Recombinant adeno-associated viral vectors for the treatment of fabry disease

Abstract

Polypeptide variants of human alpha-Gal A, codon-optimized coding sequences for the polypeptide variants and wild-type protein, and recombinant adeno-associated viral (rAAV) vectors comprising the coding sequences are provided. Also provided are chimeric promoters that can drive high expression in the liver and heart, which are suitable for use in the rAAVs of the present application.

Description

Technical Field

[0001] This disclosure pertains to the field of gene therapy. Specifically, this disclosure provides a nucleotide sequence encoding human α-galactosidase A (α-GalA) or a variant thereof, and a recombinant adeno-associated virus (rAAV) vector containing the nucleotide sequence for treating symptoms or conditions caused by α-GalA deficiency, particularly Fabry disease.

[0002] sequence list

[0003] This disclosure includes a sequence list that is part of the disclosure. Background Technology

[0004] Fabry disease (FD, OMIM 301500) is an X-linked dominant genetic disorder caused by a deficiency or absence of lysosomal α-galactosidase A (EC 3.2.1.22, α-Gal A). The deficiency or absence of α-Gal A leads to abnormal and excessive deposition of glycosphingolipids, particularly trihexysphingolipids (Gb3 or GL-3) and galactosylceramide. Gb3 accumulates in various tissues and cell types, including capillary endothelial cells, kidney cells, cardiac cells, and nerve cells. Typical symptoms of FD include burning pain in the extremities, angiokeratoma, corneal verticillate, left ventricular hypertrophy, and chronic nephrosis. Without appropriate treatment, the life expectancy for male patients is estimated at approximately 58.2 years, according to data from the Fabry Disease Registry. Women may also experience symptoms, but usually milder than men. Their life expectancy is reported to be about 75.4 years, about 5 years shorter than the average life expectancy for women.

[0005] Currently, the main available treatment for FD is enzyme replacement therapy (ERT). FD patients have two options: agarsidase beta and agarsidase a, both of which are recombinant human α-Gal A enzymes. Both drugs require intravenous injection every two weeks to relieve symptoms, which is time-consuming and costly. According to statistics, 90% of men receiving agarsidase beta and 56% of men receiving agarsidase a developed antibodies against the injected drugs, thereby reducing the therapeutic effect of ERT. A survey of patients receiving long-term ERT showed that although the risk of complications decreased with the extension of treatment time, ERT did not stop disease progression[1].

[0006] Given the limitations of ERT, there remains an unmet medical need for better and more cost-effective treatments for FD patients. AAV-based gene therapy can deliver α-Gal A coding sequences to replace defective genes and alleviate symptoms caused by α-Gal A deficiency in FD patients. A single dose can provide a durable therapeutic effect, significantly improving convenience and cost-effectiveness for FD patients. Summary of the Invention

[0007] The inventors have developed an optimized codon sequence encoding α-Gal A, as well as an α-Gal A variant with enhanced activity, thus completing this invention.

[0008] In a first aspect, this application provides a nucleotide sequence encoding a human α-Gal A polypeptide, wherein the amino acid sequence of the α-Gal A polypeptide is as shown in SEQ ID NO: 1, and the nucleotide sequence is any one of the nucleotide sequences shown in SEQ ID NOs: 3-9. In the most preferred embodiment, the nucleotide sequence encoding the human α-Gal A polypeptide is the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the human α-Gal A polypeptide is contained in an rAAV vector.

[0009] In a second aspect, this application provides a polypeptide variant of human α-Gal A, wherein the polypeptide variant has the enzymatic activity of lysosomal α-galactosidase A and has an increased number of N-linked glycosylation sites (specifically NXS / T motifs) compared to the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0010] In a more specific embodiment of the second aspect, the amino acid sequence of the human α-Gal A polypeptide variant, compared to the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, includes at least one amino acid substitution selected from the group consisting of: G274S, G274T, Q280S, Q280T, Q357S, Q357T, Q422N, Q280N, E398N, E418N, D233N, D244N, and R100N. In a preferred embodiment, the amino acid sequence of the human α-Gal A polypeptide variant, compared to the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, includes at least one amino acid substitution selected from the group consisting of: Q422N, E418N, D233N, E398N, and D244N. In a preferred embodiment of the second aspect, the human α-Gal A polypeptide variant has enhanced lysosomal α-galactosidase A activity compared to the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0011] In a specific embodiment of the second aspect, the human α-Gal A polypeptide variant comprises or consists of any of the amino acid sequences shown in SEQ ID NOs: 18-30. In a preferred embodiment, the human α-Gal A polypeptide variant comprises or consists of any of the amino acid sequences shown in SEQ ID NOs: 24 and 26-29. In a more preferred embodiment, the human α-Gal A polypeptide variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 24.

[0012] In a third aspect, this application provides a nucleotide sequence encoding a human α-Gal A polypeptide variant as described in the second aspect. In a preferred embodiment, the nucleotide sequence encoding the human α-Gal A polypeptide variant is codon-optimized for expression in humans. In a preferred embodiment, the nucleotide sequence encoding the human α-Gal A polypeptide variant is based on the nucleotide sequence encoding wild-type human α-Gal A as shown in SEQ ID NO: 5, and includes codon changes corresponding to amino acid residue substitutions.

[0013] In a specific embodiment of the third aspect, the nucleotide sequence encoding the human α-Gal A polypeptide variant comprises or consists of any of the nucleotide sequences shown in SEQ ID NOs: 31-43. In a preferred embodiment, the nucleotide sequence encoding the human α-Gal A polypeptide variant comprises or consists of any of the nucleotide sequences shown in SEQ ID NOs: 37 and 39-42. In a more preferred embodiment, the nucleotide sequence encoding the human α-Gal A polypeptide variant comprises or consists of the nucleotide sequence shown in SEQ ID NOs: 37. In some embodiments, the nucleotide sequence encoding the human α-Gal A polypeptide variant is contained in an rAAV vector.

[0014] In any aspect of this application, the rAAV vector is a single-stranded AAV (ssAAV) vector or a self-complementary AAV (scAAV) vector.

[0015] Furthermore, the inventors have developed chimeric promoters that can drive strong expression, particularly in the liver. Therefore, in a fourth aspect, this application provides a chimeric promoter comprising, from 5' to 3', a cis-regulatory element, a TTR (transthyretin) enhancer, and a TTR promoter, wherein the cis-regulatory element is derived from a gene highly expressed in the liver. In a more preferred embodiment of the fourth aspect, the chimeric promoter further comprises a 3 × HS-CRM (hepatocyte-specific cis-regulatory module) sequence. Preferably, the 3 × HS-CRM sequence is located between the cis-regulatory element and the TTR enhancer. For example, the TTR enhancer has the nucleotide sequence shown in SEQ ID NO: 12. For example, the TTR promoter has the nucleotide sequence shown in SEQ ID NO: 13. For example, the 3 × HS-CRM has the nucleotide sequence shown in SEQ ID NO: 11. For example, the cis-regulatory element has the nucleotide sequence shown in any of SEQ ID NOs: 44 and 47-56. In a specific implementation, the chimeric promoter is selected from the P4, P7, P10, P14, P15, P16, P17, P18, P19, P20, and P21 promoters of this application. Preferably, the chimeric promoter is P10, P14, P16, P18, or P21. More preferably, the chimeric promoter is P14 or P18. For example, the promoter is used in recombinant AAV vectors.

[0016] In a fifth aspect, this application provides an expression cassette comprising the nucleotide sequence encoding the human α-Gal A polypeptide as described in the first aspect, or the nucleotide sequence encoding a variant of the human α-Gal A polypeptide as described in the third aspect. In a preferred embodiment, the nucleotide sequence described in the first or third aspect is operatively linked to the chimeric promoter described in the fourth aspect.

[0017] In a sixth aspect, this application provides an expression cassette comprising the chimeric promoter and target gene (GOI) described in the fourth aspect. In a preferred embodiment, the GOI is a nucleotide sequence encoding a human α-Gal A polypeptide or a variant thereof having lysosomal α-galactosidase A enzyme activity, for example, the nucleotide sequence described in the first or third aspect.

[0018] In preferred embodiments of the fifth and sixth aspects, the expression cassette is used for expression via an rAAV vector. Preferably, the expression cassette further comprises a Kozak sequence and an SV40 polyA. For example, the SV40 polyA has the nucleotide sequence shown in SEQ ID NO:16.

[0019] In a seventh aspect, this application provides an rAAV vector comprising the nucleotide sequence described in the first or third aspect, the promoter described in the fourth aspect, or the expression cassette described in the fifth or sixth aspect. In a preferred embodiment, the rAAV vector provides desired levels of expression of a target gene (GOI), such as human α-Gal A protein or a variant thereof having lysosomal α-galactosidase A activity, in a target tissue (e.g., plasma, liver, heart, and / or kidney, especially liver). In a further embodiment, the rAAV vector comprises a 5' ITR and a 3' ITR. For example, the 5' ITR has the nucleotide sequence shown in SEQ ID NO: 10, and / or the 3' ITR has the nucleotide sequence shown in SEQ ID NO: 17. For example, the 5' ITR has the nucleotide sequence shown in SEQ ID NO: 63, and / or the 3' ITR has the nucleotide sequence shown in SEQ ID NO: 64.

