Recombinant viral vector, recombinant adeno-associated virus comprising same and use thereof in treating sialic acid storage diseases

Abstract

The present application relates to a recombinant viral vector comprising an AAV 5 '-terminal inverted repeat, an AAV 3'-terminal inverted repeat, and an expression group located between the AAV 5 '-terminal inverted repeat and the AAV 3'-terminal inverted repeat. According to the embodiment of the invention, the expression group comprises a first coding sequence and a second coding sequence which are respectively used for coding human neuraminidase 1 and human protective protein / cathepsin A. The present application also relates to a recombinant adeno-associated virus (AAV) comprising the recombinant viral vector, a pharmaceutical composition comprising the recombinant AAV, and the use thereof in the treatment of sialic acid storage diseases.

Description

Sequence Listing

[0001] This application also submits an electronic version of the sequence listing in XML format. The title of the submitted XML format sequence listing is P4311-PCT_seq listing, the production date is October 11, 2023, and the size is 42kb. The contents of the submitted XML format sequence listing are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of disease treatment. More specifically, the present application is to treat sialic acid storage disease using a novel recombinant adeno-associated virus (rAAV). Background Art

[0005] Sialidosis is a rare lysosomal storage disorder caused by a mutation in the NEU1 gene that results in a deficiency of neuraminidase 1 (also known as "sialidase"). The lack of neuraminidase 1 results in an abnormal accumulation of sialylated sugar complexes in the body. There are two types of sialidosis (i.e., type I and type II sialidosis). Type I sialidosis is caused by decreased activity of neuraminidase 1 and is a milder, late-onset disease characterized by progressive myoclonus, cerebellar ataxia, visual disturbances, and gait abnormalities. As people age, some patients develop seizures and mild cognitive impairment. People with type I sialidosis usually develop symptoms in their teens or twenties. Sialidosis type II is caused by the complete loss of neuraminidase 1 activity. It is a severe, early-onset disease that usually develops in infancy or childhood. Characteristics of sialidosis type II include skeletal dysplasia, coarse facial features, organomegaly, severe ataxia, and intellectual disability. Sialidosis is an autosomal recessive genetic disease. Both types of sialidosis present with a cherry-red macular spot.

[0006] There is currently no effective treatment for sialic acid storage disease. In 2005, mice with sialic acid storage disease were given short-term, high-dose enzyme replacement therapy (ERT); however, two weeks after ERT, the mice developed a severe immune response to the exogenous NEU1 enzyme. In view of this, the relevant field is in urgent need of a novel agent and method for treating sialic acid storage disease. Summary of the invention

[0007] The invention summary is intended to provide a simplified summary of the present application so that readers can have a basic understanding of the present application. This invention summary is not a complete overview of the present application, and it is not intended to point out the important / critical components of the embodiments of the present invention or to limit the scope of the present invention.

[0008] The first aspect of the present application is to a recombinant viral vector, which comprises an AAV 5' inverted terminal repeat (ITR) sequence, an AAV 3'ITR sequence, and an expression group located between the AAV 5'ITR and 3'ITR sequences. According to an embodiment of the present application, the expression group comprises: (a) a promoter; (b) a first coding sequence, which is operably linked to the promoter and encodes human neuraminidase 1 (NEU1); and (c) a second coding sequence, which is operably linked to the promoter, encoding human protectin / cathepsin A (PPCA).

[0009] According to certain embodiments, the AAV 5'ITR and 3'ITR sequences are derived from AAV serotype 2 (AAV2). In an exemplary embodiment, the AAV 5'ITR and 3'ITR sequences comprise the nucleotide sequences of SEQ ID NOs: 19 and 20, respectively.

[0010] Depending on the purpose of implementation, the promoter can be a human NEU1 promoter (eg, a human P2NEU1 promoter or a human P3 NEU1 promoter) or a cytomegalovirus (CMV) promoter. In a preferred embodiment, the promoter is a human NEU1P3 promoter and comprises the nucleotide sequence of SEQ ID NO:11.

[0011] According to certain embodiments, human NEU1 and human PPCA respectively comprise the amino acid sequences of SEQ ID NOs: 2 and 10. In a preferred embodiment, the first and second coding sequences respectively comprise the nucleotide sequences of SEQ ID NOs: 1 and 9.

[0012] Preferably, the expression set further comprises an internal ribosome entry site (IRES) located between the first and second coding sequences. Optionally, the expression set further comprises a third coding sequence located between the first and second coding sequences, which is used to encode the 2A polypeptide.

[0013] According to certain embodiments, the expression set further comprises a polyadenylation (polyA) sequence located at the 3' end of the second coding sequence. In a preferred embodiment, the polyA sequence is derived from human growth hormone (hGH).

[0014] According to a specific embodiment, the expression group comprises the nucleotide sequence of sequence number: 22.

[0015] The present application also provides a recombinant AAV, which structurally comprises the recombinant viral vector of the present application and an AAV shell that encapsulates the recombinant viral vector. Preferably, the AAV shell has a wide tropism for the brain and internal organs. According to certain exemplary embodiments, the AAV shell is derived from AAV serotype 9 (AAV9). In one embodiment, the AAV shell comprises the amino acid sequence of sequence number: 21.

[0016] Another aspect of the present application is to use the recombinant AAV in preparing a pharmaceutical composition and to use the recombinant AAV in treating sialidosis.

[0017] According to certain embodiments of the present application, the pharmaceutical composition comprises the recombinant AAV of the present invention and a pharmaceutically acceptable drug carrier.

[0018] The recombinant AAV or pharmaceutical composition of the present invention can be used to treat sialic acid storage disease. Accordingly, the present application also provides a method for treating sialic acid storage disease in an individual. The method of the present invention comprises administering an effective amount of recombinant AAV or pharmaceutical composition to the individual, thereby alleviating or improving the symptoms of sialic acid storage disease.

[0019] Depending on the purpose of implementation, the sialidosis may be type I sialidosis or type II sialidosis.

[0020] According to some embodiments, about 1×10 13 to 1×10 15 According to some embodiments, about 1×10 GC per kg body weight is administered to an individual. 13 to 1×10 15 The recombinant AAV of the GC is administered to the individual.

[0021] The recombinant AAV or pharmaceutical composition can be administered to a subject by any suitable route, for example, intracerebrospinal fluid (e.g., intraventricularly, intracisternal, or intrathecal), intravenous, intraperitoneal, intraarterial, or intramuscular injection. According to one embodiment, the recombinant AAV or pharmaceutical composition is injected into the subject via the cerebrospinal fluid. According to another embodiment, the recombinant AAV or pharmaceutical composition is injected into the subject via intravenous injection.

[0022] In all embodiments of the present application, the individual is a mammal; preferably a human.

[0023] After referring to the following embodiments, those skilled in the art can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation methods adopted by the present invention. DETAILED DESCRIPTION

[0024] In order to make the description of this application more detailed and complete, the following is an illustrative description of the implementation and specific examples of the present invention; however, this is not the only form of implementing or using the specific examples of the present invention. The implementation covers the features of multiple specific examples and the method steps and sequences used to construct and operate these specific examples. However, other specific examples can also be used to achieve the same or equivalent functions and step sequences.

[0025] I. Definitions

[0026] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant numerical values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of those skilled in the art. Except for the experimental examples, or unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used herein (for example, to describe the amount of material used, the length of time, temperature, operating conditions, quantitative ratios and the like) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the attached patent scope are all approximate values ​​and can be changed as needed. At least these numerical parameters should be understood as the number of significant digits indicated and the values ​​obtained by applying the general rounding method. Herein, numerical ranges are expressed from one end point to another point or between two end points; unless otherwise stated, the numerical ranges described herein include the end points.

