses in the major tissues tested, spleen, heart, kidney, lung, etc. (fig. 5B). Among them, AAV9-CB-hSGSH 1-treated group showed that SGSH enzyme activity was expressed at an excessive physiological level in spleen and heart, and SGSH enzyme activity was also increased in kidney, lung and muscle. Overall, at 28 days post-injection, the AAV9-CB-hSGSH1 treated group achieved some elevation in SGSH activity levels compared to untreated model mice, achieving supraphysiological level expression in liver, heart and spleen, and a significant increase in brain SGSH activity (fig. 5B,. p < 0.01).

In addition, to evaluate the therapeutic effect of systemic administration in MPS IIIA model mice, the degradation of substrate GAGs after injection of AAV9-CB-hSGSH1, AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 was further analyzed. The results show that the degradation degree of GAG and the increase degree of SGSH enzyme activity are in the same trend. This means that the higher the recovery of SGSH activity level, the greater the decrease in GAG accumulation that follows. Compared with untreated model group mice, the accumulation of GAG in the tested tissues of 3 treatment groups of AAV9-CB-hSGSH1, AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 is relieved, wherein the GAG in the liver of 3 treatment groups of mice is reduced to WT level, but is not shown in other tested tissues, and the virus with relatively best treatment effect is still AAV9-CB-hSGSH 1.

The results showed that there was a significant reduction in GAG content in liver, spleen, heart, kidney and lung tissues of MPS IIIA mice treated with AAV 9-CB-hsgshgsh 1. Wherein, after treatment with AAV9-CB-hSGSH1, the GAG content in the liver of MPS IIIA mice decreased to 10.02% of untreated groups (figure 6, p <0.001), which was lower than 10.96% and 15.87% after treatment with AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH 1; after treatment with AAV9-CB-hSGSH1, MPS IIIA mice had GAG levels in the spleen reduced to 5.56% of untreated group (fig. 6, # p <0.05), in the heart reduced to 27.09% of untreated group (fig. 6, # p <0.05), in the kidney reduced to 20.30% of untreated group (fig. 6, # p <0.05), and in the lung reduced to 51.56% of untreated group. The GAG content in spleen, heart, kidney and lung tissues of MPS IIIA mice treated with AAV9-MF-hSGSH1 decreased to 17.73%, 18.05%, 22.47% and 39.46% of untreated groups, respectively. MPS IIIA mice treated with AAV9-MF1-hSGSH1 showed no significant decrease in GAG content in spleen, heart and kidney tissues, respectively, 86.56%, 40.07% and 71.16%, and no decrease in GAG content in lung was observed. Remarkably, GAG levels in the brain of mice treated with AAV9-CB-hSGSH1 decreased to 25.32% of untreated levels (figure 6, p <0.01), reaching normal physiologic levels, and the AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 treated groups only decreased to 66.55% and 50.79% of untreated levels. In contrast, the therapeutic effect of AAV9-CB-hSGSH1 virus is most significant in three.

In conclusion, compared with untreated model mice, the AAV9-CB-hSGSH1, AAV9-MF-hSGSH1 and AAV9-MF1-hSGSH1 treatment groups have increased in vivo SGSH activity level to a certain extent, realize physiological level or supraphysiological level expression in detected main tissues and organs, and improve expression in brain. After treatment, the accumulation of GAG in the model mouse is improved to a certain degree, and the treatment effect is most remarkable in the liver.

It is noteworthy that although the activity of SGSH enzyme in AAV9-CB-hSGSH1 treated mice was only recovered to 1.80 times that of MPS IIIA model mice, the accumulation of substrate GAG in the treated mice brain was significantly reduced to 25.32% of untreated MPS IIIA model mice, indicating that AAV-CB-hSGSH 1-mediated distribution of SGSH in brain was uniform, and due to the cross-correction effect in the mice brain, the mature SGSH after secretion to the extracellular compartment could be absorbed by the adjacent cells through endocytosis via M6P receptor, so the recovery of less SGSH enzyme activity in the mice brain could lead to a greater degree of improvement in substrate accumulation. AAV9-CB-hSGSH1 has obvious curative recovery effect on MPS IIIA model mice.

Furthermore, in the liver, the activity of AAV9-CB-hSGSH1 and AAV9-MF1-hSGSH1 treatment group SGSH enzyme was restored to 1942.52-fold and 236.93-fold, respectively, that of MPS IIIA mice. In the brain, the SGSH enzyme activity of AAV9-CB-hSGSH1 and AAV9-MF1-hSGSH1 treated groups was restored to 1.80 and 1.54 times (p <0.05, p <0.01) that of MPS IIIA model mice, respectively. Although there was a nearly 10-fold difference in SGSH activity between the two treatment groups in the liver, the difference in brain was not obvious, suggesting that the restoration of SGSH expression in the central nervous system brain is not clearly related to somatic SGSH expression. Only 0.3 fold increased SGSH activity, an approximately 20% difference in degradation of GAGs. Further illustrating the improvement of the cross-correcting effect of SGSH well distributed in the brain.

Treatment of MPS IIIA mice with AAV9-CB/-MF2/-MF3/-MF4/-MF5-hSGSH1 vectors

The study was followed with a second panel of screens. Similarly, a 2-month-old male MPS IIIA model mouse was systemically administered at a dose of 5E13 vg/kg by tail vein injection, and a first study was conducted to screen 5 treatment groups of superior AAV9-CB-hSGSH1(n ═ 3), AAV9-MF2-hSGSH1(n ═ 3), AAV9-MF3-hSGSH1(n ═ 3), AAV9-MF4-hSGSH1(n ═ 3), and AAV9-MF5-hSGSH1(n ═ 3), and an untreated MPS IIIA model mouse (n ═ 3) of the same age and a WT mouse (n ═ 3) of the same age were used as controls. Orbital bleeds were taken 3, 7, 14, 21, 28 and 56 days after virus injection and plasma was isolated for SGSH enzyme activity assay. Mice were sacrificed 2 months after dosing and various tissues and organs of the mice were taken for analytical examination.

Plasma SGSH enzyme activity results showed that 7 days after AAV virus injection, SGSH activity reached peak 1 in the plasma of 4 other treatment groups MPS IIIA mice, except AAV9-MF 5-hsgssh 1, and decreased slightly over the next week. It can be seen that at peak 1, the recovery effect of AAV9-CB-hSGSH1 and AAV9-MF2-hSGSH1 treatment groups on SGSH activity in MPS IIIA mouse plasma was most significant. However, two weeks after virus injection, the enzymatic activity in the plasma of MPS IIIA mice in the AAV9-MF2-hSGSH1 treatment group decreased to a very low level and remained at a lower level for a later period of time (fig. 7A). AAV9-MF3-hSGSH1 and AAV9-MF4-hSGSH1 treatment group MPS IIIA mice showed an increase in plasma enzyme activity of SGSH at 7 days post-treatment, but did not return to the normal physiological level of WT mice, whereas later the SGSH enzyme activity gradually decreased to a lower value in the plasma of AAV9-MF4-hSGSH1 treatment group MPS IIIA mice (fig. 7A).

In these treatment groups, the inventors surprisingly found that the SGSH enzyme activity in the plasma of the AAV9-CB-hSGSH 1-treated MPS IIIA mice, although slightly decreased at 14 days of injection, possessed a gradual recovery of SGSH enzyme activity over the next three weeks and reached the 2 nd expression peak at 42 days after injection, achieving supra-physiological levels of SGSH expression in the plasma; furthermore, although the SGSH enzyme activity in the plasma of AAV9-MF3-hSGSH 1-treated MPS IIIA mice did not return to normal physiological levels 7 days after treatment, the SGSH enzyme activity in the plasma was steadily increased over the next five weeks of treatment and gradually approached normal physiological levels (fig. 7A). That is, at 56 days post-injection, SGSH enzyme activity was still at higher expression levels in plasma of AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 treated MPS IIIA mice, reaching 366.97-fold and 249.53-fold, respectively, of untreated MPS IIIA mice (fig. 7A).

Overall, AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 viruses have the potential to maintain plasma SGSH enzyme activity in MPS IIIA mice at physiological and even supra-physiological levels for extended periods of time, indicating that the therapeutic effects of both are more effective and stable compared to other therapeutic viral vectors. In contrast, no restoration of SGSH enzyme activity was observed in the plasma of AAV9-MF 5-hsgssh 1-treated MPS IIIA mice, suggesting to some extent that its SGSH expression on tissues was also unsatisfactory.

SGSH enzyme activity analysis in tissues shows that at 56 days after injection, the SGSH enzyme activity of 5 treatment groups of AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 in the liver of MPS IIIA mice is obviously improved (figure 7B, p is less than 0.05), the liver of the MPS IIIA mice is respectively recovered to 316.96 times, 127.60 times, 324.47 times, 287.50 times and 107.32 times of untreated MPS IIIA mice, wherein, the liver of mice treated by AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 has the most recovered SGSH enzyme activity, the treated group by AAV9-MF4-hSGSH1 has the next less, and the treated groups by AAV9-MF2-hSGSH1 and AAV9-MF5-hSGSH1 have less, however, the SGSH enzyme activity of all 5 treatment groups achieved a supraphysiological level of expression in liver, and SGSH expression in liver was consistent with that in peripheral plasma.