[0020] In an eighth aspect, this application provides an AAV viral particle comprising the rAAV vector described in the seventh aspect packaged in an AAV capsid. The AAV capsid may be derived from any one of AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVLK03, AAVS3, AAVKP1, AAVrh10, AAVNP40, AAVNP59, AAV-DJ, AAVANc80L65, AAVsL65, AAVHSC15, AAVC102, AAV204, and AAV214. The AAV capsid may be a wild-type AAV capsid of any of the above serotypes, a variant thereof, or a chimeric AAV capsid, wherein the chimeric AAV capsid has a capsid protein derived from two or more of the above-described AAV amino acid fragments, regions, or domains. In one embodiment, the AAV capsid is a hepatic capsid, such as the capsid of serotypes AAV5, AAV6, AAV8, AAV9, or variants thereof, or a chimeric AAV capsid having a capsid protein derived from amino acid fragments, regions, or domains of two or more of the aforementioned AAVs.

[0021] In a ninth aspect, this application provides a pharmaceutical composition comprising the rAAV carrier described in the seventh aspect or the viral particles described in the eighth aspect, and a pharmaceutically acceptable excipient.

[0022] In a tenth aspect, this application provides a method for treating a lysosomal storage disease (especially Fabry disease) in a subject in need, comprising administering to the subject a therapeutically effective amount of the rAAV carrier described in the seventh aspect, the rAAV particles described in the eighth aspect, or the pharmaceutical composition described in the ninth aspect.

[0023] In the eleventh aspect, this application provides the use of the rAAV carrier described in the seventh aspect, the rAAV particles described in the eighth aspect, or the pharmaceutical composition described in the ninth aspect in the treatment of lysosomal storage diseases (especially Fabry disease).

[0024] In a twelfth aspect, this application provides the use of the rAAV carrier described in the seventh aspect or the rAAV particles described in the eighth aspect in the preparation of a medicament for treating lysosomal storage diseases (especially Fabry disease). Attached Figure Description

[0025] Figure 1 A schematic diagram of a GLA expression vector used to evaluate the optimized coding sequence (CDS) is shown.

[0026] Figure 2A The concentrations of α-Gal A protein expressed in Hep3B cell supernatant with different coding sequences are shown.

[0027] Figure 2B The concentrations of α-Gal A protein expressed in Huh7 cell supernatant with different coding sequences are shown.

[0028] Figure 3 A schematic diagram of the GLA expression vector used to evaluate the α-Gal A mutant is shown.

[0029] Figure 4A The α-Gal A activity expressed by the α-Gal A mutant was demonstrated as assessed in HepG2 cells.

[0030] Figure 4B The α-Gal A activity expressed by the α-Gal A mutant was demonstrated as evaluated in Hep3B cells.

[0031] Figure 4C The α-Gal A activity expressed by the α-Gal A mutant was demonstrated in Huh7 cells.

[0032] Figure 5 A schematic diagram of two GLA expression vectors used to evaluate the α-Gal A mutant in in vivo mouse studies is shown.

[0033] Figure 6A , 6B 6C shows the in vivo evaluation results of the α-Gal A mutant M7 compared to the wild-type M0. Figure 6A The time progression of plasma α-Gal A activity was shown. Figure 6B The study showed α-Gal A activity in the liver, heart, and kidneys on day 21. Figure 6CThe time progression of plasma lyso-Gb3 concentration is shown after administration of a specified dose of rAAV.

[0034] Figure 7 A schematic diagram of the luciferase expression vector used for in vitro evaluation of promoter P0-P13 is shown.

[0035] Figure 8A The results of luciferase assays evaluating promoter P0-P13-driven expression are shown in HepG2 cells.

[0036] Figure 8B The results of a luciferase assay evaluating promoter P0-P13-driven expression are shown in Hep3B cells.

[0037] Figure 8C The results of a luciferase assay evaluating promoter P0-P13-driven expression are shown in Huh7 cells.

[0038] Figure 9 Bioluminescence imaging results are shown, comparing the expression efficiency and biodistribution of luciferase after systematic delivery of rAAV expressing luciferase driven by four different promoters (P0, P4, P7, and P10). H: Heart; Li: Liver; S: Spleen; Lu: Lung; Si: Small Intestine; K: Kidney; B: Brain; Sm: Skeletal Muscle.

[0039] Figure 10 The bar chart shows the luciferase activity in protein extracts from designated mouse tissues (H: heart; Li: liver; S: spleen; Lu: lung; Si: small intestine; K: kidney; B: brain; Sm: skeletal muscle) injected with AAV8-P0, AAV8-P4, AAV8-P7, or AAV8-P10.

[0040] Figure 11 A schematic diagram of the luciferase expression vector used for in vitro evaluation of promoter P14-P21 is shown.

[0041] Figure 12 The results of luciferase assays evaluating expression driven by promoters P14–P21 are shown in different cell types. P0 and cTNT promoters were used as controls.

[0042] Figure 13 Bioluminescence imaging results are shown, comparing the expression efficiency and biodistribution of luciferase after systematic delivery of rAAV expressing luciferase driven by four different promoters (P14, P16, P18, and P21). H: Heart; Li: Liver; S: Spleen; Lu: Lung; Si: Small Intestine; K: Kidney; B: Brain; Sm: Skeletal Muscle.

[0043] Figure 14 The bar chart shows the luciferase activity in protein extracts from designated mouse tissues (H: heart; Li: liver; S: spleen; Lu: lung; Si: small intestine; K: kidney; B: brain; Sm: skeletal muscle) injected with AAV8-P14, AAV8-P16, AAV8-P18, or AAV8-P21.

[0044] Figure 15 A schematic diagram of the α-Gal A expression vector used in the in vivo efficacy study in Example 4 is shown.

[0045] Figure 16 The study demonstrated α-Gal A activity in mouse plasma before and after rAAV treatment (days 7, 14, and 28) following administration of specified doses of AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, or AAV8-P21-M7.

[0046] Figure 17 The study demonstrated α-Gal A activity in the heart, liver, and kidney of mice that received AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, or AAV8-P21-M7 4 weeks after administration of the corresponding rAAV.

[0047] Figure 18 The results showed that different doses (1 × 10) were administered. 11 vg / kg, 1 × 10 12 vg / kg and 1 × 10 13 Time course of lyso-Gb3 concentration in mouse plasma of AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 or AAV8-P21-M7 (vg / kg).

[0048] Figure 19 The results showed that different doses (1 × 10⁻⁶) were administered. 11 vg / kg, 1 × 10 12 vg / kg and 1 × 10 13 Four weeks after administration of AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 or AAV8-P21-M7 (vg / kg), the concentration of lyso-Gb3 in the heart, liver and kidney of mice.

[0049] Figure 20AThe time progression of α-Gal A activity in mouse plasma before and after rAAV treatment (days 7, 14, 28, 56, 84, and 112) following administration of specified doses of AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, or AAV9-P14-M7 is shown.

[0050] Figure 20B The time progression of α-Gal A activity in mouse plasma before and after rAAV treatment (days 7, 14, 28, 56, 84, and 112) following administration of specified doses of AAV5-P18-M7 or AAV6-P18-M7 is shown.

[0051] Figure 21 The study demonstrated α-Gal A activity in the plasma, heart, liver, and kidney of mice receiving AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, or AAV6-P18-M7 16 weeks after administration of the specified dose of the corresponding rAAV.

[0052] Figure 22A The time course of lyso-Gb3 concentration in mouse plasma before and after rAAV treatment (days 7, 14, 28, 56, 84, and 112) following administration of specified doses of AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, or AAV9-P14-M7 is shown.

[0053] Figure 22B The time progression of lyso-Gb3 concentrations in mouse plasma before and after rAAV treatment (days 7, 14, 28, 56, 84, and 112) following administration of specified doses of AAV5-P18-M7 or AAV6-P18-M7 is shown.

[0054] Figure 23 The concentrations of lyso-Gb3 in the plasma, heart, liver, and kidney of mice receiving AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, or AAV6-P18-M7 are shown 16 weeks after administration of the corresponding dose of the specified rAAV.

[0055] Figure 24 It shows that it accepts 1 × 10 13 vg / kg AAV5-P14-M7, 1 × 10 13vg / kg AAV5-P18-M7, 1 × 10 12 vg / kg AAV8-P14-M7 or 1 × 10 12 The time course of α-Gal A activity and lyso-Gb3 concentration in mouse plasma of AAV8-P18-M7 (vg / kg) over one year. Invention Details

[0057] Unless otherwise expressly defined herein, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] As used herein, including in the appended claims, the singular forms of words such as “a”, “an”, and “the” include their corresponding plural references, unless the context clearly indicates otherwise.

[0059] In the context of this disclosure, unless otherwise stated, the term "comprise" and its variations (e.g., "comprises" and "comprising") should be understood to include the stated elements (e.g., amino acid sequences, nucleotide sequences, properties, steps, or combinations thereof), but does not exclude any other elements (e.g., amino acid sequences, nucleotide sequences, properties, and steps). As used herein, the term "comprise" or any variation thereof may be replaced by the terms "contain," "include," or sometimes "have," or equivalent variations thereof. In some embodiments, the term "comprise" also includes the case of "consisting of."

[0060] I. Definition

[0061] The term "α-galactosidase A" or "α-Gal A" refers to the EC 3.2.1.22 enzyme, which hydrolyzes the terminal non-reducing α-D-galactose residue in α-D-galactosides. α-Gal A is encoded by the GLA gene in humans. The amino acid sequence of wild-type human α-Gal A is shown in SEQ ID NO: 1.