[0027] Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as those understood and used by those skilled in the art. In addition, singular nouns used in this specification include plural forms of the nouns, and plural nouns used also include singular forms of the nouns, unless they conflict with the context.

[0028] In the present application, the term "viral vector" refers to a synthetic or artificial viral nucleic acid, in which an expression group containing a specific gene (for example, the first and second coding sequences of the present application) is encapsulated in a viral capsid or envelope, and any viral genome sequence encapsulated in the capsid or envelope is replication-defective; that is, it cannot produce progeny virions, but retains the ability to infect target cells and express specific genes in infected target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be designed as a gut-free structure, only containing transgenes connected to the signals required for amplification and packaging of the artificial genome), and these genes can be provided additionally during the preparation process. Therefore, this type of viral vector is considered to be a safe vector that can be used as a gene therapy because unless the enzymes required for viral replication are additionally provided, the progeny virions cannot replicate and infect. Alternatively, the term "viral vector" can also refer to a virus or virus particle used to deliver nucleic acid molecules into cells. In addition to nucleic acid molecules, the virus or virus particle usually also contains different viral components, as well as non-essential host components.

[0029] In this application, the term "recombinant viral vector" refers to a viral vector containing one or more exogenous gene products or sequences. Generally speaking, the exogenous gene products or sequences are introduced and replace one or more regions in the viral genome.

[0030] The term "AAV vector" in this application refers to a synthetic or artificial viral nucleic acid having an AAV 5'ITR sequence, an AAV 3'ITR sequence, and a specific gene (e.g., the first and second coding sequences of the present application) located between the AAV 5'ITR and 3'ITR sequences. In general, the specific gene is operably linked to a transcriptional regulatory element, such as a promoter, an enhancer, a polyadenylation (polyA) sequence, and optionally, one or more introns located between the exons of the gene.

[0031] The term "inverted terminal repeat" (ITR) refers to the well-known region located at the 5' and 3' ends of the AAV genome, which is a cis sequence segment required for viral genome replication and packaging into viral particles. This sequence segment also plays an indispensable and important role in the process of viral genome integration into host genome, release from host genome, and packaging of viral nucleic acid into mature virions.

[0032] In the present application, the term "operably linked" refers to an expression control sequence (e.g., the promoter of the present application) adjacent to a specific gene (e.g., the first and second coding sequences of the present application), as well as an expression control sequence that controls the specific gene in trans or remotely.

[0033] The term "derived from" in this application refers to the source of a molecule (e.g., a polynucleotide or polypeptide). When a polynucleotide or polypeptide of the present invention is "derived from" a specific source, it means that the polynucleotide or polypeptide has the same sequence as a natural polynucleotide or polypeptide of the source. Alternatively, the polynucleotide or polypeptide of the present invention may be a fragment of a natural polynucleotide or polypeptide. Polynucleotides or polypeptides derived from a specific source may be prepared from natural sequences or based on natural sequences by direct cloning, PCR amplification, or artificial synthesis.

[0034] The present application and the claimed inventive concept also include minor variations in the amino acid sequence of a polypeptide or the polynucleotide sequence of a nucleic acid, as long as the variation in the amino acid sequence / polynucleotide sequence maintains at least 80% sequence similarity, for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity. The properties of the polypeptide or polynucleotide of the present application can be changed by specific modifications without affecting its physiological activity. For example, certain amino acids can be changed and / or deleted without affecting the physiological activity of the polypeptide of the present invention. In particular, conservative amino acid substitutions are also included therein. Conservative substitutions are substitutions between amino acids with similar / related side chains. Generally speaking, the amino acids encoded by genes can be divided into four major categories: (1) acidic amino acids, namely aspartic acid and glutamic acid; (2) basic amino acids, namely lysine, arginine, and histidine; (3) non-polar amino acids, namely alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) non-charged polar amino acids, namely glycine, asparagine, glutamic acid, cysteine, serine, threonine, and tyrosine. A better classification is that serine and threonine belong to the aliphatic hydroxyl group; asparagine and glutamine belong to the amide group; alanine, valine, leucine, and isoleucine belong to the aliphatic group; and phenylalanine, tryptophan, and tyrosine belong to the aromatic group. For example, it is conceivable that replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, replacing threonine with serine, or replacing one structurally similar amino acid with another will not cause significant changes in molecular binding or protein properties, especially when the substitution position is not located in the framework region, and substitutions between amino acids will not affect the above-mentioned properties.

[0035] The "sequence similarity percentage" described in this application for amino acid sequences or nucleotide sequences refers to the percentage of amino acid residues / nucleotides of a candidate sequence that are exactly the same as the amino acid residues / nucleotides of a reference sequence. When performing the above comparison, the candidate sequence and the reference sequence can be placed side by side, and a gap can be introduced when necessary so that the two sequences form the highest sequence similarity. When calculating the similarity, the amino acid residues of conservative substitutions are regarded as different residues; the nucleotides of degenerate codons are also regarded as different nucleotides. There are many methods available in the related field for the above-mentioned side-by-side arrangement, such as publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR), etc. When performing the side-by-side arrangement, those skilled in the art can select appropriate parameters and calculation methods to obtain the best arrangement. In this specification, the sequence alignment between two amino acid sequences / nucleotide sequences is performed using the protein-protein BLAST analysis database Blastp or the nucleotide-nucleotide BLAST analysis database Blastn provided by the National Center for Biotechnology Information (NCBI) of the United States. The calculation method of the amino acid / nucleotide sequence similarity between a candidate sequence A and a reference sequence B (also referred to as the amino acid / nucleotide sequence similarity between sequence A and sequence B having a specific percentage (%)) is as follows: Where X is the number of identical amino acid residues / nucleotides obtained after aligning sequences A and B using the BLAST analysis database, and Y is the total number of amino acid residues / nucleotides in the shorter of the two sequences A and B.

[0036] As used herein, the term "treat" includes partial or complete prevention, amelioration, alleviation and / or treatment of signs, secondary signs or symptoms associated with sialic acid storage diseases. The term "treat" herein also refers to the application or administration of one or more recombinant AAV or pharmaceutical compositions of the present invention to an individual suffering from signs, secondary signs or symptoms associated with sialic acid storage diseases, so as to achieve partial or complete alleviation, alleviation, cure, delay onset, inhibit progression, reduce severity of disease, and / or reduce the occurrence of one or more signs, symptoms, or secondary signs associated with sialic acid storage diseases. Signs, secondary signs and / or symptoms associated with sialic acid storage diseases include, but are not limited to, macular cherry red spot, progressive myoclonus, ataxia, visual impairment, gait abnormality, cognitive impairment, skeletal dysplasia, coarse features and organ hypertrophy. Here, "treatment" may also refer to administration to an individual with early signs or symptoms of these diseases to reduce the risk of the individual developing signs, secondary signs and / or symptoms associated with sialidosis. A treatment is "effective" when it reduces one or more symptoms or clinical markers. Alternatively, a treatment is "effective" when it reduces, slows or stops the progression of the disease, signs or symptoms.