In brain, the activity of MPS 9-CB-hSGSH1 and AAV9-MF3-hSGSH1 treated group MPS IIIA model mouse SGSH was restored compared to untreated MPS IIIA model mouse, rising to 1.86 and 1.31 times (p <0.05) of untreated group MPS IIIA model mouse, respectively, 38.68% and 27.36% of WT mice. Unfortunately, no difference in SGSH in the brains of the 3 treated groups of mice was observed for the treated groups AAV9-MF2-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH 1.

In the spleen, it was observed that AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 treated MPS IIIA model mouse SGSH enzyme activity achieved near physiological levels of expression, returning to 42.66-fold and 40.74-fold, respectively, in the untreated model group, and that AAV9-MF2-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 treated SGSH enzyme activity was also elevated in the spleen compared to the untreated group, 4.84-fold, 12.70-fold and 12.51-fold, respectively (fig. 7B,. xp < 0.001). In the heart, the SGSH enzyme activities of mice in the treated groups of AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1 and AAV9-MF3-hSGSH1 were recovered to a large extent, and were increased to 165.56, 258.48 and 294.55 times of those in the untreated model group in turn (FIG. 7B, p <0.05), and the expression at a supraphysiological level was achieved. Similarly, in the kidney, it was found that SGSH enzyme activity was significantly elevated to 15.86, 11.36 and 15.42 fold in AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1 and AAV9-MF3-hSGSH1 treatment group mice (fig. 7B, <0.05) but did not achieve physiological levels of expression equivalent to WT mice. In the lung, AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, and AAV9-MF4-hSGSH1 mediated SGSH expression was elevated 1.5-5 fold over untreated groups (fig. 7B, p < 0.05). In muscle tissue, AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1, AAV9-MF5-hSGSH15 treatment groups restored SGSH enzyme activity to 6.65-fold, 11.02-fold, 11.61-fold, 1.84-fold, and 2.91-fold of the untreated group, with AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1 treatment groups achieving near physiological levels of expression (fig. 7B).

However, in heart, kidney and muscle tissues, SGSH activity was not effectively restored in mice treated with AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 compared to untreated model groups. Furthermore, in lung and kidney tissues, SGSH enzyme activity was slightly elevated in 5 treated mice compared to untreated mice, but was far from normal physiological levels (fig. 7B).

Next, to assess the effect of the therapeutic vector on lysosomal pathology as well, the study was conducted to quantify GAG content in harvested mouse tissues. The results showed that AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1, AAV9-MF5-hSGSH15 treated MPS IIIA mice had significantly reduced GAG content in liver, spleen and heart tissue compared to untreated MPS IIIA mice (fig. 8A, p <0.001, p <0.01, p <0.05), GAG content in liver was even lower than WT levels, MPS decreased to 11.36%, 12.48%, 7.99%, 8.52%, 9.38% of untreated MPS IIIA mice, respectively. The GAG content in the spleen of the mice in the 5 treatment groups is reduced to 15.70% -22.84% of that in the untreated group, and the GAG content in the heart is reduced to 7.86% -25.64%. In lung tissue, GAG content was reduced in 5 treated MPS IIIA mice compared to untreated mice to 33.11%, 54.30%, 45.02%, 47.10%, 49.59% respectively, but not to WT levels, with significant differences in data between AAV9-CB-hSGSH1 treated and untreated mice (figure 8A, p < 0.05). In kidney and muscle tissue, the GAG content in the treated MPS IIIA mice was slightly reduced compared to the untreated group, and the lysosomal pathology in kidney and muscle tissue was not effectively corrected in the remaining 4 treatment groups, except that the GAG content in the AAV9-MF3-hSGSH1 treatment group was reduced in muscle to 32.60% of the untreated mice.

The GAG content in the brain showed that the effect of the treatment groups AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1 was significant in improving GAG accumulation, and the GAG content in the brain of treated MPS IIIA mice decreased to 24.58% (fig. 8A, < p <0.01), 37.57% (fig. 8A, < p <0.01) and 26.95% (p <0.001), respectively, of untreated MPS IIIA mice, and there was no significant difference from WT mice (fig. 8A, < p <0.05), and the therapeutic effect could achieve normal physiological recovery. Due to the fact that the difference of the brain SGSH content of the MPS IIIA mice in the AAV9-MF3-hSGSH1 treatment group after treatment is small, analysis shows that the AAV9-MF3-hSGSH1 treatment vector has more stable and remarkable relieving and treating effects on brain lysosome pathology of the MPS IIIA mice. However, GAG content was not significantly reduced in the other 2 treatment groups, AAV9-MF4-hSGSH1, AAV9-MF5-hSGSH1, compared to the untreated group (fig. 8A).

In addition, another clinically common test index, namely the HS content in urine, was evaluated for the accumulation of mucopolysaccharides. After 56 days of MPS IIIA mice receiving treatment, the mouse urine was collected and HS content in the urine was quantified by LC-MS/MS. The results showed that HS accumulation in urine was significantly reduced in 5 treated MPS IIIA mice compared to untreated MPS IIIA mice (figure 8B, p <0.05, p <0.001), and HS levels in urine of AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 treated mice had approached normal physiological levels. The results show that the therapeutic carrier has certain therapeutic effect on mucopolysaccharide accumulation diseases in organisms.

In conclusion, MPS IIIA mice treated by AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH1, AAV9-MF4-hSGSH1 and AAV9-MF5-hSGSH1 virus vectors have relieved central nervous system and somatic diseases to different degrees at 56 days after treatment, which shows that HS accumulated in urine is effectively cleared, and SGSH expression mediated by AAV vectors in liver is obviously improved, so that lysosome pathology in liver is effectively relieved. However, in other tissues and organs, the level of SGSH expression mediated by different AAV vectors varies, resulting in different degrees of reduction of GAG accumulation. Overall evaluation of these 5 treatment groups, GAG degradation was comparable in liver and spleen tissues, although different AAV vectors mediated different degrees of SGSH expression. In kidney and lung tissues, the curative effect is not obvious, and no AAV vector with outstanding advantages exists. The 3 therapeutic groups with higher SGSH expression levels in heart and muscle tissues were AAV9-CB-hSGSH1, AAV9-MF2-hSGSH1, AAV9-MF3-hSGSH 1.

Notably, AAV 9-CB-hsgssh 1 and AAV9-MF 3-hsgssh 1 viral vectors mediated SGSH expression in the central nervous system was superior to other groups, and this advantage was amplified in terms of clearance of accumulated GAG in brain tissue by cross-correction effect, i.e. although SGSH expression in AAV 9-CB-hsgssh 1 and AAV9-MF 3-hsgssh 1 treated groups only rose in brain up to 1.86 and 1.31 times that of untreated groups, 75% and 73% of GAG clearance could already be achieved, indicating that the treatment effect of AAV9-MF3-hSGSH1 treated groups in the central nervous system was more stable and could overcome individual differences to some extent. In conclusion, the AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 vectors are more effective in the treatment of central nervous system disorders.

Example 6 therapeutic Effect study in mice with advanced disease

To evaluate the therapeutic effect of AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 viral vector having therapeutic potential for central nervous system disorders screened in this study in model mice at later stage of disease progression, 5E13 vg/kg of viral vector was injected into 7-month-old MPS IIIA male mice by tail vein injection, 2 treatment groups were set up in total, which were AAV9-CB-hSGSH1 treatment group (n ═ 3) and AAV9-MF3-hSGSH1 treatment group (n ═ 3), respectively, and untreated MPS IIIA male mice of the same age (n ═ 3) and WT male mice of the same strain of the same age (n ═ 3) were used as controls. After 5 months of long-term treatment, MPS IIIA mice were assessed for memory and learning by behavioral experiments; evaluating improvement condition of lysosome pathology in brain by mouse brain sagittal section LAMP1 immunofluorescence staining; and assessing the improvement of neuroinflammation in the brain by GFAP immunofluorescence staining of mouse sagittal brain sections to determine whether administration at late stages of disease progression improves even reversing severe pathophysiological phenotypes to some extent.

Improvement of learning and memory ability of post-aged MPS IIIA mice

At 5 months post-dose, i.e., 12 months of age of the mice, the memory and learning capacity of MPS IIIA mice was assessed by the behavioural test baynes maze. In the first 4 days, four rounds of training are performed on the mouse every day, the mouse is trained to find the escape capsule located in the target quadrant, and the time for finding the escape capsule and the failure times of the mouse are counted. The time that the mice were in the target quadrant on the platform within 90s was then counted at day 5 and day 12, respectively, showing the learning and memory abilities of the mice, respectively.

Statistical results show that the incubation period of the untreated MPS IIIA mouse searching for the escape compartment is significantly longer than that of the WT mouse within 4 days of training, while the number of false searches is more than that of the WT mouse, while the incubation period of the MPS IIIA mouse treated by AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 viral vectors is shorter than that of the untreated MPS IIIA mouse, and the number of false searches is reduced, showing a WT-like state (less fluctuation in the number of false searches), indicating that the learning ability of the MPS IIIA mouse is improved after treatment (fig. 9A).