[0062] The term "isolated nucleic acid" refers to DNA or RNA extracted from all or part of the polynucleotides in naturally occurring isolated polynucleotides, or DNA or RNA linked to a polynucleotide not linked to it in its natural state. Isolated nucleic acid molecules "containing" a specific nucleotide sequence may, in addition to that specific sequence, include operatively linked regulatory sequences that control the expression of the coding region of said nucleotide sequence. Due to codon degeneracy, those skilled in the art will understand that any particular amino acid sequence may be encoded by several different nucleotide sequences.

[0063] As used in this article, "codon-optimized coding sequence" refers to a nucleotide sequence encoding a target polypeptide (e.g., human α-Gal A polypeptide) that has been modified from a wild-type coding sequence to accommodate codon bias. Optimization can be achieved by reducing sequence complexity, adjusting GC content, modifying codon usage, and / or avoiding rare codons. Codon-optimized coding sequences typically exhibit improved translation efficiency of the target gene (GOI), thereby leading to higher protein expression levels.

[0064] The term "promoter" refers to a DNA sequence capable of initiating the transcription of a downstream gene controlled by that promoter. Promoters include, but are not limited to, constitutive promoters, cell-type-specific promoters, tissue-specific promoters, and developmental stage-specific promoters. Promoters can be naturally occurring promoters of genes, modified versions of naturally occurring promoters, or synthetic promoters.

[0065] The term "enhancer" refers to a regulatory DNA sequence that, together with the promoter, enhances the transcription of GOI in AAV.

[0066] The term "chimeric promoter" refers to a promoter element that drives the expression of a gene operatively linked to it. The chimeric promoter of this application may consist of a conventional promoter (e.g., a minimal promoter) and optional enhancers and / or cis-regulatory elements. A minimal promoter is the smallest promoter fragment required to correctly initiate transcription. A cis-regulatory element or cis-regulatory replication element (CRE) is a nucleotide fragment originating from a non-coding region that has a regulatory function on the transcription of neighboring genes.

[0067] "Operationally linked" means that a promoter or chimeric promoter is in a functionally appropriate position and / or orientation relative to a coding sequence in order to control the transcription of that coding sequence.

[0068] As used herein, the term "expression cassette" refers to a DNA component contained in a vector (e.g., an rAAV vector) that consists of a gene (e.g., the human GLA gene) and regulatory sequences to be expressed in a host cell transfected with the vector.

[0069] The term "N-linked glycosylation" refers to a type of modification that links an oligosaccharide to an amino group on the asparagine (N) side chain. N-linked glycosylation primarily occurs in NXS / T peptide sequences and occasionally in NXC, where N is asparagine, S is serine, T is threonine, C is cysteine, and X can be any amino acid except proline. Therefore, in the context of this application, the term "N-linked glycosylation site" refers to any of the aforementioned three-amino acid peptides selected from NXS, NXT, and NXC. Thus, the number of "N-linked glycosylation sites" in a particular polypeptide (e.g., a human α-Gal A polypeptide variant) is calculated by summing the number of NXS, NXT, and NXC occurrences in the polypeptide.

[0070] The term "pharmaceutical composition" refers to a composition suitable for delivery to a subject.

[0071] As used herein, the terms “administration,” “administering,” “treating,” and “treatment,” when applied to a subject (e.g., an animal, including humans) or cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with said subject, cells, tissues, organs, or biological fluid. Treatment of cells includes contact between a reagent and cells, as well as contact between a reagent and a fluid (which then contacts the cells). The terms “administration” and “treatment” also include in vitro and ex vivo treatments, such as cell treatment, which may be administered via reagents, diagnostic agents, conjugated compounds, or other cells.

[0072] II. GLA Expression Construct

[0073] Several efforts have been undertaken to reduce the therapeutic dose of rAAV for safety and cost-effectiveness: to enhance GLA enzyme activity by developing α-Gal A variants with an increased number of N-linked glycosylation sites, and to enhance the expression efficiency of GLA-expression constructs by optimizing coding sequences and evaluating chimeric promoters.

[0074] This application provides a human α-Gal A polypeptide variant with enhanced lysosomal α-galactosidase A activity. This is achieved primarily by creating new N-linked glycosylation sites in the amino acid sequence of the enzyme variant. In a preferred embodiment, compared to the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, the human α-Gal A polypeptide variant contains at least one amino acid substitution selected from Q422N, E418N, D233N, E398N, and D244N.

[0075] In some embodiments, the peptide variant of human α-Gal A comprises a single amino acid mutation selected from Q422N, E418N, D233N, E398N, and D244N, and thus comprises or consists of the amino acid sequence shown in any of SEQ ID NOs: 24 and 26-29. In a more preferred embodiment, the peptide variant of human α-Gal A comprises or consists of the amino acid sequence shown in SEQ ID NO: 24.

[0076] When expressed by the rAAV vector in the same cell type (e.g., hepatocytes or hepatocellular carcinoma cells) or the same tissue (liver, heart, kidney, or blood), the variants of this application can achieve α-Gal A activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to at least 1.5, at least 2, at least 2.5, or at least 3 times higher than wild-type human α-Gal A. Enzyme activity can be determined by any conventional method known in the art.

[0077] This application also provides codon-optimized coding sequences for wild-type human α-Gal A and human α-Gal A polypeptide variants described herein. Preferred codon-optimized coding sequences of this application are designed based on the wild-type human α-Gal A codon-optimized coding sequence shown in SEQ ID NO: 5. Therefore, the variant coding sequences of this application differ from the nucleotide sequence of SEQ ID NO: 5 only at the codons corresponding to amino acid substitutions. The substitution codons can be determined based on the codon usage of the target host cell. In a specific embodiment, the nucleotide sequence encoding the human α-Gal A variant comprises or consists of any of the nucleotide sequences shown in SEQ ID NOs: 37 and 39-42. In a more preferred embodiment, the nucleotide sequence encoding the human α-Gal A polypeptide variant comprises or consists of the nucleotide sequence shown in SEQ ID NO: 37.

[0078] In optimizing the regulatory sequence, the inventors discovered that a chimeric promoter containing a cis-regulatory element, an enhancer, and a (minimum) promoter in a specific order can significantly improve GOI expression. Specifically, the cis-regulatory element is located 5' upstream of the enhancer and promoter, rather than between the enhancer and promoter or at any other location.

[0079] In a preferred embodiment, the chimeric promoter of this application comprises or consists of (a)-(c) from the 5' to 3' direction:

[0080] (a) Cis-regulatory element,

[0081] (b) Enhancer

[0082] (c) Promoter.

[0083] The cis-regulatory elements of this application may be derived from the non-coding regions (e.g., intron regions) of genes highly expressed in target tissues (e.g., liver). Preferred cis-regulatory elements of this application have nucleotide sequences shown in any of SEQ ID NOs: 44 and 47-56.

[0084] The promoter or minimal promoter in the chimeric promoter of this application can be a promoter that drives higher expression of the target gene in the liver, and preferably also higher expression in the heart than in other tissues. For example, the promoter or minimal promoter in the chimeric promoter of this application can be a TTR promoter, such as a TTR promoter having the nucleotide sequence shown in SEQ ID NO: 13.

[0085] The enhancer in the chimeric promoter of this application can be an enhancer located upstream of the promoter in its natural state. For example, when the promoter is a TTR promoter, the enhancer in the chimeric promoter of this application can be a TTR enhancer. The TTR enhancer can have the nucleotide sequence shown in SEQ ID NO: 12.

[0086] The expression cassette may also include elements such as a Kozak sequence, a polyadenylation sequence, and a translation termination signal. For example, an SV40 polyA sequence may be added after the coding sequence of GOI. This SV40 polyA sequence may have the nucleotide sequence shown in SEQ ID NO: 16.

[0087] III. Recombinant AAV vector and viral particles

[0088] The α-Gal A coding sequence or expression cassette of this application can be constructed into a recombinant AAV (rAAV) vector to obtain rAAV particles for delivery to subjects in need.

[0089] The rAAV vector can be a self-complementary AAV (scAAV) vector or a single-stranded AAV (ssAAV) vector. The rAAV vector contains two inverted terminal repeat (ITR) sequences at both ends of the inserted expression cassette. The ITRs disclosed herein can be derived from any AAV serotype. When referring to the serotype of an AAV ITR, the word "derived from" indicates that the ITR can be an ITR of a specific serotype, or a variant containing a modified ITR of that serotype. In a preferred embodiment of this disclosure, the rAAV vector contains two ITRs derived from AAV2. For example, the scrAAV vector contains two AAV2 ITRs, or contains one wild-type AAV2 ITR and a truncated AAV2 ITR lacking the terminal resolution site (trs) and the D region. For example, the ssrAAV vector contains two AAV2 ITRs, or contains one wild-type AAV2 ITR and a truncated AAV2 ITR lacking the C or C' region. For example, the wild-type AAV2 ITR is located at the 5' end of the inserted nucleotide sequence, while the AAV2 ITR variant is located at the 3' end of the inserted nucleotide sequence; or vice versa. In one embodiment, the rAAV is scAAV, and the two ITRs contained in the rAAV have the nucleotide sequences shown in SEQ ID NO: 63 (5' ITR) and SEQ ID NO: 64 (3' ITR), respectively. In one embodiment, the rAAV is ssAAV, and the two ITRs contained in the rAAV have the nucleotide sequences shown in SEQ ID NO: 10 (5' ITR) and SEQ ID NO: 17 (3' ITR), respectively.