[0037] "Effective amount" herein refers to the amount of an ingredient sufficient to produce the desired therapeutic response. An effective amount also means that the therapeutic benefit of an ingredient outweighs its toxic or harmful effects. An effective amount of a drug does not necessarily cure a disease or symptom, but rather provides treatment for the disease or symptom, delays, inhibits or prevents the onset of the disease or symptom, or improves the symptoms associated with the disease or symptom. An effective amount can be divided into one, two or more doses in an appropriate form, so as to be administered to an individual once, twice or multiple times within a specific time. According to certain embodiments of the present application, an effective amount is administered to an individual in a single dose. The specific effective amount depends on a variety of factors, such as the specific condition to be treated, the physiological condition of the patient (e.g., the patient's weight, age or sex), the type of mammal or animal being treated, the duration of treatment, the nature of the current therapy (if any), and the specific formulation used and the structure of the compound or its derivative. For example, the effective amount can be expressed as the total weight of the drug (e.g., in grams, milligrams or micrograms) or as the ratio of the weight of the drug to the body weight (in milligrams / kilograms (mg / kg)). Alternatively, the effective amount can be expressed as the ratio of gene copy number to body weight (GC / kg). A person skilled in the art can calculate the human equivalent dose (HED) of a drug (e.g., a recombinant AAV or pharmaceutical composition of the present invention) based on the dose in an animal model. For example, a person skilled in the art can estimate the highest safe dose for human use based on the "Estimation of the Maximum Safe Starting Dose for Healthy Adult Volunteers in Initial Clinical Treatment Tests" announced by the U.S. Food and Drug Administration (FDA).

[0038] The term "subject" refers to animals, including humans, that can be treated with the recombinant AAV, pharmaceutical compositions and / or methods of the present invention. Unless otherwise specified, the term "subject" refers to both males and females.

[0039] II. Detailed description of the invention

[0040] The present application aims to provide a recombinant AAV for treating sialic acid storage disease. The recombinant AAV of the present invention is characterized in that its viral genome carries two therapeutic genes, namely, NEU1 and CTSA. After being administered to an individual suffering from sialic acid storage disease, the recombinant AAV can simultaneously express human neuraminidase 1 (hNEU1) and human protective protein / cathepsin A (hPPCA) in the individual's body, thereby alleviating or improving the symptoms of sialic acid storage disease. Therefore, the present application relates to a viral vector expressing hNEU1 and hPPCA, and a method for treating sialic acid storage disease using recombinant AAV.

[0041] (i) Recombinant AAV vector

[0042] The first aspect of the present invention relates to a recombinant AAV vector, which comprises, from the 5' end to the 3' end, (a) AAV 5'ITR sequence; (b) an expression group comprising: (b-1) a promoter; (b-2) an hNEU1 coding sequence operably linked to the promoter; and (b-3) an hPPCA coding sequence operably linked to the promoter; and (c) AAV 3'ITR sequence.

[0043] It is well known to those skilled in the art that the ITR sequence is a genetic sequence responsible for replication and assembly during the virus preparation process, and is also the only viral cis sequence required to produce rAAV. The 5'ITR and 3'ITR sequences of AAV are the minimum sequences for assembling the expression group into the AAV virus particle, which can have the same AAV source as the shell, or have a different AAV source from the shell (to prepare AAV pseudotypes). Depending on the purpose of implementation, the AAV 5'ITR and 3'ITR sequences of the recombinant AAV vector can be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8) or AAV serotype 9 (AAV9). According to certain embodiments of the present application, the AAV 5'ITR and 3'ITR sequences of the present invention are derived from AAV2 and respectively comprise nucleotide sequences having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence similarity to SEQ ID NOs: 19 and 20. Preferably, the AAV 5'ITR and 3'ITR sequences of the present invention respectively comprise nucleotide sequences having at least 90% sequence similarity to SEQ ID NOs: 19 and 20; more preferably, nucleotide sequences having at least 95% sequence similarity to SEQ ID NOs: 19 and 20. In an exemplary embodiment, the AAV 5'ITR and 3'ITR sequences of the present invention respectively comprise nucleotide sequences having 100% sequence similarity to SEQ ID NOs: 19 and 20 (i.e., comprising the nucleotide sequences of SEQ ID NOs: 19 and 20, respectively).

[0044] Depending on the purpose of implementation, the promoter can be human NEU1 promoter, CMV promoter, elongation factor 1a (EF1a) promoter, simian virus 40 (SV40) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary cancer virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, chicken type II active promoter, human H1 promoter, U6 promoter or any promoter known to stably drive gene expression in eukaryotic cells.

[0045] According to certain exemplary embodiments of the present application, the promoter is a P3 NEU1 promoter and comprises a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO: 11; preferably, a nucleotide sequence having at least 90% sequence similarity to SEQ ID NO: 11; more preferably, a nucleotide sequence having at least 95% sequence similarity to SEQ ID NO: 11. In a specific embodiment, the promoter comprises a nucleotide sequence having 100% sequence similarity to SEQ ID NO: 11 (i.e., comprising the nucleotide sequence of SEQ ID NO: 11).

[0046] According to certain exemplary embodiments of the present application, the promoter is a P2 NEU1 promoter and comprises a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO: 12; preferably, a nucleotide sequence having at least 90% sequence similarity to SEQ ID NO: 12; more preferably, a nucleotide sequence having at least 95% sequence similarity to SEQ ID NO: 12. In a specific embodiment, the promoter comprises a nucleotide sequence having 100% sequence similarity to SEQ ID NO: 12 (i.e., comprising the nucleotide sequence of SEQ ID NO: 12).

[0047] According to an alternative embodiment of the present application, the promoter is a CMV promoter and comprises a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO: 13; preferably, a nucleotide sequence having at least 90% sequence similarity to SEQ ID NO: 13; more preferably, a nucleotide sequence having at least 95% sequence similarity to SEQ ID NO: 13. In a specific embodiment, the promoter comprises a nucleotide sequence having 100% sequence similarity to SEQ ID NO: 13 (i.e., comprising the nucleotide sequence of SEQ ID NO: 13).

[0048] According to certain embodiments, the hNEU1 coding sequence comprises the nucleotide sequence of sequence number: 1, and the hNEU1 protein produced by the sequence comprises the amino acid sequence of sequence number: 2. It is conceivable that the hNEU1 coding sequence of the present invention may comprise one or more degenerate nucleotides, as long as the protein encoded by the degenerate nucleotide sequence (i.e., hNEU1 protein) retains a specific activity or function. The term "degenerate nucleotide sequence" (also referred to as "nucleotide degeneracy") refers to a sequence comprising one or more degenerate codons (compared to a reference polynucleotide molecule encoding a polypeptide). Degenerate codons comprise different nucleotide triplets but encode the same amino acid residue (e.g., GAU and GAC triplets are both used to encode Asp). In addition or optionally, the hNEU1 coding sequence of the present invention may comprise one or more optimized codons (i.e., codon optimization) to increase the expression level of hNEU1 protein in cells. Therefore, the scope of the present application also covers nucleotide sequences having degenerate nucleotides and / or optimized nucleotides, as long as the nucleotide sequence has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence similarity to SEQ ID NO: 1.

[0049] Alternatively, the hNEU1 protein may be modified so that its amino acid sequence comprises one or more mutations to reduce the self-aggregation reaction. For example, according to certain embodiments, compared to the hNEU1 protein of SEQ ID NO: 2, the modified hNEU1 protein comprises two mutations in its amino acid sequence, wherein the amino acid residues "G" at No. 248 and No. 252 in the hNEU1 protein (SEQ ID NO: 2) are both replaced with amino acid residues "S", thereby reducing the degree / content of self-aggregation. In these embodiments, the modified hNEU1 protein comprises the amino acid sequence of SEQ ID NO: 4. According to certain exemplary embodiments, the modified hNEU1 protein (SEQ ID NO: 4) is encoded by a coding sequence comprising the nucleotide sequence of SEQ ID NO: 3. As described above, the coding sequence encoding the modified hNEU1 protein may comprise one or more degenerate nucleotides and / or optimized nucleotides, as long as the protein produced thereby (i.e., the modified hNEU1 protein) retains a specific activity or function. Therefore, the scope of the present application also covers nucleotide sequences having degenerate nucleotides and / or optimized nucleotides, as long as the nucleotide sequence has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence similarity to SEQ ID NO: 3.