On

day

5, 4 groups of mice were tested and the residence time in the target quadrant was counted in 90s for mice, it can be seen that AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 treated MPS IIIA mice stayed significantly higher in the target quadrant than untreated MPS IIIA mice, 2.17 and 2.09 times higher (fig. 9B,. p < 0.059), respectively, and approached WT levels; meanwhile, the movement locus of the mice also shows the unintended movement state of the MPS IIIA mice, and the MPS IIIA mice and WT mice after treatment have more movement characteristics towards the target quadrant (figure 9C), which shows that the impaired learning ability of the MPS IIIA aged mice after 5 months of carrier drug treatment is normalized. The results of the testing of the mice at day 12 showed that the residence time of AAV 9-CB-hsgssh 1 treated MPS IIIA mice in the target quadrant was extended to 1.36 times that of untreated MPS IIIA mice (fig. 9B, <0.05), and AAV9-MF 3-hsgssh 1 treated MPS IIIA mice in the target quadrant was extended to 1.92 times that of untreated MPS IIIA mice. The trajectory of the mice on the platform also showed that the treated MPS IIIA mice tended to move within the target quadrant more than untreated MPS IIIA mice (fig. 9C), indicating that the memory capacity of the treated MPS IIIA mice was also improved.

The data show that the learning and memory abilities of the aged MPS IIIA mice are reduced to a certain extent and are shown as degenerative behavioral symptoms, after the aged MPS IIIA mice are treated by AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 virus vectors, the severely damaged learning and memory abilities of the aged MPS IIIA mice are recovered, and the 2 therapeutic virus vectors have the capacity of sufficiently improving the MPS IIIA aged mouse behavioral abnormalities, so that the development process of diseases can be effectively delayed.

Improvement of post-treatment MPS IIIA mouse intracerebral lysosomal pathology

To more specifically assess the clearance effect of AAV viral vectors on lysosomal storage disorders in aged MPS IIIA mice, LAMP1 was commonly used to indicate endosome/lysosomal membrane content by immunofluorescence staining of LAMP1 in sagittal sections of aged MPS IIIA mice 5 months after treatment. The results show that in different brain regions, untreated MPS IIIA mice had a significant increase in LAMP1 positive signal compared to WT mice, indicating that they had lysosomal GAG-stored lesions; the positive signal of MPS IIIA mouse LAMP1 was correspondingly reduced to a different extent after viral vector treatment compared to untreated MPS IIIA mice.

Specifically, it can be seen that, in cells in Cerebral cortex (CBC), Thalamus (thaalamus, TH) and Striatum (stritum, STR) regions, the positive signals for MPS IIIA mice LAMP1 in AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 treated groups were significantly less than those in untreated MPS IIIA mice (fig. 10), and the positive signals for MPS IIIA mice LAMP1 in AAV9-MF3-hSGSH1 treated group were less than those in AAV9-CB-hSGSH1 treated group, indicating that lysosomal storage in the Cerebral cortex, Thalamus and Striatum regions was improved, and the improved effect of AAV9-MF3-hSGSH1 treated group was better. Furthermore, in the medullary region of Hippocampus (hipppocampus, HP) and Cerebellum (Cerebellum, CB), MPS IIIA mice LAMP1 positive signal treated via AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 viral vectors was also significantly less than untreated MPS IIIA mice (fig. 10), whereas in the purkinje cell layer of the Cerebellum region (purkinje cells are known to be the only efferent neurons of cerebellar cortex, critical for the motor coordination ability of the individual), no reduction in MPS IIIA mice LAMP1 positive signal after treatment was found compared to untreated mice (fig. 10).

Overall, the post-treatment MPS IIIA aged mice had significantly reduced intracerebral LAMP1 signals compared to untreated MPS IIIA aged mice, suggesting that its intracerebral lysosomal pathology was corrected, and that in certain brain regions, such as the cerebral cortex, thalamus and striatal regions, the AAV9-MF3-hSGSH1 viral vector had a stronger corrective clearance of lysosomal storage pathology than the AAV9-CB-hSGSH1 viral vector.

Improvement of treating intracerebral neuroinflammation of aged MPS IIIA mice

In disease progression of MPS IIIA, astrocytosis is activated due to massive accumulation of HS in the nervous system, causing astrocytosis, resulting in the development of neuroinflammation, and Glial Fibrillary Acidic Protein (GFAP) is a marker of astrocytes. Thus, in order to visually demonstrate the relief of neuroinflammation in the brain of aged MPS IIIA mice by AAV9-CB-hSGSH1 and AAV9-MF3-hSGSH1 viral vectors after 5 months of treatment, immunofluorescent staining of GFAP in sagittal sections of the brain of aged MPS IIIA mice was performed in this study.

The results show that in several brain regions tested, the positive GFAP signals of MPS IIIA aged mice treated with AAV 9-CB-hsgssh 1 and AAV9-MF 3-hsgssh 1 were all weaker than untreated MPS IIIA aged mice (fig. 11), demonstrating that the therapeutic viral vector effectively ameliorated the conditions of astrocytosis in MPS IIIA aged mice, with the ability to ameliorate neuroinflammation in the central nervous system of model mice. Specifically, in cerebral cortex, thalamus, striatum, hippocampus and Medulla oblongata (MY) regions, the positive signal for GFAP was significantly reduced in 2 groups of treated MPS IIIA aged mice compared to untreated MPS IIIA aged mice (fig. 11). The immunofluorescent staining results for GFAP showed substantially similar results to the LAMP1 immunofluorescent staining. MPS IIIA aged mice treated by the AAV9-MF3-hSGSH1 vector have less GFAP positive signal intensity in cerebral cortex, thalamus, striatum and medulla oblongata regions than MPS IIIA aged mice treated by the AAV9-CB-hSGSH1, which shows that the AAV9-MF3-hSGSH1 viral vector can more effectively treat the situation of astrocytosis in the brain of the MPS IIIA aged mice and relieve neuroinflammation than the AAV9-CB-hSGSH1 viral vector; in addition, in the hippocampal region, different from the similar level correction effect of two virus vectors on lysosome pathology, the number of GFAP positive cells of MPS IIIA aged mice treated by AAV9-MF3-hSGSH1 vector is slightly less than that of AAV9-CB-hSGSH1 vector, and a better treatment effect is presented; furthermore, the positive signal intensity of GFAP did not differ significantly between the treated and untreated groups in the purkinje cell layer and medulla of the cerebellar region (fig. 11).

In summary, AAV9-MF3-hSGSH1 and AAV9-CB-hSGSH1 therapeutic vectors were able to penetrate the BBB of 7-month old MPS IIIA aged male mice and widely express functional SGSH in the brain, reduce GAG accumulation in multiple brain cells, correct lysosomal accumulation pathology, and effectively reduce neuroinflammatory responses induced by abnormal astrocytes.

All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated by reference herein in their entirety.

While the disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure taken in conjunction with the specific embodiments thereof, and that the specific embodiments of the disclosure are not intended to be limiting. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the disclosure.

Sequence listing

<110> Shanghai baren Biotech Co., Ltd

<120> transgenic expression cassette for treatment of mucopolysaccharidosis type IIIA