[0090] The rAAV genome is packaged into an AAV capsid. The AAV capsid is composed of three viral proteins, namely VP1, VP2, and VP3. The capsid of this application may be derived from any AAV serotype known in the art or to be identified in the future. The word "derived from" means that the capsid may be a wild-type capsid of a particular AAV serotype or a variant thereof, for example, a variant containing one or more amino acid modifications in one or more of VP1, VP2, or VP3. The capsid of this application may be a chimeric AAV capsid containing capsid proteins that are capsid proteins containing amino acid fragments, regions, or domains derived from two or more AAV serotypes. The capsid and ITR of this application may be derived from the same serotype of AAV or from different serotypes of AAV. For example, the capsid may be suitable for intravenous (IV) delivery (e.g., intravenous injection) to peripheral tissues. In the context of this application, the term "peripheral tissues" refers to any tissue other than the brain or spinal cord. In some preferred embodiments, the AAV vector comprises a capsid, variants thereof, or chimeric AAV capsid of serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVLK03, AAVS3, AAVKP1, AAVNP40, AAVNP59, AAV-DJ, AAVsL65, AAVHSC15, AAVC102, AAV204, AAV214, AAV PHP.B, AAV2.7m8, or AAVAnc80L65. In a more preferred embodiment, the capsid is an AAV5, AAV6, AAV8, or AAV9 capsid, variants thereof, or a chimeric capsid derived from two or more of AAV5, AAV6, AAV8, and AAV9. In a more preferred embodiment, the coating is an AAV5 or AAV8 coating, a variant thereof, or a chimeric coating derived from AAV5 or AAV8.

[0091] IV. Therapeutic Uses

[0092] This application also provides a pharmaceutical composition comprising the isolated nucleic acid (containing a coding sequence), an rAAV vector or viral particle as described in this application, and a pharmaceutically acceptable excipient. Conventional pharmaceutically acceptable excipients are known in the art, and may be solid or liquid excipients. In one embodiment, the pharmaceutical composition may be a liquid for injection.

[0093] In some embodiments, the rAAV carrier of this application can be administered to a subject via systemic or local administration. In some embodiments, the rAAV carrier of this application can be delivered to peripheral tissues or organs via any parenteral or enteral route, such as delivery to peripheral blood. For example, the rAAV carrier of this application can be administered via intravenous (IV), intramuscular (IM), subcutaneous (SC), intraarterial, intraperitoneal (IP), intradermal, transdermal, oral, nasal, or rectal routes. For example, rAAV can be delivered by injection. In some embodiments, the rAAV carrier can be delivered via a combination of more than one delivery route.

[0094] The rAAV vector can be administered via a single dose or multiple doses. In one specific implementation, the rAAV vector is administered via a single injection.

[0095] The rAAV carrier or pharmaceutical composition may be used to treat diseases or conditions associated with or caused by lysosomal α-galactosidase A deficiency or absence, such as lysosomal storage diseases like Fabry disease.

[0096] The therapeutically effective dose range of the rAAV vector is approximately 1 × 10⁻⁶. 11 vg / kg to approximately 5 × 10 14 vg / kg. Preferably, the rAAV vector can be in a concentration of about 1 × 10⁻⁶. 12 To approximately 5 × 10 14 The dose is administered at a rate of vg / kg. For example, the rAAV carrier can be administered at a dose of at least about 1 × 10⁻⁶. 11 At least approximately 5 × 10 11 At least approximately 1 × 10 12 At least approximately 5 × 10 12 At least approximately 1 × 10 13 At least approximately 5 × 10 13 At least approximately 1 × 10 14 vg / kg, or at least about 5 × 10 14 The dose is administered at a rate of vg / kg. For example, the rAAV carrier can be administered at a dose of approximately 1 × 10⁻⁶. 11 Approximately 5 × 10 11 Approximately 1 × 10 12 Approximately 5 × 10 12 Approximately 1 × 10 13 Approximately 5 × 10 13 Approximately 1 × 10 14 vg / kg, or approximately 5 × 10 14 Administer at a dose of vg / kg.

[0097] By administering the rAAV of this application, one or more of the following therapeutic effects can be achieved: (1) increased α-Gal A levels; (2) enhanced α-Gal A activity; (3) reduced or eliminated glycosphingolipids (particularly deacetylated Gb3 (lyso-Gb3), trihexysylsphingolipids (Gb3 or GL-3), or galactosylceramide). For example, the increase or decrease is determined by comparing the level or activity with that before administration of rAAV to the same subject. For example, increased α-Gal A levels, enhanced α-Gal A activity, and / or reduced or eliminated glycosphingolipids (such as lyso-Gb3) are observed in one or more tissues in the subject's plasma, heart, liver, and kidneys. For example, compared to the amount and / or activity of α-Gal A in the same subject prior to rAAV administration, the amount and / or activity of α-Gal A increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 10,000-fold, at least about 20,000-fold, at least about 50,000-fold, or even more. For example, compared to the level in the same subject prior to rAAV administration, the level of glycosphingolipids (especially deacetylated Gb3 (lyso-Gb3) or trihexysylsphingolipids (Gb3 or GL-3)) decreased by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In some implementations, glycosphingolipids (especially deacetylated Gb3 (lyso-Gb3) or trihexysylsphingolipids (Gb3 or GL-3)) in the subject's plasma can be substantially eliminated after rAAV administration.

[0098] In some embodiments, one or more therapeutic effects can be observed immediately after rAAV administration. In some embodiments, one or more therapeutic effects can be observed at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, or longer after rAAV administration. Example

[0099] To facilitate understanding and implementation of the present invention, its advantages will be described in more detail with reference to embodiments and accompanying drawings. However, it should be understood that the following embodiments are merely illustrative and are not intended to limit the scope of the invention. The scope of the invention should be defined by the claims.

[0100] The HepG2 cells used in this example were purchased from ATCC. All other cell types were purchased from Pronosei Life Sciences Co., Ltd.

[0101] In the embodiments, rAAV was prepared as scAAV for in vitro coding sequence evaluation, and ssAAV for in vivo and in vitro evaluation of α-Gal A mutants, chimeric promoters and final constructs.

[0102] Example 1. Optimization of coding sequences for GLA expression

[0103] To achieve robust GLA expression, an optimized coding sequence (CDS) for GLA (GLAop CDS) was designed and synthesized (see Table 1).

[0104] Table 1. Summary of GLA codon-optimized CDS

[0105]

[0106] (C0 refers to wild-type GLA CDS, and C1-C7 are optimized CDS.)

[0107] The nucleotide sequences of C0-C7 are shown below.

[0108] SEQ ID NO: 2, Wild-type GLA nucleotide sequence (C0)

[0109]

[0110] SEQ ID NO: 3,GLA C1

[0111]

[0112] SEQ ID NO: 4,GLA C2

[0113]

[0114] SEQ ID NO: 5,GLA C3

[0115]

[0116] SEQ ID NO: 6,GLA C4

[0117]

[0118] SEQ ID NO: 7,GLA C5

[0119]

[0120] SEQ ID NO: 8,GLA C6

[0121]

[0122] SEQ ID NO: 9,GLA C7

[0123]

[0124] Construct the optimized encoding sequence to have Figure 1 The plasmid vector with the structure shown was used to evaluate GOI expression. This plasmid vector contains the following elements between two ITRs: 3×HS-CRM (SEQ ID NO: 11), a TTR enhancer (SEQ ID NO: 12), a TTR promoter (SEQ ID NO: 13), a chimeric intron (SEQ ID NO: 14), an optimized GLA coding sequence (GLAop CDS) (one of SEQ ID NOs: 2-9), WPRE (SEQ ID NO: 15), and SV40 poly A (SEQ ID NO: 16). Sequence information for each element is shown in Table 2 below.

[0125] Table 2. Sequence information of different sequence motifs in GLA expression vectors used to evaluate codon-optimized CDS.

[0126]

[0127] Hep3B and Huh7 cells were maintained in DMEM + 10% FBS and passaged every 3 days. The day before transfection, cells were cultured at 1.7 × 10⁻⁶ cells / day. 4 1.48 × 10⁶ cells / well (Hep3B cells) or 1.48 × 10⁶ cells / well 4 Cells were seeded at a density of 10 cells / well (Huh7 cells) in 96-well plates. Cells were transfected with plasmids containing C0-C7 using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008) according to the user guide. 72 hours after transfection, the supernatant was collected and α-Gal A concentration was detected by ELISA (Sinosure, SEK12078). ELISA results are shown in Figure 2 and Table 3. The results indicate that C3 produced the highest GLA expression. Interestingly, as shown in Table 1, the C3 sequence had the lowest CpG number, despite having the highest GC content percentage. Given that CpG in AAV vectors can trigger immune responses and lead to exogenous gene silencing [3,4], a lower CpG number is more advantageous in clinical applications. Therefore, C3 is a promising candidate sequence for providing more durable and potent GLA expression.

[0128] Table 3. Concentrations of α-Gal A detected in the supernatants of Hep3B and Huh7.

[0129]

[0130] Example 2. α-Gal A mutant with enhanced enzyme activity

[0131] One limitation of treatment for Fabry disease is the low efficiency of cross-absorption of α-Gal A protein by relevant tissues (e.g., heart and kidney). Expressed α-Gal A enters receptor cells via mannose-6-phosphate (M6P) receptors (M6PR) [5]. Although M6PR is widely distributed in a variety of tissues and cells, its expression level is limited, which restricts the amount of α-Gal A entering the heart or kidney, resulting in insufficient therapeutic efficacy.