[0050] It is also optional that the hNEU1 coding sequence may include introns in its nucleotide sequence. Depending on the desired purpose, the intron may be derived from the hNEU1 gene (i.e., the hNEU1 intron; SEQ ID NO: 15) or other genes other than the hNEU1 gene (e.g., the beta-globulin intron; SEQ ID NO: 14). According to certain exemplary embodiments, the hNEU1 coding sequence includes the hNEU1 intron (SEQ ID NO: 15) in its nucleotide sequence. In these embodiments, the hNEU1 coding sequence includes the nucleotide sequence of SEQ ID NO: 5. As known to those skilled in the art, an intron refers to a nucleotide sequence in a nucleic acid that is removed through the RNA splicing process and therefore will not be expressed or acted on in the mature RNA and its translated protein product. The nucleotide sequence of an intron may include one or more variations (e.g., nucleotide insertions, deletions and / or substitutions) without affecting the activity or function of the protein product. Therefore, the scope of the present application also encompasses hNEU1 coding sequences having one or more variations in the intron region. According to different embodiments of the present application, the hNEU1 coding sequence may comprise a nucleotide sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to SEQ ID NO: 5.

[0051] According to certain embodiments, the hPPCA coding sequence comprises the nucleotide sequence of SEQ ID NO: 6, and the hPPCA protein produced by the sequence comprises the amino acid sequence of SEQ ID NO: 7. As described above, one skilled in the art may replace the nucleotides of the hPPCA coding sequence with one or more degenerate codons or optimized codons according to the desired purpose, as long as the protein encoded by the degenerate nucleotide sequence or the optimized sequence (i.e., the hPPCA protein of the present invention) maintains a specific activity or function. Therefore, the scope of the present application also includes nucleotide sequences having degenerate nucleotides and / or optimized nucleotides, as long as the nucleotide sequence has at least 85% sequence similarity with SEQ ID NO: 6, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity with SEQ ID NO: 6.

[0052] According to certain preferred embodiments, the leader sequence in the hPPCA coding sequence (SEQ ID NO: 6) is replaced with a leader sequence (also referred to as a "signal peptide" or "signal sequence") derived from human albumin (hAlb; SEQ ID NO: 8). In these embodiments, the hAlb-PPCA coding sequence comprises the nucleotide sequence of SEQ ID NO: 9, and the hAlb-hPPCA protein produced by the sequence comprises the amino acid sequence of SEQ ID NO: 10. Based on the above-mentioned nucleotide degeneracy and codon optimization, the scope of the present application also covers hAlb-PPCA coding sequences with variants (e.g., nucleotide sequences having at least 85% sequence similarity to SEQ ID NO: 9). It is conceivable that a person skilled in the art can replace the leader sequence in the hPPCA coding sequence with an appropriate leader sequence (e.g., a leader sequence that can direct a protein to a secretory pathway; for example, the leader sequence of CD33, interleukin-2, or insulin).

[0053] Preferably, the expression set of the present application further comprises a bicistronic component, which is located between the hNEU1 coding sequence and the hPPCA coding sequence.

[0054] The bicistronic component can be an IRES, a 2A polypeptide, or any sequence or polypeptide known to express multiple proteins from a single transcript. IRES is a sequence that recruits ribosomes and performs cap-independent translation. Operationally, IRES acts as a connector to connect two coding sequences in a bicistronic vector, allowing the cell to translate two proteins. According to certain exemplary embodiments, the bicistronic component is an IRES and comprises a nucleotide sequence of sequence number: 16. According to certain alternative embodiments, the bicistronic component is an IRES and comprises a nucleotide sequence of sequence number: 17. The 2A polypeptide, also known as a "2A self-cleaving polypeptide," is a polypeptide having 18 to 22 amino acid residues that can induce ribosome jumping during the process of cellular protein translation. Exemplary 2A polypeptides known to those skilled in the art include, but are not limited to, T2A ("EGRGSLLTCGDVEENPGP"; SEQ ID NO: 23), P2A ("ATNFSLLKQAGDVEENPGP"; SEQ ID NO: 24), E2A ("QCTNYALLKLAGDVESNPGP"; SEQ ID NO: 25), and F2A ("VKQTLNFDLLKLAGDVESNPGP"; SEQ ID NO: 26).

[0055] According to a preferred embodiment, the expression group of the present application comprises, from the 5' end to the 3' end, the human NEU1 P3 promoter (SEQ ID NO: 11), the hNEU1 coding sequence (SEQ ID NO: 1), IRES (SEQ ID NO: 16), and the hAlb-PPCA coding sequence (SEQ ID NO: 9). In this embodiment, the expression group comprises the nucleotide sequence of SEQ ID NO: 22. According to different embodiments of the present application, the expression group may comprise one or more degenerate nucleotides and / or optimized nucleotides in its nucleotide sequence without affecting the activity or function of the protein product (i.e., hNEU1 and hPPCA). Accordingly, the scope of the present application also covers the expression group whose nucleotide sequence has 85% sequence similarity with SEQ ID NO: 22.

[0056] According to certain alternative embodiments, the expression set of the present application may include human NEU1P3 promoter (SEQ ID NO: 11), hAlb-PPCA coding sequence (SEQ ID NO: 9), IRES (SEQ ID NO: 16), and hNEU1 coding sequence (SEQ ID NO: 1) from 5' to 3' end.

[0057] Optionally, the expression vector of the present application further comprises a beta-globulin intron (SEQ ID NO: 14), which is located between the promoter and the hNEU1 coding sequence.

[0058] Preferably, the expression vector of the present application further comprises a polyA sequence, which is located at the 3' end of the hPPCA coding sequence.

[0059] The polyA sequence (i.e., the addition of a polyadenylic acid tail to the RNA transcript) is important for the nuclear export, translation and stability of mRNA. According to certain exemplary embodiments, the polyA sequence is derived from human growth hormone (hGH). In these embodiments, the polyA sequence comprises a nucleotide sequence having at least 85% sequence similarity to sequence number: 18; preferably, it comprises a nucleotide sequence having at least 90% sequence similarity to sequence number: 18; more preferably, it comprises a nucleotide sequence having at least 95% sequence similarity to sequence number: 18. In a specific embodiment, the polyA sequence comprises the nucleotide sequence of sequence number: 18. It is conceivable that those skilled in the art can select an appropriate polyA sequence according to the purpose of implementation; for example, the polyA sequence of bovine growth hormone (bGH), SV40, rabbit beta globulin or herpes simplex virus type I thymidine kinase (HSV-TK).

[0060] Optionally, the recombinant AAV vector of the present invention further comprises an enhancer adjacent to the promoter. According to one embodiment, the recombinant AAV vector further comprises the CMV major immediate early enhancer (MIE) to stimulate the expression of the hNEU1 coding sequence and the hPPCA coding sequence.

[0061] Optionally, the recombinant AAV vector of the present invention further comprises a transcription start site, a stop site and / or an effective RNA processing signal; for example, a cleavage signal, a regulatory element that can increase gene expression (e.g., a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a sequence that can stabilize cytoplasmic mRNA, a sequence that can increase translation efficiency (e.g., a Kozak consensus sequence), a sequence that can increase protein stability, and / or a sequence that can increase secretion of the encoded product when necessary.