<130> PCNCNN223719G

<160> 21

<170> PatentIn version 3.5

<210> 1

<211> 1515

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of hSGSH1

<400> 1

gccaccatga gctgtcctgt gcctgcctgt tgtgctctgc tgctggttct gggcctgtgc 60

agagccagac ctagaaatgc actgctcctg ctggctgatg atggtggctt tgagtctggg 120

gcctacaaca actctgccat tgccacacct cacctggatg ccctggctag aagaagcctg 180

ctgttcagaa atgccttcac ctctgtgtcc agctgcagcc cttctagagc cagcctgctg 240

acaggactgc cccagcatca gaatgggatg tatggcctgc accaggatgt gcaccacttc 300

aacagctttg acaaagtcag atccctgcca ctgctgctga gccaggctgg tgttagaaca 360

ggcatcattg gcaagaaaca tgtgggccct gagacagtgt acccctttga ctttgcctac 420

actgaagaga atggctctgt gctgcaagtg ggcagaaaca tcaccaggat caagctgctt 480

gtcagaaagt tcctgcagac ccaggatgac agacccttct tcctgtatgt ggccttccat 540

gatcctcaca gatgtggcca ctctcagccc cagtatggca ccttctgtga aaagtttgga 600

aatggagagt ctggcatggg cagaatccct gactggaccc ctcaggccta tgatcccctg 660

gatgtgctgg tgccctactt tgtgcccaac acaccagctg ccagagctga tctggctgcc 720

cagtacacca cagtgggaag aatggaccag ggtgttggcc tggtcctgca agagcttaga 780

gatgcagggg tgctgaatga taccctggtc atctttacct ctgacaatgg catccccttt 840

ccaagtggca ggaccaacct gtactggcct ggaacagctg agcccctgct ggtgtctagc 900

cctgagcacc ctaagagatg gggccaagtg tctgaggcct atgtgtccct gctggatctg 960

acccctacca tcctggactg gttcagcatc ccctatccta gctatgccat ctttggcagc 1020

aagaccatcc acctgacagg cagaagtctg ctgcctgctc tggaagctga acccctgtgg 1080

gccacagtgt ttggcagcca gtctcaccat gaagtgacaa tgagctaccc catgagaagt 1140

gtgcagcata gacacttcag actggtccac aacctgaact tcaagatgcc ttttccaatt 1200

gaccaggact tctatgtctc cccaaccttc caggacctgc tgaacagaac cactgcaggc 1260

cagcctacag gctggtacaa ggacctgaga cactactact atagggccag atgggagctg 1320

tatgacagat ccagagatcc tcatgagaca cagaacctgg ccacagatcc cagatttgcc 1380

cagctgctgg aaatgctgag agatcagctg gccaaatggc agtgggagac acatgaccct 1440

tgggtctgcg ctcctgatgg tgttctggaa gagaagctgt cccctcagtg ccagcctctg 1500

cacaatgaac tgtga 1515

<210> 2

<211> 1515

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of hSGSH2

<400> 2

gccaccatgt cttgtcctgt tccagcttgt tgtgccctgc tgctggtgct gggactgtgt 60

agagccagac ctagaaatgc tctgctgctc ctggctgatg atggtggctt tgagtctggg 120

gcctacaaca actctgccat tgccacacct cacctggatg ccctggccag aagaagcctg 180

ctgttcagaa atgccttcac ctctgtgtcc agctgcagcc cttctagagc cagcctgctg 240

acaggactgc ctcagcacca gaatgggatg tatggcctgc accaggatgt gcaccacttc 300

aacagctttg acaaagtcag atccctgcct ctgctcctga gccaggctgg tgttagaaca 360

ggcatcattg gcaagaaaca tgtgggccct gagacagtgt acccctttga ctttgcctac 420

actgaagaga atggctctgt gctgcaagtg ggcagaaaca tcaccaggat caagctgctt 480

gtcagaaagt tcctgcagac ccaggatgac agacccttct tcctgtatgt ggccttccat 540

gatcctcaca gatgtggcca ctctcagccc cagtatggca ccttctgtga aaagtttgga 600

aatggagagt ctggcatggg cagaatccct gactggaccc ctcaggccta tgatcccctg 660

gatgtgctgg tgccctactt tgtgcccaac acaccagctg ccagagctga tctggctgcc 720

cagtacacca cagtgggaag aatggaccag ggtgttggcc tggtcctgca agagcttaga 780

gatgctgggg tgctgaatga taccctggtc atctttacct ctgacaatgg catccccttt 840

ccaagtggca ggaccaacct gtactggcct ggaacagctg agcccctgct ggtgtctagc 900

cctgagcacc ctaagagatg gggccaagtg tctgaggcct atgtgtccct gctggatctg 960

acccctacca tcctggactg gttcagcatc ccctatccta gctatgccat ctttggcagc 1020

aagaccatcc acctgacagg cagatctctg ctgcctgctc tggaagctga acccctgtgg 1080

gccacagtgt ttggcagcca gtctcaccat gaagtgacca tgagctaccc catgagatct 1140

gtgcagcata gacacttcag actggtccac aacctgaact tcaagatgcc ttttccaatt 1200

gaccaggact tctatgtctc cccaaccttc caggacctgc tgaacagaac cacagcaggc 1260

cagcctacag gctggtacaa ggacctgaga cactactact acagagccag atgggagctg 1320

tatgacagat ccagagatcc tcatgagaca cagaacctgg ccacagatcc cagatttgct 1380

cagctgctgg aaatgctgag ggaccagctg gccaaatggc agtgggagac acatgaccct 1440

tgggtctgcg ctcctgatgg tgttctggaa gagaagctgt cccctcagtg ccagcctctg 1500

cacaatgagc tgtga 1515

<210> 3

<211> 620

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CB promoter

<400> 3

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc ccgcccattg 60

acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 120

tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 180

agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 240

atgaccttat gggactttcc tacttggcag tacatctact cgaggccacg ttctgcttca 300

ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat 360

tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg 420

gcgaggggcg gggcggggcg aggcggaaag gtgcggcggc agccaatcaa agcggcgcgc 480

tccgaaagtt tccttttatg gcaaggcggc ggcggcggcg gccctataaa aagcaaaccg 540

cgcggcgggc gggagcggga tcagccaccg cggtggcggc ctaaagtcga cgaggaactg 600

aaaaaccaga aagttaactg 620

<210> 4

<211> 631

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MF promoter

<400> 4

cccttcagat taaaaataac tgaggtaagg gcctgggtag gggaggtggt gtgagacgct 60

cctgtctctc ctctatctgc ccatcggccc tttggggagg aggaatgtgc ccaaggacta 120

aaaaaaggcc atggagccag aggggcgagg gcaacagacc tttcatgggc aaaccttggg 180

gccctgctgt gcagagggcc ctgcgtatga gtgcaagtgg gttttaggac caggatgagg 240

cggggtgggg gtgcctacct gacgaccgac cccgacccac tggacaagca cccaaccccc 300

attccccaaa ttgcgcatcc cctatcagag agggggaggg gaaacaggat gcggcgaggc 360

gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg 420

cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact 480

ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg gcccagccgg accgcaccac 540

gcgaggcgcg agataggggg gcacgggcgc gaccatctgc gctgcggcgc cggcgactca 600

gcgctgcctc agtctgcggt gggcagcgga g 631

<210> 5

<211> 864

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MF1 promoter

<400> 5

cccttcagat taaaaataac tgaggtaagg gcctgggtag gggaggtggt gtgagacgct 60

cctgtctctc ctctatctgc ccatcggccc tttggggagg aggaatgtgc ccaaggacta 120

aaaaaaggcc atggagccag aggggcgagg gcaacagacc tttcatgggc aaaccttggg 180

gccctgctgc tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag 240

gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc 300

cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg 360

cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc 420

gctgcagagg gccctgcgta tgagtgcaag tgggttttag gaccaggatg aggcggggtg 480

ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc cccattcccc 540

aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg 600

cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc gcgcgccacc 660

gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa actccccttc 720

ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc 780

gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc 840

ctcagtctgc ggtgggcagc ggag 864

<210> 6

<211> 865

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MF2 promoter

<400> 6

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc ccgcccattg 60

acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 120

tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 180

agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 240

atgaccttat gggactttcc tacttggcag tacatctaca ccggtctgca gagggccctg 300

cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac 360

gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct 420

atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc 480

accgcggaca gtgccttcgc ccccgcctgg cggcgcgcaa gctttcgagg ccacgttctg 540

cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta 600

attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg 660

gcggggcgag gggcggggcg gggcgaggcg gaaaggtgcg gcggcagcca atcaaagcgg 720

cgcgctccga aagtttcctt ttatggcaag gcggcggcgg cggcggccct ataaaaagca 780

aaccgcgcgg cgggcgggag cgggatcagc caccgcggtg gcggcctaaa gtcgacgagg 840

aactgaaaaa ccagaaagtt aactg 865

<210> 7

<211> 853

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MF3 promoter

<400> 7

ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60

gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120

aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180

actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgccgttaca 240

taacttacgg taaatggccc gcctggctga ccgcccaacg accccgccca ttgacgtcaa 300

taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 360

agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 420

cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 480

tatgggactt tcctacttgg cagtacatct actcgaggcc acgttctgct tcactctccc 540

catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc 600

agcgatgggg gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg 660

gcggggcggg gcgaggcgga aaggtgcggc ggcagccaat caaagcggcg cgctccgaaa 720

gtttcctttt atggcaaggc ggcggcggcg gcggccctat aaaaagcaaa ccgcgcggcg 780

ggcgggagcg ggatcagcca ccgcggtggc ggcctaaagt cgacgaggaa ctgaaaaacc 840

agaaagttaa ctg 853

<210> 8

<211> 825

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MF4 promoter

<400> 8

aggttaattt ttaaaaagca gtcaaaagtc caagtggccc ttggcagcat ttactctctc 60

tgtttactct ggttaataat ctcaggagta caaacattcc agatccaggt taatttttaa 120

aaaaagagtg ctctagtttt gcaatacagg ctgcagaggg ccctgcgtat gagtgcaagt 180

gggttttagg accaggatga ggcggggtgg gggtgcctac ctgacgaccg accccgaccc 240

actggacaag cacccaaccc ccattcccca aattgcgcat cccctatcag agagggggag 300

gggaaacagg atgcggcgag gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc 360