[0132] M6Ps are involved in the phosphorylation of protein glycosylation sites, and each glycosylation site has multiple M6Ps. Therefore, α-Gal A mutants with increased N-linked glycosylation sites were designed to increase the total number of M6Ps in the expressed α-Gal A protein. Thirteen α-Gal A mutants, each containing a single amino acid substitution, were designed, and their sequence information is shown in Table 4, including the amino acid sequence and the corresponding nucleotide sequence. The nucleotide sequence selected as the mutant template was C3 (SEQ ID NO: 5), as shown in Example 1.

[0133] according to Figure 3 The diagram shown illustrates the construction of recombinant AAV vectors for each mutant. Specifically, the vectors contain the following elements between the two ITRs: 3×HS-CRM (SEQ ID NO: 11), a TTR enhancer (SEQ ID NO: 12), a TTR promoter (SEQ ID NO: 13), the coding sequence of the wild-type protein or any mutant (see Table 4), and SV40 poly A (SEQ ID NO: 16).

[0134] Table 4. Overview of α-Gal A mutants

[0135]

[0136] Table 5. Regulatory motif sequence information in GLA expression vectors used to evaluate α-Gal A mutants.

[0137]

[0138] HepG2, Hep3B, and Huh7 cells were maintained in DMEM + 10% FBS and passaged every 3 days. The day before transfection, cells were seeded into 96-well plates at a density of 1.48 × 10⁶ cells per well. 4 1.7 × 10⁶ cells (HepG2 or Huh7 cells) or 1.7 × 10⁶ cells per well 4 One cell line (Hep3B cells). Plasmid transfection was performed using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008) according to the user guide.

[0139] 72 hours after transfection, the supernatant was collected to determine α-Gal A activity. α-Gal A activity was assessed using a fluorescence-based enzyme activity assay. Specifically, the supernatant was diluted 10-fold. 10 µL of the diluted supernatant was mixed with 20 µL of 10 mM 4-methylumbelliferyl α-D-galactopyranoside (4MU-α-GAL) dissolved in assay buffer (0.1 M sodium citrate, 0.05 M Na₂HPO₄, and 100 mM N-acetyl-D-galactosamine) and incubated at 37°C for 2 hours. The reaction was terminated by adding 100 µL of 0.5 M glycine (pH = 10.2). Fluorescence intensity was measured using a microplate reader (Em = 365 nm, Ex = 450 nm). The activity results are shown in Figure 4 and Table 6. Of all the mutant and wild-type sequences tested, mutant M7 performed best in HepG2 and Hep3B cells, and second best in Huh7 cells. Therefore, M7 was selected for further analysis.

[0140] Table 6. Activity of α-Gal A mutant in the supernatant of HepG2, Hep3B and Huh7 cells.

[0141]

[0142] To further evaluate the α-Gal A mutant M7, M0 and M7 were cloned into a liver-specific AAV expression construct for in vivo evaluation. The structure of the construct is as follows: Figure 5 As shown (AAV8-P10-M0 and AAV8-P10-M7). Specifically, the vector contains the following elements between the two ITRs: CRE I (SEQ ID NO: 44), 3×HS-CRM (SEQ ID NO: 11), TTR enhancer (SEQ ID NO: 12), TTR promoter (SEQ ID NO: 13), wild-type protein coding sequence (SEQ ID NO: 5) or mutant coding sequence (SEQ ID NO: 37), and SV40 poly A (SEQ ID NO: 16). Sequence information is also shown in Table 7.

[0143] Table 7. Sequence information of regulatory motifs in GLA expression vectors used for in vivo evaluation of α-Gal A mutant M7.

[0144]

[0145] AAV8-P10-M0 and AAV8-P10-M7 were injected intravenously (IV) into GLA - / YIn vivo in wild-type (WT) mice (n=6), at varying doses. α-Gal A activity in plasma and tissues was assessed. Results are shown in... Figure 6A -B and Table 8-9.

[0146] Table 8. α-Gal A activity in the plasma of mice receiving AAV8-P10-M0 or AAV8-P10-M7.

[0147]

[0148]

[0149] Table 9. α-Gal A activity in the heart, liver, and kidney of mice receiving AAV8-P10-M0 or AAV8-P10-M7 4 weeks after rAAV administration.

[0150]

[0151] Plasma lyso-Gb3 is considered a promising biomarker for clinical monitoring of Fabry disease. Plasma lyso-Gb3 concentrations were assessed by liquid chromatography-mass spectrometry (LC-MS) before and after rAAV administration (days 7, 14, 21, and 28). Results are shown below. Figure 6C See Table 10.

[0152] Table 10. Plasma lyso-Gb3 concentrations in mice receiving AAV8-P10-M0 or AAV8-P10-M7.

[0153]

[0154] (UD, Not detected. Limit of quantitation (LLOQ) = 0.4 ng / mL)

[0155] In summary, the results indicate that M7 achieved higher α-Gal A enzyme activity in the liver and heart, and therefore also achieved a better lyso-Gb3 reduction effect in plasma.

[0156] Example 3. Screening for chimeric promoters that drive stronger GLA expression

[0157] Chimeric promoters based on the TTR promoter were designed to enhance GLA expression in the liver. Cis-regulatory elements (CREs) were selected from genes highly expressed in human liver [6]. The selected CREs were inserted into different positions of P1 to generate P2-P13. A liver-specific promoter P0 was used as a control. A schematic diagram of the luciferase expression constructs containing P0-P13 is shown below. Figure 7 As shown. Sequence information is shown in Table 11.

[0158] Table 11. Sequence information of luciferase expression vectors used for in vitro evaluation of P0-P13.

[0159]

[0160] HepG2, Hep3B, and Huh7 cells were maintained in DMEM + 10% FBS and passaged every 3 days. The day before transfection, cells were seeded into 96-well plates at a density of 1.48 × 10⁶ cells per well. 4 1.7 × 10⁶ cells (HepG2 or Huh7 cells) or 1.7 × 10⁶ cells 4 One cell line (Hep3B cells). Plasmid transfection was performed using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008) according to the user guide.

[0161] Luciferase activity was measured 72 hours after transfection using the Bright-Lite luciferase detection system (Vazyme, DD1204). The relative light units (RLU) results are shown in Figure 8 and Table 12-14.

[0162] Table 12. Relative light units (RLU) of P0-P13 in HepG2 cells.

[0163]

[0164] Table 13. Relative light units (RLU) of P0-P13 in Hep3B cells.

[0165]

[0166] Table 14. Relative light units (RLU) of P0-P13 in Huh7 cells.

[0167]

[0168] The results showed that stronger expression was achieved when the CRE was located at the 5' end of the 3×HS-CRM, as constructs containing P4, P7, and P10 produced higher levels of protein expression. Therefore, AAV8-capped rAAV vectors were prepared for P4, P7, and P10, with P0 as a reference (AAV8-P0, AAV8-P4, AAV8-P7, and AAV8-P10) to evaluate these three promoters in vivo.

[0169] AAV8-P0, AAV8-P4, AAV8-P7, and AAV8-P10 were intravenously injected into ICR mice at doses of 1×10⁻⁶. 11 and 1×10 12vg / mouse (n = 3). Three weeks after rAAV administration, bioluminescent imaging was used to assess luciferase activity in different tissues. Figure 9 The results showed that P0, P4, P7, and P10 all mediated liver-specific biodistribution. Among the three promoters evaluated, P10 mediated the strongest luciferase expression. Luciferase activity in proteins extracted from various tissues was expressed as RLU per 20 µg of total protein, as shown in the figures. Figure 10 As shown in Table 15.

[0170] Table 15. Relative light units (RLU) of mouse tissues receiving AAV8-P0, AAV8-P4, AAV8-P7, or AAV8-P10 3 weeks after rAAV administration.

[0171]

[0172] (H: Heart; Li: Liver; S: Spleen; Lu: Lung; Si: Small Intestine; K: Kidney; B: Brain; Sm: Skeletal Muscle)

[0173] To enhance GLA expression in the heart, another series of enhancers was introduced into P1 to generate the chimeric promoter P14-P21. For the design of P14-P21, heart-specific enhancers were identified from reference [7]. Due to the size limitations of the AAV genome, excessively large enhancers were truncated based on data from Enhancer 2.0. P0 and cTNT were used as controls. A schematic diagram of the construct containing P14-P21 is shown below. Figure 11 The sequence information is shown in Table 16.

[0174] Table 16. Sequence information of regulatory motifs in luciferase expression vectors used for in vitro evaluation of P14-P21.

[0175]

[0176] HepG2, Hep3B, Huh7, AC16, IHC-SV40 (IHC), H9C2, and 769P cells were passaged every 3 days. One day before transfection, cells were seeded into 96-well plates at a density of 1.48 × 10⁶ cells per well. 4 1.7 × 10⁶ cells (HepG2 or Huh7 cells) or 1.7 × 10⁶ cells 4 Cell types (Hep3B, AC16, IHC, H9C2, or 769P cells). Following the user guide, plasmid transfection was performed using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008).

[0177] Luciferase activity was measured 72 hours after transfection using a Bright-Lite luciferase detection system (Vazyme, DD1204). Relative optical units (RLU) results are shown below. Figure 12 See Table 17.