[0062] The size of the recombinant AAV vector can be changed according to regulatory sequences such as promoters, promoters, introns and poly A. According to certain embodiments, the size of the recombinant AAV vector is about 4.4 kilobase pairs (kb) to 5.3 kb. According to an operating example, the size of the recombinant AAV vector is about 4.7 kb.

[0063] (ii) Recombinant AAV

[0064] The recombinant AAV vector described in part (i) of the present application can be used to prepare a recombinant AAV, wherein the recombinant AAV vector, a helper plasmid (an AAV plasmid carrying adenovirus-related genes such as E2A, E4 and VA that enable AAV replication) and a Rep-Cap plasmid (a plasmid carrying Rep and Cap genes that enable viral replication and assembly) are co-transfected into a host cell (e.g., HEK293 cells) to prepare the recombinant AAV. The obtained recombinant AAV comprises an AAV capsid and a recombinant AAV vector encapsidated in the AAV capsid. The method of co-transfecting three plasmids into host cells to prepare recombinant AAV is a technique known to those skilled in the art; for the sake of brevity, it will not be repeated here.

[0065] The amino acid sequence of the AAV capsid is determined by the Cap sequence of the Rep-Cap plasmid. Preferably, the AAV capsid has a broad tropism for the brain and internal organs. According to certain preferred embodiments, the AAV capsid is derived from AAV9. In one embodiment, the AAV capsid comprises the amino acid sequence of SEQ ID NO: 21.

[0066] (iii) Pharmaceutical compositions or drugs comprising recombinant AAV

[0067] According to the embodiments of the present application, recombinant AAV can treat sialic acid storage disease by simultaneously expressing hNEU1 and hPPCA without causing protein aggregation. Therefore, another aspect of the present application is the use of recombinant AAV in preparing a pharmaceutical composition or drug for treating sialic acid storage disease. The pharmaceutical composition or drug comprises the recombinant AAV described in (ii) of the present application and a pharmaceutically acceptable drug carrier.

[0068] A pharmaceutically acceptable drug carrier can be any pharmaceutically acceptable material or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or coating material, used to carry an active ingredient (e.g., the recombinant AAV of the present invention), or to transport an active ingredient (e.g., the recombinant AAV of the present invention) from one organ or part of the body to another organ or part of the body. The drug carrier must be "acceptable", that is, compatible with other ingredients in the formulation; at the same time, it minimizes the degradation of the active ingredient and minimizes the side effects to the individual. Depending on the purpose of implementation, the pharmaceutical composition or drug of the present application may also include one or more pharmaceutically acceptable additives, including surfactants, buffers, diluents, stabilizers, emulsifiers, dispersants, suspending agents and preservatives.

[0069] The pharmaceutically acceptable drug carrier can be selected according to the administration mode of the pharmaceutical composition or drug. The pharmaceutical composition or drug of the present invention can be administered to the subject by intracerebrospinal fluid or intravenous injection.

[0070] The pharmaceutical composition or drug for injection can be prepared as a sterile water-soluble, sterile non-aqueous solution, suspension or emulsion. Exemplary non-aqueous solutions include, but are not limited to, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and organic esters for injection such as ethyl oleate. Exemplary aqueous solutions include water, emulsions and suspensions, such as physiological saline or buffered media. Common drug carriers suitable for parenteral administration include sodium chloride solution, Ringer's dextrose, glucose, lactated Ringer's injection or fixed oil, while drug carriers suitable for intravenous administration generally include liquids, nutritional supplements and electrolytes (e.g., electrolytes associated with Ringer's dextrose), etc. According to certain operating embodiments, the drug carrier is phosphate buffered saline (PBS).

[0071] According to certain embodiments, the pharmaceutical composition or drug of the present invention is prepared in the form of a liquid suspension suitable for administration into a subject suffering from sialidosis by injection into the cerebrospinal fluid or intravenously. In certain embodiments, each milliliter of the suspension contains at least 1×10 13 GC recombinant AAV; preferably, each ml of the suspension contains 1×10 13 to 1×10 15 GC of recombinant AAV. The GC of recombinant AAV can be determined by conventional methods for quantifying viruses, such as quantitative PCR (qPCR), digital droplet PCR (ddPCR), or AAV vector genome (vg) titration.

[0072] (iv) Use of recombinant AAV, pharmaceutical composition or drug for treating sialic acid storage disease

[0073] Another aspect of the present application is a method for treating sialidosis in an individual, comprising administering to the individual an effective amount of the recombinant AAV, pharmaceutical composition or medicament of the present invention.

[0074] The recombinant AAV, pharmaceutical composition or medicament of the invention can be administered to a subject by any suitable route; for example, intracerebrospinal fluid, intravenous, intraperitoneal, intraarterial or intramuscular injection.

[0075] According to certain embodiments of the present application, about 1×10 13 to 1×10 15 The recombinant AAV of GC is administered to an individual; for example, 1×10 13 , 1.5×10 13 , 2×10 13 , 2.5×10 13 , 3×10 13 , 3.5×10 13 , 4×10 13 , 4.5×10 13 , 5×10 13 , 5.5×10 13 , 6×10 13 , 6.5×10 13 ,7×10 13 , 7.5×10 13 , 8×10 13 , 8.5×10 13 ,9×10 13 , 9.5×10 13 , 1×10 14 , 1.5×10 14 , 2×10 14 , 2.5×10 14 , 3×10 14 , 3.5×10 14 , 4×10 14 , 4.5×10 14 , 5×10 14 , 5.5×10 14 , 6×10 14 , 6.5×10 14 ,7×10 14 , 7.5×10 14 , 8×10 14 , 8.5×10 14 ,9×10 14 , 9.5×10 14 or 1×10 15 According to certain alternative embodiments, about 1×10 13to 1×10 15 The recombinant AAV of GC is administered to an individual. As can be imagined, the amount of recombinant AAV administered to an individual will vary with clinical factors such as age, gender, underlying disease, treatment plan, course of treatment, and infection. Those skilled in the art or clinical medical personnel can adjust or optimize the administration dose of recombinant AAV according to actual needs.

[0076] According to certain embodiments, a single dose of recombinant AAV, pharmaceutical composition, or drug is administered to a subject. In one embodiment, approximately 1.0×10 50 μg of recombinant AAV, pharmaceutical composition, or drug is administered to a subject using a lumbar puncture, a cisternal puncture, or a microcatheter inserted into the spinal canal from the lumbar region to the cisterna magna. 13 GC to about 1.0×10 per kg brain weight 15 The recombinant AAV, pharmaceutical composition or drug of the GC is administered to the cerebrospinal fluid (CSF) of the subject. In another embodiment, CSF and intravenous administration can be combined, wherein the CSF and intravenous administration can be performed simultaneously or sequentially to the subject.

[0077] The amount of virus particles administered to an individual will vary depending on the yield of AAV in the cell culture system. According to certain embodiments, the average yield of AAV is about 50%, wherein about 50% of the virus particles have the vector genome encapsidated in the capsid, i.e., about 50% intact particles. Thus, for 1×10 14 For an exemplary dose of GC, the total particle dose is about 2×10 14 GC.

[0078] According to certain embodiments of the present application, administration of recombinant AAV may be performed in an individual (e.g., Neu1 - / - hNEU1 and hPPCA are effectively expressed in the brain of mice, thereby producing a therapeutic effect on the individual's sialidosis.

[0079] According to certain embodiments, the sialidosis is sialidosis type I. According to certain embodiments, the sialidosis is sialidosis type II.

[0080] The subject that can be treated by the method of the present invention is a mammal, for example, a human, mouse, rat, guinea pig, hamster, monkey, pig, dog, cat, horse, sheep, goat, cow and rabbit. Preferably, the subject is a human.