ttcgcccccg cctggcggcg cgctgcagag ggccctgcgt atgagtgcaa gtgggtttta 420

ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac ccactggaca 480

agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg aggggaaaca 540

ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg ccttcgcccc 600

cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg tcactcgccg 660

gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag 720

ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg 780

gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggag 825

<210> 9

<211> 736

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MF5 promoter

<400> 9

aggttaattt ttaaaaagca gtcaaaagtc caagtggccc ttggcagcat ttactctctc 60

tgtttactct ggttaataat ctcaggagta caaacattcc agatccaggt taatttttaa 120

aaaaagagtg ctctagtttt gcaatacagg accggtctgc agagggccct gcgtatgagt 180

gcaagtgggt tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc 240

cgacccactg gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag 300

ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac 360

agtgccttcg cccccgcctg gcggcgcgca agctttcgag gccacgttct gcttcactct 420

ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 480

tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga 540

ggggcggggc ggggcgaggc ggaaaggtgc ggcggcagcc aatcaaagcg gcgcgctccg 600

aaagtttcct tttatggcaa ggcggcggcg gcggcggccc tataaaaagc aaaccgcgcg 660

gcgggcggga gcgggatcag ccaccgcggt ggcggcctaa agtcgacgag gaactgaaaa 720

accagaaagt taactg 736

<210> 10

<211> 1509

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of original hSGSH

<400> 10

atgagctgcc ccgtgcccgc ctgctgcgcg ctgctgctag tcctggggct ctgccgggcg 60

cgtccccgga acgcactgct gctcctcgcg gatgacggag gctttgagag tggcgcgtac 120

aacaacagcg ccatcgccac cccgcacctg gacgccttgg cccgccgcag cctcctcttt 180

cgcaatgcct tcacctcggt cagcagctgc tctcccagcc gcgccagcct cctcactggc 240

ctgccccagc atcagaatgg gatgtacggg ctgcaccagg acgtgcacca cttcaactcc 300

ttcgacaagg tgcggagcct gccgctgctg ctcagccaag ctggtgtgcg cacaggcatc 360

atcgggaaga agcacgtggg gccggagacc gtgtacccgt ttgactttgc gtacacggag 420

gagaatggct ccgtcctcca ggtggggcgg aacatcacta gaattaagct gctcgtccgg 480

aaattcctgc agactcagga tgaccggcct ttcttcctct acgtcgcctt ccacgacccc 540

caccgctgtg ggcactccca gccccagtac ggaaccttct gtgagaagtt tggcaacgga 600

gagagcggca tgggtcgtat cccagactgg accccccagg cctacgaccc actggacgtg 660

ctggtgcctt acttcgtccc caacaccccg gcagcccgag ccgacctggc cgctcagtac 720

accaccgtcg gccgcatgga ccaaggagtt ggactggtgc tccaggagct gcgtgacgcc 780

ggtgtcctga acgacacact ggtgatcttc acgtccgaca acgggatccc cttccccagc 840

ggcaggacca acctgtactg gccgggcact gctgaaccct tactggtgtc atccccggag 900

cacccaaaac gctggggcca agtcagcgag gcctacgtga gcctcctaga cctcacgccc 960

accatcttgg attggttctc gatcccgtac cccagctacg ccatctttgg ctcgaagacc 1020

atccacctca ctggccggtc cctcctgccg gcgctggagg ccgagcccct ctgggccacc 1080

gtctttggca gccagagcca ccacgaggtc accatgtcct accccatgcg ctccgtgcag 1140

caccggcact tccgcctcgt gcacaacctc aacttcaaga tgccctttcc catcgaccag 1200

gacttctacg tctcacccac cttccaggac ctcctgaacc gcaccacagc tggtcagccc 1260

acgggctggt acaaggacct ccgtcattac tactaccggg cgcgctggga gctctacgac 1320

cggagccggg acccccacga gacccagaac ctggccaccg acccgcgctt tgctcagctt 1380

ctggagatgc ttcgggacca gctggccaag tggcagtggg agacccacga cccctgggtg 1440

tgcgcccccg acggcgtcct ggaggagaag ctctctcccc agtgccagcc cctccacaat 1500

gagctgtga 1509

<210> 11

<211> 97

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SV40 intron

<400> 11

gtaagttaag tctttttgtc ttttatttca ggtcccggat ccggtggtgg tgcaaatcaa 60

agaactgctc ctcagtggat gttgccttta cttctag 97

<210> 12

<211> 109

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> left ITR

<400> 12

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggcca 109

<210> 13

<211> 145

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Right ITR

<400> 13

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ccttaattaa cctaa 145

<210> 14

<211> 208

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> bGH PolyA

<400> 14

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagacaa tagcaggcat gctgggga 208

<210> 15

<211> 2934

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAAV2.1-CB-SV40-hSGSH1

<400> 15

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg tacctctggt cgttacataa cttacggtaa 240

atggcccgcc tggctgaccg cccaacgacc ccgcccattg acgtcaataa tgacgtatgt 300

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 420

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 480

tacttggcag tacatctact cgaggccacg ttctgcttca ctctccccat ctcccccccc 540

tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg 600

gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg 660

aggcggaaag gtgcggcggc agccaatcaa agcggcgcgc tccgaaagtt tccttttatg 720

gcaaggcggc ggcggcggcg gccctataaa aagcaaaccg cgcggcgggc gggagcggga 780

tcagccaccg cggtggcggc ctaaagtcga cgaggaactg aaaaaccaga aagttaactg 840

gtaagttaag tctttttgtc ttttatttca ggtcccggat ccggtggtgg tgcaaatcaa 900

agaactgctc ctcagtggat gttgccttta cttctaggcc tgtacggaag tgttacttct 960

gctctaaaag ctgcggaatt gtacccgcgg ccggccgcca ccatgagctg tcctgtgcct 1020

gcctgttgtg ctctgctgct ggttctgggc ctgtgcagag ccagacctag aaatgcactg 1080

ctcctgctgg ctgatgatgg tggctttgag tctggggcct acaacaactc tgccattgcc 1140

acacctcacc tggatgccct ggctagaaga agcctgctgt tcagaaatgc cttcacctct 1200

gtgtccagct gcagcccttc tagagccagc ctgctgacag gactgcccca gcatcagaat 1260

gggatgtatg gcctgcacca ggatgtgcac cacttcaaca gctttgacaa agtcagatcc 1320

ctgccactgc tgctgagcca ggctggtgtt agaacaggca tcattggcaa gaaacatgtg 1380

ggccctgaga cagtgtaccc ctttgacttt gcctacactg aagagaatgg ctctgtgctg 1440

caagtgggca gaaacatcac caggatcaag ctgcttgtca gaaagttcct gcagacccag 1500

gatgacagac ccttcttcct gtatgtggcc ttccatgatc ctcacagatg tggccactct 1560

cagccccagt atggcacctt ctgtgaaaag tttggaaatg gagagtctgg catgggcaga 1620

atccctgact ggacccctca ggcctatgat cccctggatg tgctggtgcc ctactttgtg 1680

cccaacacac cagctgccag agctgatctg gctgcccagt acaccacagt gggaagaatg 1740

gaccagggtg ttggcctggt cctgcaagag cttagagatg caggggtgct gaatgatacc 1800

ctggtcatct ttacctctga caatggcatc ccctttccaa gtggcaggac caacctgtac 1860

tggcctggaa cagctgagcc cctgctggtg tctagccctg agcaccctaa gagatggggc 1920

caagtgtctg aggcctatgt gtccctgctg gatctgaccc ctaccatcct ggactggttc 1980

agcatcccct atcctagcta tgccatcttt ggcagcaaga ccatccacct gacaggcaga 2040

agtctgctgc ctgctctgga agctgaaccc ctgtgggcca cagtgtttgg cagccagtct 2100

caccatgaag tgacaatgag ctaccccatg agaagtgtgc agcatagaca cttcagactg 2160

gtccacaacc tgaacttcaa gatgcctttt ccaattgacc aggacttcta tgtctcccca 2220

accttccagg acctgctgaa cagaaccact gcaggccagc ctacaggctg gtacaaggac 2280

ctgagacact actactatag ggccagatgg gagctgtatg acagatccag agatcctcat 2340

gagacacaga acctggccac agatcccaga tttgcccagc tgctggaaat gctgagagat 2400

cagctggcca aatggcagtg ggagacacat gacccttggg tctgcgctcc tgatggtgtt 2460

ctggaagaga agctgtcccc tcagtgccag cctctgcaca atgaactgtg attcgaatta 2520

aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 2580

cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 2640

gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 2700

gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggactc gagtagataa 2760

gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc 2820

ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 2880

ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc cttaattaac ctaa 2934

<210> 16

<211> 2914

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAAV2.1-MF-SV40-hSGSH1

<400> 16

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg tacccccttc agattaaaaa taactgaggt 240