[0178] Table 17. Relative light units (RLU) of cell lines transfected with P14-P21, P0 and cTNT.

[0179]

[0180] The results showed that P14, P16, P18, and P21 enhanced luciferase expression not only in cardiac cell lines but also in liver and kidney cell lines. Therefore, an AAV8 capsid rAAV containing these four chimeric promoters (AAV8-P14, AAV8-P16, AAV8-P18, and AAV8-P21) was constructed for in vivo evaluation.

[0181] AAV8-P14, AAV8-P16, AAV8-P18, and AAV8-P21 were intravenously injected into ICR mice at doses of 1 × 10⁻⁶. 11 and 1 × 10 12 vg / mouse (n = 3). Three weeks after rAAV administration, bioluminescence imaging was used to assess luciferase activity in different tissues. Figure 13 The results showed that P14, P16, P18, and P21 all mediated liver-specific biodistribution. Among them, P14 and P18 mediated stronger luciferase expression. Luciferase activity in proteins extracted from various tissues was expressed as RLU per 20 µg of total protein ( Figure 14 (and Table 18).

[0182] Table 18. Relative light units (RLU) of mouse tissues injected with AAV8-P14, AAV8-P16, AAV8-P18 or AAV8-P21.

[0183]

[0184] (H: Heart; Li: Liver; S: Spleen; Lu: Lung; Si: Small Intestine; K: Kidney; B: Brain; Sm: Skeletal Muscle)

[0185] Example 4. In vivo efficacy study of the selected construct

[0186] We combined the selected promoter with an optimized α-Gal A mutant containing codon-optimized sequences to generate another round of constructs. Schematic diagrams of these constructs are shown below. Figure 15 The sequence information is shown in Table 19.

[0187] Table 19. Sequence information of the regulatory motifs of the α-Gal A expression vector used in the in vivo efficacy study.

[0188]

[0189] AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, and AAV8-P21-M7 were injected intravenously into GLA. - / Y Mice (n = 3). GLA received PBS. - / Y Mice and WT mice were used as controls. Plasma α-Gal A activity was assessed, and the results are as follows: Figure 16 As shown in Table 20.

[0190] As shown in Table 20, when given 1 × 10 11 Fourteen days after administration of AAV (vg / kg), the α-Gal A activity in the AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, and AAV8-P21-M7 groups increased by 1,001.6-fold, 1,381.9-fold, 529.3-fold, 28,875.9-fold, and 722.5-fold, respectively, and was 233.2-fold, 321.8-fold, 123.3-fold, 6,723.7-fold, and 168.2-fold higher than that of the WT group. After administration of 1 × 10⁻⁶ g / kg AAV, the α-Gal A activity increased by 1,001.6-fold, 1,381.9-fold, 529.3-fold, 28,875.9-fold, and 722.5-fold, respectively. 13 Fourteen days after administration of AAV at a dose of vg / kg, the α-Gal A activity in the AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, and AAV8-P21-M7 groups increased by 38,149.0-fold, 261,649.1-fold, 24,461.2-fold, 515,638.0-fold, and 30,157.0-fold, respectively, which were 8,882.9-fold, 60,924.5-fold, 5,695.7-fold, 120,065.4-fold, and 7,022.0-fold higher than that of the WT group.

[0191] Table 20. Plasma α-Gal A activity in mice receiving AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 or AAV8-P21-M7.

[0192] Four weeks after rAAV administration, α-Gal A activity in various peripheral tissues was assessed, and the results were as follows: Figure 17 As shown in Table 21. Given 1 × 10 13Following administration of AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, and AAV8-P21-M7 at a dose of vg / kg, α-Gal A activity in cardiac tissue was found to increase significantly by 1,653.2-fold, 1,470.1-fold, 209.6-fold, 4,506.1-fold, and 119.5-fold, respectively. Correspondingly, α-Gal A activity in the liver increased by 880.4-fold, 2,289.3-fold, 914.8-fold, 2,105.3-fold, and 573.0-fold, respectively. α-Gal A activity in the kidneys increased by 36.3-fold, 470.3-fold, 6.4-fold, 233.6-fold, and 24.6-fold, respectively.

[0193] Table 21. α-Gal A activity in the heart, liver, and kidney of mice receiving AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, or AAV8-P21-M7 4 weeks after rAAV administration.

[0194]

[0195] The concentrations of Lyso-Gb3 in plasma, heart, liver, and kidney were also assessed by LC-MS. Results are as follows: Figure 18-19 And as shown in Tables 22-23. When given 1 × 10 13 Four weeks after administration of rAAV at doses of vg / kg to AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, and AAV8-P21-M7, plasma Lyso-Gb3 decreased by 98.7%, 98.9%, 98.6%, 99.1%, and 98.8%, respectively. Correspondingly, cardiac Lyso-Gb3 decreased by 99.1%, 99.2%, 98.8%, 99.4%, and 99.1%, respectively; hepatic Lyso-Gb3 decreased by 99.5%, 99.5%, 99.2%, 99.6%, and 99.5%, respectively; and renal Lyso-Gb3 decreased by 98.6%, 99.0%, 97.7%, 99.5%, and 97.9%, respectively. These results indicate a significant therapeutic effect even four weeks after rAAV administration.

[0196] Table 22. Plasma Lyso-Gb3 concentrations in mice receiving AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, or AAV8-P21-M7.

[0197]

[0198] Table 23. Lyso-Gb3 concentrations in the heart, liver, and kidneys of mice receiving AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7, or AAV8-P21-M7 4 weeks after rAAV administration.

[0199] Example 5.4 Comparative Study of In Vivo Therapeutic Effects of Four AAV Serotypes

[0200] This embodiment evaluates the effect of different serotype capsids on in vivo efficacy. P14-M7 was packaged into AAV5, AAV6, AAV8, and AAV9, respectively. Simultaneously, P18-M7 was packaged into AAV5 and AAV6, respectively. Schematic diagrams of the constructs P14-M7 and P18-M7 are shown below. Figure 15 The sequence information is shown in Table 19.

[0201] AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, and AAV6-P18-M7 were administered intravenously to 15-week-old GLA infants. - / Y In mice (n = 3), the doses of AAV5-P14-M7 and AAV5-P18-M7 were 1 × 10⁻⁶. 12 vg / kg (low dose), 1 × 10 13 vg / kg (medium dose) and 5 × 10 13 vg / kg (high dose). The doses of AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, and AAV6-P18-M7 are 1 × 10 11 vg / kg (low dose), 1 × 10 12 vg / kg (medium dose) and 1 × 10 13 vg / kg (high dose). GLA receiving PBS - / Y Mice and WT mice were used as controls.

[0202] Plasma α-Gal A activity was tracked up to 16 weeks post-dose. Results are shown in Figure 20A , 20B21 and Table 24. Plasma α-Gal A activity increased after AAV administration, reaching a plateau approximately 14 days post-administration. In the high-dose group, at 14 days post-administration, the α-Gal A activity of AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, and AAV6-P18-M7 increased by 38434.3 times, 203.5 times, 303784.6 times, 183069.0 times, 106195.3 times, and 176325.0 times, respectively, which were 9709.7 times, 51.4 times, 76745.6 times, 46249.0 times, 26828.3 times, and 46401.3 times higher than WT, respectively.

[0203] Table 24. Plasma α-Gal A activity in mice receiving AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 or AAV6-P18-M7.

[0204]

[0205] α-Gal A activity in the heart, liver, and kidneys was assessed 16 weeks after rAAV administration. Results are as follows: Figure 21 As shown in Table 25.

[0206] Significantly increased α-Gal A activity was observed in peripheral tissues. In the high-dose group, 16 weeks post-dose, GLA levels were increased by AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, and AAV6-P18-M7. - / Y The α-Gal A activity in mouse hearts reached 1.1, 1.1, 23.1, 44.0, 30.1, and 15.4 times that of the mean tachycardia (WT), respectively. The α-Gal A activity in the liver reached 107.7, 1.0, 230.5, 110.3, 258.2, and 657.7 times that of the WT, respectively. The α-Gal A activity in the kidneys reached 18.8, 1.0, 70.1, 22.1, 21.3, and 20.1 times that of the WT, respectively.

[0207] Table 25. α-Gal A activity in the heart, liver and kidney of mice 16 weeks after administration of rAAV of AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 or AAV6-P18-M7.

[0208]

[0209] The concentrations of Lyso-Gb3 in plasma, heart, liver, and kidney were also assessed by LC-MS. Results are as follows: Figure 22A , 22B As shown in Tables 23 and 26-27.

[0210] Sixteen weeks after administration of moderate doses of AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, or AAV6-P18-M7, plasma lyso-Gb3 levels decreased by 98.5%, 55.5%, 98.6%, 96.8%, 98.2%, and 89.0% compared to baseline, respectively. Cardiac lyso-Gb3 levels decreased by 98.9%, 59.2%, 99.3%, 98.9%, 99.4%, and 92.3%, respectively. Liver lyso-Gb3 levels decreased by 99.4%, 82.0%, 99.6%, 99.5%, 99.6%, and 97.9%, respectively. Renal lyso-Gb3 levels decreased by 98.0%, 65.7%, 99.2%, 97.3%, 98.5%, and 92.4%, respectively. The above results indicate that rAAV administration has a significant therapeutic effect 16 weeks after administration.

[0211] Table 26. Plasma lyso-Gb3 concentrations in mice that received AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, or AAV6-P18-M7.