[0081] A number of experimental examples are presented below to illustrate certain aspects of the present invention, so as to facilitate those skilled in the art to practice the present invention, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art can fully utilize and practice the present invention without excessive interpretation after reading the description presented herein. All public documents cited herein are considered to be part of this specification in their entirety. Example

[0082] Materials and Methods

[0083] Neu1 knock-out (KO) mice - / - )

[0084] Using CRISPR / Cas9 technology to generate Neu1 - / - In brief, Cas9 mRNA and two guide RNAs (sgRNAs; sequence numbers: 27 and 28) targeting the first and third coding exons of the mouse Neu1 gene were injected into fertilized eggs of wild-type C57BL / 6J mice. These embryonic mice were sequenced to determine whether their genomes had deletions, and then bred with C57BL / 6J mice to pass on the allele to offspring. According to the sequencing analysis results, Neu1 - / - The mouse Neu1 gene deletion occurred between middle exon 1 and middle exon 3, resulting in the fusion of exons 1 and 3. The mice were cultured in a 12-hour light and dark cycle and provided with sufficient drinking water and food.

[0085] Neu1 - / - The phenotype of the mouse model is similar to that of the knockout mouse model of sialidosis published by Natalie de Geest et al. (Systemic and neurologic abnormalities distinguish the lysosomaldisorders sialidosis and galactosialidosis in mice, Hum Mol Genet., 2002; 11(12): 1455-64), in which Neu1 is expressed in wild-type mice compared with wild-type mice. - / - The mice were smaller in size and had shorter skulls (results not shown). Histological analysis showed that 8-month-old Neu1 - / - The mice showed vacuoles in the kidneys, bone marrow, and liver (results not shown). - / - The mice began to show slow weight gain and motor dysfunction starting at 4 months of age and usually died before 9 months of age (results not shown).

[0086] Construction of recombinant AAV (AAV-P3-NP)

[0087] All sequences are derived from pAAV-MCS or prepared by oligonucleotide synthesis technology. AAV2 vector is used to construct a gene therapy vector AAV-P3-hNEU1-IRES-hPPCA (AAV-P3-NP), which contains human NEU1 cDNA driven by P3 NEU1 promoter, and human PPCA cDNA containing human albumin leader polypeptide sequence connected to the human NEU1 cDNA by IRES. The prepared hNEU1-hPPCA expression group has ITRs derived from AAV2 at both ends. At the same time, the polyadenylation signal of hGH is constructed into the transgene. According to the sequencing results, the hNEU1-hPPCA expression group of the present invention contains the nucleotide sequence of sequence number: 22.

[0088] COS-1 cells were used to determine the expression of the constructs. Briefly, COS-1 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). The expression plasmid was transfected into COS-1 cells cultured in 6 cm culture dishes at 3000 μg / mL. 48 hours after transfection, cells were fixed with polyformaldehyde for immunofluorescence staining or lysed for enzyme activity assay. For Western blot analysis, cells were disrupted by ultrasound treatment, centrifuged, and the supernatant and cell pellet were analyzed. Denatured proteins were separated by 10% polyacrylamide gel electrophoresis and transferred to nylon paper, followed by the addition of primary and secondary antibodies conjugated to horseradish peroxidase (HRP).

[0089] To prepare recombinant AAV, a bicistronic vector, a helper plasmid (with genes from adenovirus such as E2A, E4, and VA that promote AAV replication) and a Rep-Cap plasmid (with genes such as Rep and Cap that promote viral replication and assembly) were co-transfected into HEK293 cells. The transfected cells were cultured at 37°C for 72 hours. After centrifugation at 1500g for 5 minutes, the cell pellet was resuspended in 0.5 ml of cell lysis buffer (10mM Tris-HCl (pH8.5), 150mM NaCl) and frozen / thawed three times using a dry ice / ethanol bath and a 37°C water bath to obtain a crude lysate. The crude lysate was centrifuged at 3000g for 10 minutes. The supernatant was transferred to a new tube and purified to obtain recombinant AAV.

[0090] Animal testing

[0091] This study used two approaches to perform gene therapy. During intracerebroventricular (ICV) injection, 2 μl of recombinant AAV was injected into the bilateral ventricles of newborn mice (postnatal day 0, P0), with an injection dose of 2×10 10GC. For intravenous (IV) injection, 30 μl of recombinant AAV was injected into the facial vein of newborn mice (postnatal day 2, P2) at a dose of 5 × 10 11 GC. During the virus injection, the mice were anesthetized on ice. The viral vector was injected into the facial vein using an insulin needle or The viral vector was injected into the space between the lambdoid suture tip and the bregma by syringe for ICV administration. After injection, the mice were placed under a warm lamp until the skin color returned to normal.

[0092] For functional analysis, a roller test was performed on a 3 cm diameter shaft at 5 speeds (including 16, 20, 24, 28 and 32 rpm), with each speed tested 3 times, and the mice were allowed to rest for 5 minutes between each test. The results of each test were averaged. The grip strength of the animal's limbs was measured with a grip dynamometer. Each test was tested 3 times, and the best test result was recorded.

[0093] Immunohistochemistry (IHC) staining

[0094] The mice were sacrificed by perfusion with PBS containing 4% paraformaldehyde. After antigen retrieval and cell permeabilization, 25 μm frozen coronal sections (Bregma -1.5 to -2.0 mm, including the cerebral cortex, hippocampus and midbrain) were subjected to IHC staining. Dorsal root ganglia (DRG) were isolated, pooled and processed in a similar manner. Tissue sections were treated with primary antibodies diluted in blocking buffer and placed at 4°C until the next day. They were then washed and fluorescently conjugated secondary antibodies were added and reacted at room temperature for 30 minutes. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) at 0.1 μg / ml in PBS for 5 minutes at room temperature. Staining results were detected by confocal microscopy and analyzed by software.

[0095] To count cells expressing LAMP1 or GFAP (LAMP1 or GFAP positive cells), whole brain sections were imaged (20x magnification) using an upright fluorescence and bright field system. Images were analyzed using software. Briefly, after adjusting brightness / contrast and aligning the images, regions of interest (ROIs) were manually defined, while signal region gating was used to isolate specific areas with significant fluorescence signals.

[0096] statistics

[0097] The number of animals in each experimental design was the same, unless animals died or were sacrificed during the experimental analysis, each group had n ≥ 5. T-tests were used to evaluate the differences between the two groups.

[0098] Example 1 Confirmation of hNEU1, hPPCA and bicistronic expression groups

[0099] To analyze the sialidase activity of hNEU1, three hNEU1 expression plasmids were constructed, including hNEU1 plasmid, hAlb-hNEU1 plasmid and mutant hNEU1 plasmid, wherein the hNEU1 plasmid carries the original hNEU1 cDNA (sequence number: 1); the hAlb-hNEU1 plasmid carries the hNEU1 cDNA with an albumin leader sequence (hAlb-hNEU1), and the mutant hNEU1 plasmid carries the hNEU1 cDNA with a mutation to reduce the self-aggregation reaction (mutant hNEU1; sequence number: 3). The three plasmids were transfected into COS-1 cells to determine the sialidase activity in the transfected cells. The data indicated that the original hNEU1 cDNA expressed moderate sialidase activity (Table 1). Since overexpression of hNEU1 will cause self-aggregation reaction, the present invention does not need to increase the expression amount of NEU1 protein through codon optimization.

