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WO2025092951A1 - Vecteurs viraux adéno-associés recombinés pour le traitement de la maladie de fabry - Google Patents

Vecteurs viraux adéno-associés recombinés pour le traitement de la maladie de fabry Download PDF

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Publication number
WO2025092951A1
WO2025092951A1 PCT/CN2024/129227 CN2024129227W WO2025092951A1 WO 2025092951 A1 WO2025092951 A1 WO 2025092951A1 CN 2024129227 W CN2024129227 W CN 2024129227W WO 2025092951 A1 WO2025092951 A1 WO 2025092951A1
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seq
nucleotide sequence
gal
aav8
amino acid
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Lu GUO
Shin-Shay Tian
Xiaoping Zhao
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Shanghai Vitalgen Biopharma Co Ltd
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Shanghai Vitalgen Biopharma Co Ltd
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the present disclosure belongs to the field of gene therapy. Specifically, the present disclosure provides nucleotide sequences encoding human alpha-galactosidase-A ( ⁇ -Gal A) or variants thereof, and recombinant adeno-associated viral (rAAV) vectors comprising the nucleotide sequence for treating a disorder or condition caused by the deficiency of ⁇ -Gal A, especially Fabry disease.
  • ⁇ -Gal A human alpha-galactosidase-A
  • rAAV adeno-associated viral
  • the present disclosure includes a sequence listing as a part of the disclosure.
  • Fabry disease (FD, OMIM 301500) is an X-linked, dominantly inherited genetic disorder caused by the deficiency or absence of lysosomal ⁇ -galactosidase A (EC 3.2.1.22, ⁇ -Gal A) .
  • Deficiency or absence of ⁇ -Gal A results in abnormal and excessive deposition of glycosphingolipids, particularly globotriaosylceramide (Gb3 or GL-3) and galactosylceramide.
  • Gb3 accumulation occurs in a variety of tissues and cell types, including capillary endothelial, renal, cardiac and nerve cells.
  • Classical symptoms for FD include burning pain in the arms and legs, angiokeratoma, cornea verticillate, left ventricular hypertrophy and chronic nephrosis. Without proper treatment, the life expectancy of male patients was estimated to be around 58.2 years old based on the data from Fabry Registry. Women can also have symptoms, but usually milder than those for men and life expectancy is reported to be around 75.4 years old, about 5 years shorter than the general woman’s life expectancy.
  • An AAV-based gene therapy will deliver the ⁇ -Gal A coding sequence to replace the defective gene and relieve the symptoms resulting from the deficiency of ⁇ -Gal A in FD patients, providing an endurable therapy after a single dose to be significantly more convenient and cost effective for FD patients.
  • the present inventors have developed codon-optimized sequences encoding ⁇ -Gal A, as well as ⁇ -Gal A variants having increased activity thereof, thus completing the invention.
  • the present application provides a nucleotide sequence encoding for human ⁇ -Gal A polypeptide, wherein the amino acid sequence of the ⁇ -Gal A polypeptide is shown in SEQ ID NO: 1, and the nucleotide sequence is any one of the nucleotide sequences as shown in SEQ ID NOs: 3-9.
  • the nucleotide sequence encoding for human ⁇ -Gal A polypeptide is the nucleotide sequence as shown in SEQ ID NO: 5.
  • the nucleotide sequence encoding for human ⁇ -Gal A polypeptide is comprised in a rAAV vector.
  • the present application provides a polypeptide variant of human ⁇ -Gal A, wherein the polypeptide variant has an enzyme activity of lysosomal ⁇ -galactosidase A, and an increased number of N-linked glycosylation sites (specifically, NXS/T motifs) as compared to the wild-type human ⁇ -Gal A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.
  • the amino acid sequence of the polypeptide variant of human ⁇ -Gal A comprises at least one amino acid substitution selected from a group consisting of G274S, G274T, Q280S, Q280T, Q357S, Q357T, Q422N, Q280N, E398N, E418N, D233N, D244N, and R100N, as compared to the wild-type human ⁇ -Gal A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.
  • the polypeptide variant of human ⁇ -Gal A comprises at least one amino acid substitution selected from a group consisting of Q422N, E418N, D233N, E398N, and D244N, as compared to the wild-type human ⁇ -Gal A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.
  • the polypeptide variant of human ⁇ -Gal A has an increased enzyme activity of lysosomal ⁇ -galactosidase A, as compared to the wild-type human ⁇ -Gal A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.
  • the polypeptide variant of human ⁇ -Gal A comprises or consists of the amino acid sequence as shown in any one of SEQ ID NOs: 18-30. In preferred embodiments, the polypeptide variant of human ⁇ -Gal A comprises or consists of the amino acid sequence as shown in any one of SEQ ID NOs: 24, and 26-29. In more preferred embodiments, the polypeptide variant of human ⁇ -Gal A comprises or consists of the amino acid sequence as shown in SEQ ID NO: 24.