[0212]

[0213] Table 27. lyso-Gb3 concentrations in the heart, liver, and kidneys of mice 16 weeks after administration of rAAVs of AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7, or AAV6-P18-M7.

[0214]

[0215] Example 6. Long-term in vivo efficacy study

[0216] AAV5-P14-M7, AAV5-P18-M7, AAV8-P14-M7, and AAV8-P18-M7 were administered intravenously to 15-week-old GLA cells. - / Y In mice (n = 3), the administered concentrations of AAV5-P14-M7 and AAV5-P18-M7 were 1 × 10⁻⁶. 13 The dosage concentration of AAV8-P14-M7 and AAV8-P18-M7 is 1 × 10⁻⁶ g / kg. 12 vg / kg. GLA treated with PBS - / Y Mice and WT mice were used as controls.

[0217] Plasma α-Gal A activity and lyso-Gb3 concentration were monitored for up to 52 weeks after administration. Results are as follows: Figure 24 As shown in Tables 28 and 29, plasma α-Gal A activity increased shortly after AAV administration and remained at a high plateau level for more than 52 weeks. After 52 weeks of administration, the α-Gal A activities in the AAV5-P14-M7, AAV5-P18-M7, AAV8-P14-M7, and AAV8-P18-M7 groups remained at high levels, 125.8 times, 2514.6 times, 591.6 times, and 2299.7 times higher than those in the WT group, respectively. After 52 weeks of administration, all four groups receiving rAAV treatment maintained near-complete clearance of plasma lyso-Gb3.

[0218] The above results indicate that α-Gal A activity is increased in a long-term therapeutic manner and lyso-Gb3 is cleared.

[0219] Table 28. Accept 1 × 10 13 vg / kg AAV5-P14-M7, 1 × 10 13 vg / kg AAV5-P18-M7, 1 × 10 12 vg / kg AAV8-P14-M7 or 1 × 10 12 Mean plasma α-Gal A activity in mice with vg / kg AAV8-P18-M7.

[0220]

[0221] Table 29. Accept 1 × 10 13 vg / kg AAV5-P14-M7, 1 × 10 13 vg / kg AAV5-P18-M7, 1 × 10 12 vg / kg AAV8-P14-M7 or 1 × 10 12Mean plasma lyso-Gb3 concentration in mice with AAV8-P18-M7 (vg / kg).

[0222]

[0223] (UD, Not detected. Limit of quantitation (LLOQ) = 0.1 ng / mL)

[0224] Sequence information

[0225] SEQ ID NO: 1, Wild-type GLA amino acid sequence

[0226] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQ ADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKP NYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQ DPLGKQGYQLRQGDNFEVWERPLSGLAWAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL

[0227] SEQ ID NOs: 2-9, 11-16 are listed above.

[0228] SEQ ID NO: 10, 5' ITR

[0229] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0230] SEQ ID NO: 17, 3' ITR

[0231] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0232] SEQ ID NO: 18, Amino acid sequence of M1 (single amino acid substitutions are underlined)

[0233] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNF S LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0234] SEQ ID NO: 19, Amino acid sequence of M2

[0235] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNF T LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0236] SEQ ID NO: 20, Amino acid sequence of M3

[0237] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQ S VTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0238] SEQ ID NO: 21, Amino acid sequence of M4

[0239] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQ T VTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0240] SEQ ID NO: 22, Amino acid sequence of M5

[0241] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINR SEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0242] SEQ ID NO: 23, Amino acid sequence of M6

[0243] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINR T EIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0244] SEQ ID NO: 24, Amino acid sequence of M7

[0245] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTM N MSLKDLL*

[0246] SEQ ID NO: 25, Amino acid sequence of M8

[0247] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQ N VTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0248] SEQ ID NO: 26, Amino acid sequence of M9

[0249] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFY N WTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0250] SEQ ID NO: 27, Amino acid sequence of M10

[0251] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQL NNTMQMSLKDLL*

[0252] SEQ ID NO: 28, Amino acid sequence of M11

[0253] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADI N DSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0254] SEQ ID NO: 29, Amino acid sequence of M12

[0255] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSIL NWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0256] SEQ ID NO: 30, Amino acid sequence of M13

[0257] MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQ N DSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL*

[0258] SEQ ID NO: 31, Nucleotide sequence of M1

[0259]

[0260] SEQ ID NO: 32, nucleotide sequence of M2

[0261]

[0262] SEQ ID NO: 33, nucleotide sequence of M3

[0263]

[0264] nucleotide sequence of SEQ ID NO: 34, M4

[0265]

[0266] nucleotide sequence of SEQ ID NO: 35, M5

[0267]

[0268] nucleotide sequence of SEQ ID NO: 36, M6

[0269]

[0270] nucleotide sequence of SEQ ID NO: 37, M7

[0271]

[0272] nucleotide sequence of SEQ ID NO: 38, M8

[0273]

[0274] SEQ ID NO: 39, nucleotide sequence of M9

[0275]

[0276] Nucleotide sequence of SEQ ID NO: 40, M10

[0277]

[0278] nucleotide sequence of SEQ ID NO: 41, M11

[0279]

[0280] nucleotide sequence of SEQ ID NO: 42, M12

[0281]

[0282] nucleotide sequence of SEQ ID NO: 43, M13

[0283]

[0284] SEQ ID NO: 44,CRE I

[0285]

[0286] SEQ ID NO: 45, Modified 5’ UTR

[0287] GAACACCACCAAGCACAGGGTACAGGTCTCAGTAATTATTGTCAAATTTATGTGGATTTGCTTTTAAACAATATCTTCCATTTACTGAGTGTTTATGTGGAAGAACTGTACTAAATTTTAATGCATTTCTTTATTCCTATTCTTAAAACCTTCCAGCAAGGTGGCTCTACCACCCTCTTTTCCCAGCTTCAGGAGCAGTTGTGCTAATAGCTGGAGAACACCAGGCTGGATTTAAACCCAGATCACTCTTACATTTGCTCTTTACCTGCTGTGCTCAGCCTTCACCTGCCCTCTAGCTGTAGTTTTCTGAAGTCAGCTCACAGCAAGGCAGTGTGCTTAGAGGTTAACAGAAGGGAAAACAACAACAACAAAAATCTAAATGAGAATCCTGACTGTTTCAGCTGGGGGTAAGGGGGGCTGATTATTCATATAATTGTTATACCAGACTGTCCCAGGCTTAGTCCAATTGCAGAGAACTCCCTTCCCAGGCTTCTGAGAGTCCTGGAAGTGCCTAAACCTGTCTAATGGACTGGGCTTGGGTGGCCAGTGGCTCCCTGGCTTCTTCCCTTTACCCAGGGCTGGCAGCCAAGTGGTGCCTCCTGCCTCCCCCACACCCTCCCTCAGCCCCTCCCCTCCTGCCCATCCTGGGCAGGTGACCTGGAGCATCCAGCAGGCTGCCCTGGCCTCCTGCTTCAGGACAAGGCCCACTAGGGGCCTTACTGTGCTGAGATGCACCAGGCAAGAGACACCCTTTGTAACTCTCTTCTCCTCCCTAGTGCCAGGTTAAAACCTTCAGCCCCAGGTGCTGTTTGCAAACCTGCCTGTACCTGAGGCCCTAAAAAGCCAGAGACCTCACTCCCTGGGA

[0288] SEQ ID NO: 46, Nucleotide sequence of luciferase

[0289]

[0290] SEQ ID NO: 47,CRE D

[0291]

[0292] SEQ ID NO: 48,CRE G

[0293]

[0294] SEQ ID NO: 49,hs1750-5

[0295]

[0296] SEQ ID NO: 50,hs1750-3

[0297]

[0298] SEQ ID NO: 51,hs1751-5

[0299]

[0300] SEQ ID NO: 52,hs1751-3

[0301]

[0302] SEQ ID NO: 53,hs1760

[0303]

[0304] SEQ ID NO: 54,hs1958-5

[0305]

[0306] SEQ ID NO: 55,hs1959

[0307]

[0308] SEQ ID NO: 56,hs1971-3

[0309]

[0310] SEQ ID NO: 57, cTNT

[0311] TGTAGTTAATGATTAACCCGCCATGCTACTTATCTACCAGGGTAATGGGGATCCTCTAGAACTATAGCTAGAATTCGCCCTTACGGCCCCCCCTCGAGGTCGGGATAAAAGCAGTCTGGGCTTTCACATGACAGCATCTGGGGCTGCGGCAGAGGGTCG GGTCCGAAGCGCTGCCTTATCAGCGTCCCCAGCCCTGGGAGGTGACAGCTGGCTGGCTTGTGTCAGCCCCTCGGGCACTCACGTATCTCCGTCCGACGGGTTTAAAATAGCAAAACTCTGAGGCCACACAATAGCTTGGGCTTATATGGGCTCCTGTGGG GGAAGGGGGAGCACGGAGGGGGCCGGGGCCGCTGCTGCCAAAATAGCAGCTCACAAGTGTTGCATTCCTCTCTGGGCGCCGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGTGGGCGCCGGGGGGACCTTAAAGCCTCTGCCCCCCCAAGGAGCCCTT CCCAGACAGCCGCCGGCACCCACCGCTCCGTGGGACGATCCCCGAAGCTCTAGAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAG

[0312] SEQ ID NO: 58, P10-M7

[0313]

[0314] SEQ ID NO: 59,P14-M7

[0315]

[0316] SEQ ID NO: 60,P16-M7

[0317]

[0318] SEQ ID NO: 61,P18-M7

[0319]

[0320] SEQ ID NO: 62,P21-M7

[0321]

[0322] References:

[0323] 1. Rombach SM, Smid BE, Bouwman MG, Linthorst GE, Dijkgraaf MG, Hollak CE. Long term enzyme replacement therapy for fabry disease: Effectiveness on kidney, heart and brain. Orphanet journal of rare diseases. 2013; 8: 47.