aagggcctgg gtaggggagg tggtgtgaga cgctcctgtc tctcctctat ctgcccatcg 300

gccctttggg gaggaggaat gtgcccaagg actaaaaaaa ggccatggag ccagaggggc 360

gagggcaaca gacctttcat gggcaaacct tggggccctg ctgtgcagag ggccctgcgt 420

atgagtgcaa gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac 480

cgaccccgac ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc 540

agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc 600

gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg 660

cgcgctgacg tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt 720

ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg 780

gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag 840

cggaggtaag ttaagtcttt ttgtctttta tttcaggtcc cggatccggt ggtggtgcaa 900

atcaaagaac tgctcctcag tggatgttgc ctttacttct aggagtcgtg tcgtgcctga 960

gagcgcaggg ccggccgcca ccatgagctg tcctgtgcct gcctgttgtg ctctgctgct 1020

ggttctgggc ctgtgcagag ccagacctag aaatgcactg ctcctgctgg ctgatgatgg 1080

tggctttgag tctggggcct acaacaactc tgccattgcc acacctcacc tggatgccct 1140

ggctagaaga agcctgctgt tcagaaatgc cttcacctct gtgtccagct gcagcccttc 1200

tagagccagc ctgctgacag gactgcccca gcatcagaat gggatgtatg gcctgcacca 1260

ggatgtgcac cacttcaaca gctttgacaa agtcagatcc ctgccactgc tgctgagcca 1320

ggctggtgtt agaacaggca tcattggcaa gaaacatgtg ggccctgaga cagtgtaccc 1380

ctttgacttt gcctacactg aagagaatgg ctctgtgctg caagtgggca gaaacatcac 1440

caggatcaag ctgcttgtca gaaagttcct gcagacccag gatgacagac ccttcttcct 1500

gtatgtggcc ttccatgatc ctcacagatg tggccactct cagccccagt atggcacctt 1560

ctgtgaaaag tttggaaatg gagagtctgg catgggcaga atccctgact ggacccctca 1620

ggcctatgat cccctggatg tgctggtgcc ctactttgtg cccaacacac cagctgccag 1680

agctgatctg gctgcccagt acaccacagt gggaagaatg gaccagggtg ttggcctggt 1740

cctgcaagag cttagagatg caggggtgct gaatgatacc ctggtcatct ttacctctga 1800

caatggcatc ccctttccaa gtggcaggac caacctgtac tggcctggaa cagctgagcc 1860

cctgctggtg tctagccctg agcaccctaa gagatggggc caagtgtctg aggcctatgt 1920

gtccctgctg gatctgaccc ctaccatcct ggactggttc agcatcccct atcctagcta 1980

tgccatcttt ggcagcaaga ccatccacct gacaggcaga agtctgctgc ctgctctgga 2040

agctgaaccc ctgtgggcca cagtgtttgg cagccagtct caccatgaag tgacaatgag 2100

ctaccccatg agaagtgtgc agcatagaca cttcagactg gtccacaacc tgaacttcaa 2160

gatgcctttt ccaattgacc aggacttcta tgtctcccca accttccagg acctgctgaa 2220

cagaaccact gcaggccagc ctacaggctg gtacaaggac ctgagacact actactatag 2280

ggccagatgg gagctgtatg acagatccag agatcctcat gagacacaga acctggccac 2340

agatcccaga tttgcccagc tgctggaaat gctgagagat cagctggcca aatggcagtg 2400

ggagacacat gacccttggg tctgcgctcc tgatggtgtt ctggaagaga agctgtcccc 2460

tcagtgccag cctctgcaca atgaactgtg attcgaatta aaccgctgat cagcctcgac 2520

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 2580

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 2640

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 2700

ggaagacaat agcaggcatg ctggggactc gagtagataa gtagcatggc gggttaatca 2760

ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 2820

tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 2880

tgagcgagcg agcgcgcagc cttaattaac ctaa 2914

<210> 17

<211> 3147

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAAV2.1-MF1-SV40-hSGSH1

<400> 17

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg tacccccttc agattaaaaa taactgaggt 240

aagggcctgg gtaggggagg tggtgtgaga cgctcctgtc tctcctctat ctgcccatcg 300

gccctttggg gaggaggaat gtgcccaagg actaaaaaaa ggccatggag ccagaggggc 360

gagggcaaca gacctttcat gggcaaacct tggggccctg ctgctgcaga gggccctgcg 420

tatgagtgca agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga 480

ccgaccccga cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat 540

cagagagggg gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac 600

cgcggacagt gccttcgccc ccgcctggcg gcgcgctgca gagggccctg cgtatgagtg 660

caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc 720

gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg 780

gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca 840

gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg 900

acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc 960

gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac 1020

catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggaggt 1080

aagttaagtc tttttgtctt ttatttcagg tcccggatcc ggtggtggtg caaatcaaag 1140

aactgctcct cagtggatgt tgcctttact tctaggagtc gtgtcgtgcc tgagagcgca 1200

gggccggccg ccaccatgag ctgtcctgtg cctgcctgtt gtgctctgct gctggttctg 1260

ggcctgtgca gagccagacc tagaaatgca ctgctcctgc tggctgatga tggtggcttt 1320

gagtctgggg cctacaacaa ctctgccatt gccacacctc acctggatgc cctggctaga 1380

agaagcctgc tgttcagaaa tgccttcacc tctgtgtcca gctgcagccc ttctagagcc 1440

agcctgctga caggactgcc ccagcatcag aatgggatgt atggcctgca ccaggatgtg 1500

caccacttca acagctttga caaagtcaga tccctgccac tgctgctgag ccaggctggt 1560

gttagaacag gcatcattgg caagaaacat gtgggccctg agacagtgta cccctttgac 1620

tttgcctaca ctgaagagaa tggctctgtg ctgcaagtgg gcagaaacat caccaggatc 1680

aagctgcttg tcagaaagtt cctgcagacc caggatgaca gacccttctt cctgtatgtg 1740

gccttccatg atcctcacag atgtggccac tctcagcccc agtatggcac cttctgtgaa 1800

aagtttggaa atggagagtc tggcatgggc agaatccctg actggacccc tcaggcctat 1860

gatcccctgg atgtgctggt gccctacttt gtgcccaaca caccagctgc cagagctgat 1920

ctggctgccc agtacaccac agtgggaaga atggaccagg gtgttggcct ggtcctgcaa 1980

gagcttagag atgcaggggt gctgaatgat accctggtca tctttacctc tgacaatggc 2040

atcccctttc caagtggcag gaccaacctg tactggcctg gaacagctga gcccctgctg 2100

gtgtctagcc ctgagcaccc taagagatgg ggccaagtgt ctgaggccta tgtgtccctg 2160

ctggatctga cccctaccat cctggactgg ttcagcatcc cctatcctag ctatgccatc 2220

tttggcagca agaccatcca cctgacaggc agaagtctgc tgcctgctct ggaagctgaa 2280

cccctgtggg ccacagtgtt tggcagccag tctcaccatg aagtgacaat gagctacccc 2340

atgagaagtg tgcagcatag acacttcaga ctggtccaca acctgaactt caagatgcct 2400

tttccaattg accaggactt ctatgtctcc ccaaccttcc aggacctgct gaacagaacc 2460

actgcaggcc agcctacagg ctggtacaag gacctgagac actactacta tagggccaga 2520

tgggagctgt atgacagatc cagagatcct catgagacac agaacctggc cacagatccc 2580

agatttgccc agctgctgga aatgctgaga gatcagctgg ccaaatggca gtgggagaca 2640

catgaccctt gggtctgcgc tcctgatggt gttctggaag agaagctgtc ccctcagtgc 2700

cagcctctgc acaatgaact gtgattcgaa ttaaaccgct gatcagcctc gactgtgcct 2760

tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 2820

gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 2880

tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 2940

aatagcaggc atgctgggga ctcgagtaga taagtagcat ggcgggttaa tcattaacta 3000

caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga 3060

ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga 3120

gcgagcgcgc agccttaatt aacctaa 3147

<210> 18

<211> 3175

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAAV2.1-MF2-SV40-hSGSH1

<400> 18

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg tacctccgtt acataactta cggtaaatgg 240