[0100] Table 1 Expression of different hNEU1 constructs

[0101] In addition, four hPPCA expression plasmids were constructed in this study, including hPPCA plasmid, hAlb-hPPCA plasmid, coPPCA plasmid and hAlb-coPPCA plasmid. The hPPCA plasmid carries the original hPPCA cDNA (SEQ ID NO: 6), the hAlb-hPPCA plasmid carries the hPPCA cDNA with an albumin leader sequence (hAlb-hPPCA; SEQ ID NO: 9), the coPPCA plasmid carries the codon-optimized hPPCA (coPPCA), and the hAlb-coPPCA plasmid carries the codon-optimized hPPCA with an albumin leader sequence (hAlb-coPPCA). The four hPPCA plasmids were co-transfected with the hNEU1 plasmid or the hAlb-hNEU1 plasmid into COS-1 cells, and the sialidase activity in the transfected cells was determined. As shown in the results in Table 2, hPPCA with an albumin leader polypeptide (ie, hAlb-hPPCA) produced the highest sialidase activity compared to other hPPCA constructs (Table 2).

[0102] Table 2 Sialidase activity of cells transfected with different hNEU1 and hPPCA constructs

[0103] Next, hNEU1 was expressed alone or co-expressed with hPPCA in COS-1 cells. IHC results indicated that overexpression of hNEU1 caused cell damage and resulted in changes in cell morphology, while co-expression of hPPCA protected COS-1 cells from damage caused by hNEU1 and improved cell morphology (results not shown). In COS-1 cells transfected with NEU1 expression plasmids, most of the expressed NEU1 protein was present in the cell precipitate (results not shown). However, when the NEU1 expression plasmid was co-transfected with the hPPCA expression plasmid into the cells, the expressed NEU1 protein was present in the soluble fraction (results not shown). These data confirm that expression of hPPCA can improve the solubility of the hNEU1 protein.

[0104] After confirming the expression and activity of hNEU1 and hPPCA of the present invention, a bicistronic vector containing the hNEU1 coding sequence and the hAlb-hPPCA coding sequence was constructed according to the process described in "Materials and Methods", wherein the hNEU1 coding sequence and the hAlb-hPPCA coding sequence were connected by an IRES component, and the expression of both sequences was driven by the P3 promoter. The results in Table 3 confirmed that high sialidase activity was produced in COS-1 cells after transfection of the bicistronic vector into the cells. Insertion of an alternative IRES (IRES1; SEQ ID NO: 17) or an intron into the hNEU1 coding sequence did not further improve the sialidase activity (Table 4).

[0105] Table 3 Sialidase activity of cells transfected with different constructs hNEU1: hNEU1 plastid. hAlb-hPPCA: hAlb-hPPCA plasmid.

[0106] Table 4 Sialidase activity of cells transfected with different constructs NEU1-IRES-PPCA: a bicistronic vector comprising the hNEU1 coding sequence and the hAlb-hPPCA coding sequence, wherein the two coding sequences are connected by IRES of SEQ ID NO: 16. NEU-IRES1-PPCA: a bicistronic vector comprising the hNEU1 coding sequence and the hAlb-hPPCA coding sequence, wherein the two coding sequences are connected by IRES of SEQ ID NO: 17.

[0107] The data in Table 5 further demonstrate that the P3 promoter can be used to drive the expression of hNEU1 and hPPCA in COS-1 and N2A cells.

[0108] Table 5 Sialidase activity of cells transfected with different constructs

[0109] Next, as described in "Materials and Methods", the bicistronic vector, helper plasmid and Rep-Cap plasmid were co-transfected into HEK293 cells to prepare recombinant AAV ("AAV-P3-NP"). The data in Table 6 indicate that the viral genome containing the hNEU1-hPPCA expression group can be successfully encapsidated in the AAV9 capsid.

[0110] Table 6 Viral amount of specific recombinant AAV AAV-P3-NP: A recombinant AAV obtained by co-transfection of a bicistronic vector, a helper plasmid and a Rep-Cap plasmid, wherein the bicistronic vector contains an hNEU1-hPPCA expression group driven by the P3 promoter. AAV-CMV-NP: A recombinant AAV obtained by co-transfection of a bicistronic vector, a helper plasmid and a Rep-Cap plasmid, wherein the bicistronic vector contains an hNEU1-hPPCA expression group driven by a CMV promoter. AAV-P3-GFP: A recombinant AAV obtained by co-transfection of GFP vector, helper plasmid and Rep-Cap plasmid, where the GFP vector contains the GFP gene driven by the P3 promoter.

[0111] Example 2 In vivo test

[0112] 2.1 AAV-P3-NP of the present invention in Neu1 - / - Expression in mice

[0113] Recombinant AAV-P3-NP was injected intracerebroventricularly (ICV) or intravenously (IV) into neonatal Neu1 - / - In mice. Three months after the injection, the brain tissue of the mice was isolated. IHC analysis was performed on the brain tissue sections. According to the results of IHC analysis, in untreated Neu1 - / - PPCA signals were detected in mouse brain tissue, but NEU1 signals were not detected; and a large amount of LAMP1 accumulated in IbaI-expressing (IbaI-positive) microglial cells (results not shown). In contrast, hNEU1 was widely expressed in ICV-injected Neu1 - / -Its expression can be observed in the brain tissues of mice, including the cerebral cortex, hippocampus, putamen, brainstem and cerebellum (results not shown). In addition, the analysis results also showed that the perinuclear plasm of microglial cells showed a positive reaction to hNEU1 staining, while the expression signal of hNEU1 could not be detected in astrocytes (results not shown). This study also performed hPPCA staining analysis; the results showed that the staining signal of hPPCA was weaker than that of hNEU1 (results not shown). If the tissues were double stained for hNEU1 and hPPCA at the same time, it was found that the two proteins were co-expressed in Neu1 - / - In addition, the results of IHC analysis confirmed that most of the hNEU1-positive cells were NeuN-positive cells, suggesting that after ICV administration of the AAV-P3-NP of the present invention, hNEU1 is mainly expressed in neurons (results not shown).

[0114] These data demonstrate that the specificity of AAV-P3-NP is similar to the natural biodistribution of NEU1 protein in the brain.

[0115] 2.2 Effect of AAV-P3-NP of the present invention on Neu1 - / - Effects in mice

[0116] This example will evaluate whether the AAV-P3-NP of the present invention can be used as a therapeutic agent for treating sialic acid storage disease. As described in Materials and Methods, Neu1 was injected by ICV or IV. - / - Mice (a mouse model of sialic acid storage disease) were administered with the AAV-P3-NP of the present invention. PBS was used as a negative control group in this experiment. Biochemical analysis, histological analysis and functional testing were used to determine the therapeutic efficacy of the AAV-P3-NP of the present invention on sialic acid storage disease.

[0117] 2.2.1 Biochemical and histological analysis

[0118] One-month-old wild-type, Neu1 - / - or ICV-injected Neu1 - / - The results showed that compared with wild-type mice, Neu1 - / - The activity of sialidase in the mouse brain was severely reduced, while the activity of sialidase in the liver was partially deficient; ICV injection of the present invention AAV-P3-NP normalized the activity of sialidase in the brain (results not shown). In addition, compared with wild-type mice, Neu1 - / - The brain and liver of mice have high β-galactosidase activity, and ICV injection of the AAV-P3-NP of the present invention reduces the activity of β-galactosidase in the brain and liver of mice (results not shown).