[0324] 2. Chuah MK, Petrus I, De Bleser P, Le Guiner C, Gernoux G, Adjali O et al. Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates. Molecular Therapy. 2014; 22: 1605-1613.

[0325] 3. Bertolini TB, Shirley JL, Zolotukhin I, Li X, Kaisho T, Xiao W et al. Effect of cpg depletion of vector genome on cd8+ t cell responses in aav gene therapy. Frontiers in Immunology. 2021; 12.

[0326] 4.Konkle BA, Walsh CE, Escobar MA, Josephson NC, Young G, VonDrygalski A et al.Bax 335 hemophilia b gene therapy clinical trial results:Potential impact of cpg sequences on gene expression.Blood, The Journal ofthe American Society of Hematology.2021; 137: 763-774.

[0327] 5.Gary-Bobo M, Nirdé P, Jeanjean A, Morère A, Garcia M. Mannose 6-phosphate receptor targeting and its applications in human diseases.Currentmedicinal chemistry.2007; 14: 2945-2953.

[0328] 6.Yamashita T, Hashimoto S-I, Kaneko S, Nagai S, Toyoda N, Suzuki Tet al.Comprehensive gene expression profile of a normal humanliver.Biochemical and biophysical research communications.2000; 269: 110-116.

[0329] 7.May D, Blow MJ, Kaplan T, Mcculley DJ, Jensen BC, Akiyama JA etal.Large-scale discovery of enhancers from human heart tissue.Naturegenetics.2012; 44: 89-93。

Claims

1. A nucleotide sequence encoding a human α-Gal A polypeptide, wherein the amino acid sequence of the α-Gal A polypeptide is as shown in SEQ ID NO: 1, and the nucleotide sequence is any one of the nucleotide sequences shown in SEQ ID NOs: 3-9.

2. The nucleotide sequence of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO:

5.

3. A polypeptide variant of a human α-Gal A polypeptide, wherein the polypeptide variant has lysosomal α-galactosidase A enzymatic activity and has an increased number of N-linked glycosylation sites compared with the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO:

1.

4. The polypeptide variant of claim 3, wherein, compared with the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, its amino acid sequence comprises at least one amino acid substitution selected from the group consisting of: G274S, G274T, Q280S, Q280T, Q357S, Q357T, Q422N, Q280N, E398N, E418N, D233N, D244N, and R100N.

5. The polypeptide variant of claim 4, compared with the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid substitution selected from the group consisting of: Q422N, E418N, D233N, E398N, and D244N.

6. The polypeptide variant according to any one of claims 3-5, having enhanced lysosomal α-galactosidase A activity compared to the wild-type human α-Gal A polypeptide having the amino acid sequence shown in SEQ ID NO:

1.

7. The polypeptide variant of any one of claims 3-6, comprising or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 18-30.

8. The polypeptide variant of claim 7, comprising or consisting of any of the amino acid sequences shown in SEQ ID NOs: 24 and 26-29.

9. The polypeptide variant of claim 8, comprising or consisting of the amino acid sequence shown in SEQ ID NO:

24.

10. A nucleotide sequence encoding a polypeptide variant as described in any one of claims 3-9.

11. The nucleotide sequence of claim 10, codon-optimized for expression of α-Gal A protein in human cells.

12. The nucleotide sequence of claim 10 or 11, based on the nucleotide sequence shown in SEQ ID NO: 5 encoding wild-type human α-Gal A, wherein codons for amino acid residues that are different from those in the variant of wild-type human α-Gal A are replaced.

13. The nucleotide sequence of any one of claims 10-12, comprising or consisting of any of the nucleotide sequences shown in SEQ ID NOs: 31-43.

14. The nucleotide sequence of claim 13, comprising or consisting of any of the nucleotide sequences shown in SEQ ID NOs: 37 and 39-42.

15. The nucleotide sequence of claim 14, comprising or consisting of the nucleotide sequence shown in SEQ ID NO:

37.

16. A chimeric promoter comprising, from 5' to 3', a cis-regulatory element, a 3×HS-CRM, a TTR enhancer, and a TTR promoter, wherein the cis-regulatory element is derived from a gene highly expressed in the liver.

17. The chimeric promoter of claim 16, wherein the TTR enhancer has the nucleotide sequence shown in SEQ ID NO:

12.

18. The chimeric promoter of claim 16 or 17, wherein the TTR promoter has the nucleotide sequence shown in SEQ ID NO:

13.

19. The chimeric promoter of any one of claims 16-18, wherein the 3×HS-CRM sequence has the nucleotide sequence shown in SEQ ID NO:

11.

20. The chimeric promoter of any one of claims 16-19, wherein the cis-regulatory element has a nucleotide sequence shown in any one of SEQ ID NOs: 44 and 47-56.

21. The chimeric promoter according to any one of claims 16-20, wherein the chimeric promoter is selected from the P4, P7, P10, P14, P15, P16, P17, P18, P19, P20 or P21 promoters.

22. The chimeric promoter of claim 21, wherein the chimeric promoter is a P10, P14, P16, P18 or P21 promoter.

23. The chimeric promoter of claim 22, used in recombinant AAV vectors.

24. An expression cassette comprising the nucleotide sequence as described in any one of claims 1-2 and 10-15.

25. The expression cassette of claim 24, wherein the nucleotide sequence of any one of claims 1-2 and 10-15 is operatively linked to the chimeric promoter of any one of claims 16-23.

26. An expression cassette comprising a chimeric promoter as described in any one of claims 16-23, operatively linked to a target gene.

27. The expression cassette of claim 26, wherein the target gene is a nucleotide sequence encoding a human α-Gal A polypeptide or a variant thereof having the enzymatic activity of lysosomal α-galactosidase A.

28. The expression cassette as described in any one of claims 24-27, for expression via an rAAV vector.

29. The expression box of claim 28, further comprising SV40 polyA.

30. The expression cassette of claim 29, wherein the SV40 polyA has the nucleotide sequence shown in SEQ ID NO:

16.

31. A recombinant AAV (rAAV) vector comprising a nucleotide sequence as described in any one of claims 1-2 and 10-15, a chimeric promoter as described in any one of claims 16-23, or an expression cassette as described in any one of claims 24-30.

32. The rAAV vector as described in claim 31 is a single-stranded AAV (ssAAV) vector or a self-complementary AAV (scAAV) vector.

33. The rAAV carrier as claimed in claim 31 or 32, further comprising a 5' ITR and a 3' ITR.

34. The rAAV vector of claim 33, wherein the 5' ITR has the nucleotide sequence shown in SEQ ID NO: 10, and / or the 3' ITR has the nucleotide sequence shown in SEQ ID NO:

17.

35. The rAAV vector of claim 33, wherein the 5' ITR has the nucleotide sequence shown in SEQ ID NO: 63, and / or the 3' ITR has the nucleotide sequence shown in SEQ ID NO:

64.

36. An AAV virus particle comprising an rAAV vector as described in any one of claims 31-35, packaged in an AAV capsid.

37. The AAV viral particle of claim 36, wherein the AAV capsid is derived from an AAV capsid selected from the following AAV serotypes: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVLK03, AAVS3, AAVKP1, AAVrh10, AAVNP40, AAVNP59, AAV-DJ, AAVANc80L65, AAVsL65, AAVHSC15, AAVC102, AAV204, AAV214, AAV PHP.B, AAV2.7m8, or variants thereof, or a chimeric AAV capsid having a capsid protein derived from two or more of the above-mentioned AAVs, including amino acid fragments, regions, or domains.

38. The AAV virus particle of claim 36 or 37, wherein the AAV capsid is a hepatic capsid.

39. The AAV viral particle of claim 38, wherein the AAV capsid is a capsid of AAV5, AAV6, AAV8, AAV9, a variant thereof, or a chimeric AAV capsid, wherein the chimeric AAV capsid has a capsid protein derived from two or more of the above-mentioned AAVs, amino acid fragments, regions, or domains.

40. A pharmaceutical composition comprising an rAAV vector as described in any one of claims 31-35 or a viral particle as described in any one of claims 36-39, and a pharmaceutically acceptable excipient.

41. A method of treating lysosomal storage disease in a subject in need, comprising administering to the subject a therapeutically effective amount of the rAAV vector as described in any one of claims 31-35, the viral particles as described in any one of claims 36-39, or the pharmaceutical composition as described in claim 40.

42. The method of claim 41, wherein the lysosomal storage disease is Fabry disease.

43. Use of the rAAV vector of any one of claims 31-35, the viral particle of any one of claims 36-39, or the pharmaceutical composition of any one of claims 40 in the treatment of lysosomal storage diseases.

44. The use as described in claim 43, wherein the lysosomal storage disease is Fabry disease.

45. Use of the rAAV vector of any one of claims 31-35 or the viral particle of any one of claims 36-39 in the preparation of a medicament for treating lysosomal storage diseases.

46. ​​The use as claimed in claim 45, wherein the lysosomal storage disease is Fabry disease.

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