cccgcctggc tgaccgccca acgaccccgc ccattgacgt caataatgac gtatgttccc 300

atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact 360

gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat 420

gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact 480

tggcagtaca tctacaccgg tctgcagagg gccctgcgta tgagtgcaag tgggttttag 540

gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa 600

gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag 660

gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc cttcgccccc 720

gcctggcggc gcgcaagctt tcgaggccac gttctgcttc actctcccca tctccccccc 780

ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 840

gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 900

gaggcggaaa ggtgcggcgg cagccaatca aagcggcgcg ctccgaaagt ttccttttat 960

ggcaaggcgg cggcggcggc ggccctataa aaagcaaacc gcgcggcggg cgggagcggg 1020

atcagccacc gcggtggcgg cctaaagtcg acgaggaact gaaaaaccag aaagttaact 1080

ggtaagttaa gtctttttgt cttttatttc aggtcccgga tccggtggtg gtgcaaatca 1140

aagaactgct cctcagtgga tgttgccttt acttctaggc ctgtacggaa gtgttacttc 1200

tgctctaaaa gctgcggaat tgtacccgcg gccggccgcc accatgagct gtcctgtgcc 1260

tgcctgttgt gctctgctgc tggttctggg cctgtgcaga gccagaccta gaaatgcact 1320

gctcctgctg gctgatgatg gtggctttga gtctggggcc tacaacaact ctgccattgc 1380

cacacctcac ctggatgccc tggctagaag aagcctgctg ttcagaaatg ccttcacctc 1440

tgtgtccagc tgcagccctt ctagagccag cctgctgaca ggactgcccc agcatcagaa 1500

tgggatgtat ggcctgcacc aggatgtgca ccacttcaac agctttgaca aagtcagatc 1560

cctgccactg ctgctgagcc aggctggtgt tagaacaggc atcattggca agaaacatgt 1620

gggccctgag acagtgtacc cctttgactt tgcctacact gaagagaatg gctctgtgct 1680

gcaagtgggc agaaacatca ccaggatcaa gctgcttgtc agaaagttcc tgcagaccca 1740

ggatgacaga cccttcttcc tgtatgtggc cttccatgat cctcacagat gtggccactc 1800

tcagccccag tatggcacct tctgtgaaaa gtttggaaat ggagagtctg gcatgggcag 1860

aatccctgac tggacccctc aggcctatga tcccctggat gtgctggtgc cctactttgt 1920

gcccaacaca ccagctgcca gagctgatct ggctgcccag tacaccacag tgggaagaat 1980

ggaccagggt gttggcctgg tcctgcaaga gcttagagat gcaggggtgc tgaatgatac 2040

cctggtcatc tttacctctg acaatggcat cccctttcca agtggcagga ccaacctgta 2100

ctggcctgga acagctgagc ccctgctggt gtctagccct gagcacccta agagatgggg 2160

ccaagtgtct gaggcctatg tgtccctgct ggatctgacc cctaccatcc tggactggtt 2220

cagcatcccc tatcctagct atgccatctt tggcagcaag accatccacc tgacaggcag 2280

aagtctgctg cctgctctgg aagctgaacc cctgtgggcc acagtgtttg gcagccagtc 2340

tcaccatgaa gtgacaatga gctaccccat gagaagtgtg cagcatagac acttcagact 2400

ggtccacaac ctgaacttca agatgccttt tccaattgac caggacttct atgtctcccc 2460

aaccttccag gacctgctga acagaaccac tgcaggccag cctacaggct ggtacaagga 2520

cctgagacac tactactata gggccagatg ggagctgtat gacagatcca gagatcctca 2580

tgagacacag aacctggcca cagatcccag atttgcccag ctgctggaaa tgctgagaga 2640

tcagctggcc aaatggcagt gggagacaca tgacccttgg gtctgcgctc ctgatggtgt 2700

tctggaagag aagctgtccc ctcagtgcca gcctctgcac aatgaactgt gattcgaatt 2760

aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 2820

ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 2880

ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 2940

ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggact cgagtagata 3000

agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc 3060

cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 3120

gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ccttaattaa cctaa 3175

<210> 19

<211> 3163

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAAV2.1-MF3-SV40-hSGSH1

<400> 19

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg tacctcctgc agagggccct gcgtatgagt 240

gcaagtgggt tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc 300

cgacccactg gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag 360

ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac 420

agtgccttcg cccccgcctg gcggcgcgcc gttacataac ttacggtaaa tggcccgcct 480

ggctgaccgc ccaacgaccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 540

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 600

tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 660

aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 720

acatctactc gaggccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc 780

aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg 840

ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggaaagg 900

tgcggcggca gccaatcaaa gcggcgcgct ccgaaagttt ccttttatgg caaggcggcg 960

gcggcggcgg ccctataaaa agcaaaccgc gcggcgggcg ggagcgggat cagccaccgc 1020

ggtggcggcc taaagtcgac gaggaactga aaaaccagaa agttaactgg taagttaagt 1080

ctttttgtct tttatttcag gtcccggatc cggtggtggt gcaaatcaaa gaactgctcc 1140

tcagtggatg ttgcctttac ttctaggcct gtacggaagt gttacttctg ctctaaaagc 1200

tgcggaattg tacccgcggc cggccgccac catgagctgt cctgtgcctg cctgttgtgc 1260

tctgctgctg gttctgggcc tgtgcagagc cagacctaga aatgcactgc tcctgctggc 1320

tgatgatggt ggctttgagt ctggggccta caacaactct gccattgcca cacctcacct 1380

ggatgccctg gctagaagaa gcctgctgtt cagaaatgcc ttcacctctg tgtccagctg 1440

cagcccttct agagccagcc tgctgacagg actgccccag catcagaatg ggatgtatgg 1500

cctgcaccag gatgtgcacc acttcaacag ctttgacaaa gtcagatccc tgccactgct 1560

gctgagccag gctggtgtta gaacaggcat cattggcaag aaacatgtgg gccctgagac 1620

agtgtacccc tttgactttg cctacactga agagaatggc tctgtgctgc aagtgggcag 1680

aaacatcacc aggatcaagc tgcttgtcag aaagttcctg cagacccagg atgacagacc 1740

cttcttcctg tatgtggcct tccatgatcc tcacagatgt ggccactctc agccccagta 1800

tggcaccttc tgtgaaaagt ttggaaatgg agagtctggc atgggcagaa tccctgactg 1860

gacccctcag gcctatgatc ccctggatgt gctggtgccc tactttgtgc ccaacacacc 1920

agctgccaga gctgatctgg ctgcccagta caccacagtg ggaagaatgg accagggtgt 1980

tggcctggtc ctgcaagagc ttagagatgc aggggtgctg aatgataccc tggtcatctt 2040

tacctctgac aatggcatcc cctttccaag tggcaggacc aacctgtact ggcctggaac 2100

agctgagccc ctgctggtgt ctagccctga gcaccctaag agatggggcc aagtgtctga 2160

ggcctatgtg tccctgctgg atctgacccc taccatcctg gactggttca gcatccccta 2220

tcctagctat gccatctttg gcagcaagac catccacctg acaggcagaa gtctgctgcc 2280

tgctctggaa gctgaacccc tgtgggccac agtgtttggc agccagtctc accatgaagt 2340

gacaatgagc taccccatga gaagtgtgca gcatagacac ttcagactgg tccacaacct 2400

gaacttcaag atgccttttc caattgacca ggacttctat gtctccccaa ccttccagga 2460

cctgctgaac agaaccactg caggccagcc tacaggctgg tacaaggacc tgagacacta 2520

ctactatagg gccagatggg agctgtatga cagatccaga gatcctcatg agacacagaa 2580

cctggccaca gatcccagat ttgcccagct gctggaaatg ctgagagatc agctggccaa 2640

atggcagtgg gagacacatg acccttgggt ctgcgctcct gatggtgttc tggaagagaa 2700

gctgtcccct cagtgccagc ctctgcacaa tgaactgtga ttcgaattaa accgctgatc 2760

agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 2820

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 2880

gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 2940

ggaggattgg gaagacaata gcaggcatgc tggggactcg agtagataag tagcatggcg 3000

ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg 3060

ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg 3120

cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc taa 3163

<210> 20

<211> 3108

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAAV2.1-MF4-SV40-hSGSH1

<400> 20

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg taccaggtta atttttaaaa agcagtcaaa 240