[0119] Neu1 injected IV or ICV at 8 months of age - / - Long-term therapeutic efficacy of gene therapy on brain pathology was observed in mice. Immunofluorescence analysis of LAMP1, NEU1 and GFAP was performed in brain regions such as the cortex, hippocampus and midbrain. The results showed that in Neu1 - / - Increased numbers of astrocytes (stained with GFAP) were observed in all regions of the mouse brain (a pathological phenomenon called "astrocytosis", a common pathological phenomenon in brain lysosomal storage diseases), while Neu1 - / - The number of microglia in the mouse brain did not change significantly (results not shown). According to the analysis results, the morphology of these astrocytes did not change, and LAMP1 (a membrane marker for lysosomes) was not accumulated in the astrocytes. In addition, IHC analysis using anti-LAMP1 antibodies showed that there were clearly filled lysosomes in microglia throughout the brain, and these microglia were round (a characteristic of activation; results not shown). AAV-P3-NP administered by ICV or IV injection inhibited Neu1 - / - The proliferation of stellate cells in mice (results not shown). - / - Activated microglia with swelling and larger cell volume in the mouse brain, Neu1 treated with AAV-P3-NP - / - Microglia in the mouse brain had normal morphology, and no LAMP1 staining signals were observed in the brain (results not shown). - / - In mice, NEU1 signals increased, while LAMP1 and GFAP signals decreased. Cell counts of whole brain tissue sections indicated that ICV or IV injection of the AAV-P3-NP of the present invention reduced the number of LAMP1-positive cells, and ICV injection of the AAV-P3-NP of the present invention further reduced the number of GFAP-positive cells (results not shown). These results confirm that AAV-P3-NP treatment can improve Neu1 - / - Pathological changes in the mouse brain.

[0120] This study also analyzed untreated 8-month-old Neu1 - / - Dorsal root ganglion (DRG) slice specimens of mice. The results showed that more than half of the neurons in DRG showed significant LAMP1 signals (results not shown). - / -In mice, the proportion of LAMP1-positive neurons decreased (results not shown). We further used antibodies specific for peripherin (a marker of pain receptors) or neurofilaments (a marker of mechanical receptors) to detect the expression of LAMP1 in wild-type and Neu1 - / - Mouse slice specimens were stained and analyzed. - / - How is the intensity of LAMP1 staining in mice? Are neurons positive for peripherin or neurofilaments different from those in wild-type and Neu1 - / - Similar shapes and numbers were observed in mice (results not shown).

[0121] 2.2.2 Functional Testing

[0122] Compared with wild-type control mice, Neu1 - / The mice began to gain less weight at 4 months of age and performed worse in the roller test at 24 and 32 rpm (results not shown). - / - Mice have impaired grip strength from an early age (results not shown). Intraventricular or intravenous injection of AAV-P3-NP can improve Neu1 - / - The body weight, performance of the roller test and the grip strength of the mice. According to the analysis data of the roller test, compared with the untreated Neu1 - / - Mice, treated with ICV injection of Neu1 - / - Mice at 7 months of age showed better locomotion at 28 rpm (p<0.01), and at 8 months of age showed better locomotion at 24 rpm (p<0.001), 28 rpm (p<0.0001), and 32 rpm (p<0.0001). - / - Mice showed better locomotion at 24 rpm (p<0.05) at 6 months of age, at 28 rpm (p<0.001) and 32 rpm (p<0.05) at 7 months of age, and at both 28 rpm (p<0.01) and 32 rpm (p<0.001) at 8 months of age. - / - Mice, received intravenous injection of Neu1 - / - Mice had better grip strength at 2 months (p<0.01), 3 months (p<0.0001), 4 months (p<0.001), 5 months (p<0.05), and 7 months (p<0.05). - / -Mice showed better gripping power at 3 months of age (p<0.05). It is worth noting that recombinant AAV does not replicate with cell division; therefore, the AAV-P3-NP of the present invention administered during the neonatal period will disappear in large quantities after cell proliferation in muscle and other body tissues. Therefore, the efficacy of improving motor ability is speculated to be caused by the treatment results of the nervous system.

[0123] The body weight was then analyzed based on the sex of the mice. - / - The female mice were lighter and less responsive to treatment (results not shown). The female mice also performed worse in the tumble test (32 rpm) (results not shown). Due to insufficient sample size after grouping, further statistical analysis was not possible.

[0124] These data demonstrate that administration of the present AAV-P3-NP can improve Neu1 - / - The pathological changes, body weight and motor ability of mice (a known animal model of sialidosis, which develops systemic and neurodegenerative symptoms similar to those of human sialidosis patients) were observed. Therefore, the AAV-P3-NP of the present invention can be used as a potential drug for the treatment of sialidosis.

[0125] Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those skilled in the art may make various changes and modifications thereto without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on that defined by the accompanying patent application.

Claims

1. A recombinant viral vector comprising an AAV 5' inverted terminal repeat (ITR) sequence, an AAV 3' ITR sequence, and an expression group, wherein the expression group is located between the AAV 5' ITR and 3' ITR sequences and comprises: (a) a promoter; (b) a first coding sequence, which is operably linked to the promoter and encodes a human neuraminidase 1 (NEU1); and (c) a second coding sequence, which is operably linked to the promoter and encodes a human protective protein / cathepsin A (PPCA).

2. The recombinant viral vector of claim 1, wherein the AAV 5'ITR and 3'ITR sequences are derived from AAV serotype 2 (AAV2).

3. The recombinant viral vector of claim 2, wherein the AAV 5'ITR and 3'ITR sequences comprise the nucleotide sequences of SEQ ID NOs: 19 and 20, respectively. The recombinant viral vector according to claim 1 , wherein the promoter is a human NEU1 promoter or a cytomegalovirus (CMV) promoter.

5. The recombinant viral vector of claim 4, wherein the promoter comprises the nucleotide sequence of sequence number:

11. 6 . The recombinant viral vector of claim 1 , wherein the human NEU1 and human PPCA comprise the amino acid sequences of SEQ ID NOs: 2 and 10, respectively.

7. The recombinant viral vector of claim 6, wherein the first and second coding sequences comprise the nucleotide sequences of sequence numbers: 1 and 9, respectively.

8. The recombinant viral vector of claim 1, wherein the expression vector further comprises an internal ribosome entry site (IRES) located between the first and second coding sequences.

9. The recombinant viral vector of claim 1, wherein the expression group further comprises a third coding sequence located between the first and second coding sequences, wherein the third coding sequence is operably linked to the promoter to encode a 2A polypeptide.

10. The recombinant viral vector of claim 1, wherein the expression vector further comprises a polyadenylation (polyA) sequence located at the 3' end of the second coding sequence. The recombinant viral vector of claim 10 , wherein the polyA sequence is derived from human growth hormone (hGH). 12 . The recombinant viral vector of claim 1 , wherein the expression group comprises the nucleotide sequence of SEQ ID NO:

22.

13. A recombinant adeno-associated virus (AAV) comprises: an AAV capsid, and a recombinant viral vector as described in claim 1 encapsidated in the AAV capsid.

14. The recombinant AAV of claim 13, wherein the AAV capsid is derived from AAV serotype 9 (AAV9).

15. The recombinant AAV of claim 14, wherein the AAV shell comprises the amino acid sequence of sequence number:

21.

16. A pharmaceutical composition comprising the recombinant AAV as claimed in claim 13 and a pharmaceutically acceptable drug carrier.

17. A method of treating sialidosis in an individual, comprising administering to the individual the recombinant AAV of claim 13 or the pharmaceutical composition of claim 16 to reduce or delay symptoms of sialidosis.

18. The method of claim 17, wherein about 1×10 13 to 1×10 15 The recombinant AAV has a gene copy number.

19. The method of claim 17, wherein about 1×10 13 to 1×10 15 The recombinant AAV has a gene copy number.

20. The method of claim 17, wherein the recombinant AAV or pharmaceutical composition is injected into the subject intravenously or intravenously.

21. The method of claim 17, wherein the individual is a human.

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