agtccaagtg gcccttggca gcatttactc tctctgttta ctctggttaa taatctcagg 300

agtacaaaca ttccagatcc aggttaattt ttaaaaaaag agtgctctag ttttgcaata 360

caggctgcag agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg 420

gtgggggtgc ctacctgacg accgaccccg acccactgga caagcaccca acccccattc 480

cccaaattgc gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt 540

gcgcactgcc agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgctgc 600

agagggccct gcgtatgagt gcaagtgggt tttaggacca ggatgaggcg gggtgggggt 660

gcctacctga cgaccgaccc cgacccactg gacaagcacc caacccccat tccccaaatt 720

gcgcatcccc tatcagagag ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg 780

ccagcttcag caccgcggac agtgccttcg cccccgcctg gcggcgcgcg ccaccgccgc 840

ctcagcactg aaggcgcgct gacgtcactc gccggtcccc cgcaaactcc ccttcccggc 900

caccttggtc gcgtccgcgc cgccgccggc ccagccggac cgcaccacgc gaggcgcgag 960

ataggggggc acgggcgcga ccatctgcgc tgcggcgccg gcgactcagc gctgcctcag 1020

tctgcggtgg gcagcggagg taagttaagt ctttttgtct tttatttcag gtcccggatc 1080

cggtggtggt gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggagt 1140

cgtgtcgtgc ctgagagcgc agggccggcc gccaccatga gctgtcctgt gcctgcctgt 1200

tgtgctctgc tgctggttct gggcctgtgc agagccagac ctagaaatgc actgctcctg 1260

ctggctgatg atggtggctt tgagtctggg gcctacaaca actctgccat tgccacacct 1320

cacctggatg ccctggctag aagaagcctg ctgttcagaa atgccttcac ctctgtgtcc 1380

agctgcagcc cttctagagc cagcctgctg acaggactgc cccagcatca gaatgggatg 1440

tatggcctgc accaggatgt gcaccacttc aacagctttg acaaagtcag atccctgcca 1500

ctgctgctga gccaggctgg tgttagaaca ggcatcattg gcaagaaaca tgtgggccct 1560

gagacagtgt acccctttga ctttgcctac actgaagaga atggctctgt gctgcaagtg 1620

ggcagaaaca tcaccaggat caagctgctt gtcagaaagt tcctgcagac ccaggatgac 1680

agacccttct tcctgtatgt ggccttccat gatcctcaca gatgtggcca ctctcagccc 1740

cagtatggca ccttctgtga aaagtttgga aatggagagt ctggcatggg cagaatccct 1800

gactggaccc ctcaggccta tgatcccctg gatgtgctgg tgccctactt tgtgcccaac 1860

acaccagctg ccagagctga tctggctgcc cagtacacca cagtgggaag aatggaccag 1920

ggtgttggcc tggtcctgca agagcttaga gatgcagggg tgctgaatga taccctggtc 1980

atctttacct ctgacaatgg catccccttt ccaagtggca ggaccaacct gtactggcct 2040

ggaacagctg agcccctgct ggtgtctagc cctgagcacc ctaagagatg gggccaagtg 2100

tctgaggcct atgtgtccct gctggatctg acccctacca tcctggactg gttcagcatc 2160

ccctatccta gctatgccat ctttggcagc aagaccatcc acctgacagg cagaagtctg 2220

ctgcctgctc tggaagctga acccctgtgg gccacagtgt ttggcagcca gtctcaccat 2280

gaagtgacaa tgagctaccc catgagaagt gtgcagcata gacacttcag actggtccac 2340

aacctgaact tcaagatgcc ttttccaatt gaccaggact tctatgtctc cccaaccttc 2400

caggacctgc tgaacagaac cactgcaggc cagcctacag gctggtacaa ggacctgaga 2460

cactactact atagggccag atgggagctg tatgacagat ccagagatcc tcatgagaca 2520

cagaacctgg ccacagatcc cagatttgcc cagctgctgg aaatgctgag agatcagctg 2580

gccaaatggc agtgggagac acatgaccct tgggtctgcg ctcctgatgg tgttctggaa 2640

gagaagctgt cccctcagtg ccagcctctg cacaatgaac tgtgattcga attaaaccgc 2700

tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 2760

ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 2820

gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 2880

aagggggagg attgggaaga caatagcagg catgctgggg actcgagtag ataagtagca 2940

tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct 3000

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 3060

ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaa 3108

<210> 21

<211> 3044

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAAV2.1-MF5-SV40-hSGSH1

<400> 21

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg taccaggtta atttttaaaa agcagtcaaa 240

agtccaagtg gcccttggca gcatttactc tctctgttta ctctggttaa taatctcagg 300

agtacaaaca ttccagatcc aggttaattt ttaaaaaaag agtgctctag ttttgcaata 360

caggaccggt ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga 420

ggcggggtgg gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc 480

ccattcccca aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag 540

gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg 600

cgcaagcttt cgaggccacg ttctgcttca ctctccccat ctcccccccc tccccacccc 660

caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg 720

gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggaaag 780

gtgcggcggc agccaatcaa agcggcgcgc tccgaaagtt tccttttatg gcaaggcggc 840

ggcggcggcg gccctataaa aagcaaaccg cgcggcgggc gggagcggga tcagccaccg 900

cggtggcggc ctaaagtcga cgaggaactg aaaaaccaga aagttaactg gtaagttaag 960

tctttttgtc ttttatttca ggtcccggat ccggtggtgg tgcaaatcaa agaactgctc 1020

ctcagtggat gttgccttta cttctaggcc tgtacggaag tgttacttct gctctaaaag 1080

ctgcggaatt gtacccgcgg ccggccgcca ccatgagctg tcctgtgcct gcctgttgtg 1140

ctctgctgct ggttctgggc ctgtgcagag ccagacctag aaatgcactg ctcctgctgg 1200

ctgatgatgg tggctttgag tctggggcct acaacaactc tgccattgcc acacctcacc 1260

tggatgccct ggctagaaga agcctgctgt tcagaaatgc cttcacctct gtgtccagct 1320

gcagcccttc tagagccagc ctgctgacag gactgcccca gcatcagaat gggatgtatg 1380

gcctgcacca ggatgtgcac cacttcaaca gctttgacaa agtcagatcc ctgccactgc 1440

tgctgagcca ggctggtgtt agaacaggca tcattggcaa gaaacatgtg ggccctgaga 1500

cagtgtaccc ctttgacttt gcctacactg aagagaatgg ctctgtgctg caagtgggca 1560

gaaacatcac caggatcaag ctgcttgtca gaaagttcct gcagacccag gatgacagac 1620

ccttcttcct gtatgtggcc ttccatgatc ctcacagatg tggccactct cagccccagt 1680

atggcacctt ctgtgaaaag tttggaaatg gagagtctgg catgggcaga atccctgact 1740

ggacccctca ggcctatgat cccctggatg tgctggtgcc ctactttgtg cccaacacac 1800

cagctgccag agctgatctg gctgcccagt acaccacagt gggaagaatg gaccagggtg 1860

ttggcctggt cctgcaagag cttagagatg caggggtgct gaatgatacc ctggtcatct 1920

ttacctctga caatggcatc ccctttccaa gtggcaggac caacctgtac tggcctggaa 1980

cagctgagcc cctgctggtg tctagccctg agcaccctaa gagatggggc caagtgtctg 2040

aggcctatgt gtccctgctg gatctgaccc ctaccatcct ggactggttc agcatcccct 2100

atcctagcta tgccatcttt ggcagcaaga ccatccacct gacaggcaga agtctgctgc 2160

ctgctctgga agctgaaccc ctgtgggcca cagtgtttgg cagccagtct caccatgaag 2220

tgacaatgag ctaccccatg agaagtgtgc agcatagaca cttcagactg gtccacaacc 2280

tgaacttcaa gatgcctttt ccaattgacc aggacttcta tgtctcccca accttccagg 2340

acctgctgaa cagaaccact gcaggccagc ctacaggctg gtacaaggac ctgagacact 2400

actactatag ggccagatgg gagctgtatg acagatccag agatcctcat gagacacaga 2460

acctggccac agatcccaga tttgcccagc tgctggaaat gctgagagat cagctggcca 2520

aatggcagtg ggagacacat gacccttggg tctgcgctcc tgatggtgtt ctggaagaga 2580

agctgtcccc tcagtgccag cctctgcaca atgaactgtg attcgaatta aaccgctgat 2640

cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 2700

ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 2760

cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 2820

gggaggattg ggaagacaat agcaggcatg ctggggactc gagtagataa gtagcatggc 2880

gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc 2940

gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg 3000

gcggcctcag tgagcgagcg agcgcgcagc cttaattaac ctaa 3044

Claims

1. A transgenic expression cassette comprising:

a promoter selected from SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, preferably selected from SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 9, more preferably SEQ ID NO 3 or SEQ ID NO 7, most preferably SEQ ID NO 7; and

a nucleic acid molecule encoding a glucosamine sulfohydrolase (SGSH) having a nucleotide sequence which has at least 80% identity, preferably at least 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence set forth in SEQ ID NO. 1.

2. The transgenic expression cassette of claim 1, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID No. 1; preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

3. The transgenic expression cassette of claim 1 or 2, wherein the transgenic expression cassette further comprises regulatory elements, such as two ITRs at either end; and/or an origin of replication; and/or simian virus 40 intron; and/or a polyadenylation signal.

4. The transgenic expression cassette of any one of claims 1 to 3, wherein the nucleotide sequence of the transgenic expression cassette is as set forth in SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21 is shown; preferably, the nucleotide sequence of the transgenic expression cassette is as shown in SEQ ID NO: 15. SEQ ID NO: 18. the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21 is shown in the figure; more preferably, the nucleotide sequence of the transgenic expression cassette is as set forth in SEQ ID NO: 15 or SEQ ID NO: 19 is shown in the figure; most preferably, the nucleotide sequence of the transgenic expression cassette is as set forth in SEQ ID NO: 19, respectively.

5. A nucleic acid molecule encoding a glucosamine sulfohydrolase (SGSH), wherein the nucleotide sequence of said nucleic acid molecule has at least 80% identity, preferably at least 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence depicted in SEQ ID NO. 1.

6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO 1; preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

7. A combinatorial promoter selected from the group consisting of: the MF2 promoter consisting of CMV enhancer, SYN1 enhancer, and chicken β -actin promoter; the MF3 promoter, consisting of the SYN1 enhancer, CMV enhancer, and chicken β -actin promoter; and MF5 promoter consisting of LXP2.1 enhancer, SYN1 enhancer, and chicken β -actin promoter; preferably, the combinatorial promoter is the MF3 promoter.

8. The combination type promoter according to claim 7, wherein the nucleotide sequence of the MF2 promoter is shown as SEQ ID NO. 6, the nucleotide sequence of the MF3 promoter is shown as SEQ ID NO. 7, and the nucleotide sequence of the MF5 promoter is shown as SEQ ID NO. 9.

9. A gene delivery system, comprising: the transgenic expression cassette of any one of claims 1 to 4 and an AAV capsid protein.

10. The gene delivery system of claim 9, wherein the AAV capsid protein is a native AAV capsid protein or an artificially engineered AAV capsid protein; preferably, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, AAVrh10, AAVrh39, AAVrh43, AAV32.33, AAV3B, AAVv66, AAVXL32, and AAV. php.b.

11. Use of a transgenic expression cassette according to any one of claims 1 to 4 or a gene delivery system according to claim 9 or 10 for the manufacture of a medicament for the treatment of mucopolysaccharidosis type IIIA.

12. A medicament, comprising: one selected from the group consisting of the transgenic expression cassette of any one of claims 1 to 4, the nucleic acid molecule of claim 5 or 6, and the gene delivery system of claim 9 or 10, and an excipient.

13. The medicament of claim 12, wherein the medicament is administered by a systemic route or a local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration, and intralesional administration; preferably, the medicament is administered by a systemic route, for example intravenously.