  • the present application provides a nucleotide sequence encoding for the polypeptide variant of human ⁇ -Gal A of the second aspect.
  • the nucleotide sequence encoding for the polypeptide variant of human ⁇ -Gal A is codon-optimized for the expression in human.
  • the nucleotide sequence encoding for the polypeptide variant of human ⁇ -Gal A is based on the nucleotide sequence as shown in SEQ ID NO: 5 that encodes for the wild-type human ⁇ -Gal A, containing codon changes for the amino acid residue substituted.
  • the nucleotide sequence encoding for the polypeptide variant of human ⁇ -Gal A comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 31-43.
  • the nucleotide sequence encoding for the polypeptide variant of the human ⁇ -Gal A comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 37 and 39-42.
  • the nucleotide sequence encoding for the polypeptide variant of human ⁇ -Gal A comprises or consists of a nucleotide sequence as shown in SEQ ID NOs: 37.
  • the nucleotide sequence encoding for the polypeptide variant of human ⁇ -Gal A polypeptide is comprised in a rAAV vector.
  • the rAAV vector is a single-stranded AAV (ssAAV) vector or self-complementary AAV (scAAV) vector.
  • the present 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 having high expression in the liver.
  • the chimeric promoter further comprises a 3 ⁇ HS-CRM (hepatocyte-specific cis-regulatory module) sequence.
  • the 3 ⁇ HS-CRM sequence is located between the cis-regulatory element and the TTR enhancer.
  • the TTR enhancer has a nucleotide sequence as shown in SEQ ID NO: 12.
  • the TTR promoter has a nucleotide sequence as shown in SEQ ID NO: 13.
  • the 3 ⁇ HS-CRM has a nucleotide sequence of SEQ ID NO: 11.
  • the cis-regulatory element has a nucleotide sequence as shown in any one of SEQ ID NOs: 44, and 47-56.
  • the chimeric promoter is selected from P4, P7, P10, P14, P15, P16, P17, P18, P19, P20 and P21 promoters of the present application.
  • the chimeric promoter is P10, P14, P16, P18 or P21. More preferably, the chimeric promoter is P14 or P18.
  • the promoter is used in a recombinant AAV vector.
  • the present application provides an expression cassette, comprising the nucleotide sequence of the first aspect that encodes for the human ⁇ -Gal A polypeptide, or the nucleotide sequence of the third aspect that encodes for the polypeptide variant of the human ⁇ -Gal A.
  • the nucleotide sequence of the first or third aspect is operatively linked to the chimeric promoter of the fourth aspect.
  • the present application provides an expression cassette, comprising the chimeric promoter of the fourth aspect and a gene of interest (GOI) .
  • the GOI is a nucleotide sequence encoding for the human ⁇ -Gal A polypeptide or variant thereof enzyme activity of lysosomal ⁇ -galactosidase A, e.g., the nucleotide sequence of the first aspect or the third aspect.
  • the expression cassette is used for expression via rAAV vectors.
  • the expression cassette further comprises a KOZAK sequence and a SV40 polyA.
  • the SV40 polyA has a nucleotide sequence of SEQ ID NO: 16.
  • the present application provides a rAAV vector, comprising the nucleotide sequence of the first aspect or the third aspect, the promoter of the fourth aspect, or the expression cassette of the fifth aspect or sixth aspect.
  • the rAAV vector provides a desirable expression level of the GOI, e.g., the human ⁇ -Gal A protein or variant thereof with enzyme activity of lysosomal ⁇ -galactosidase A in target tissues, e.g., plasma, liver, heart and/or kidney, especially liver.
  • the rAAV vector comprises a 5’ ITR and a 3’ ITR.
  • the 5’ ITR has a nucleotide sequence of SEQ ID NO: 10
  • the 3’ ITR has a nucleotide sequence of SEQ ID NO: 17.
  • the 5’ ITR has a nucleotide sequence of SEQ ID NO: 63
  • the 3’ ITR has a nucleotide sequence of SEQ ID NO: 64.
  • the present application provides an AAV viral particle comprising the rAAV vector of the seventh aspect packaged into an AAV capsid.
  • the AAV capsid can 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, AAV214.
  • the AAV capsid can be a wild-type AAV capsid of any of the aforesaid serotypes, a variant thereof, or a chimeric AAV having capsid proteins with amino acid fragments, regions, or domains derived from two or more of the aforesaid AAVs.
  • the AAV capsid is a capsid with liver tropism, such as the capsid of serotype AAV5, AAV6, AAV8, AAV9, a variant thereof, or a chimeric AAV capsid having capsid proteins with amino acid fragments, regions, or domains derived from two or more of the aforesaid AAVs.
  • the present application provides a pharmaceutical composition comprising the rAAV vectors of the seventh aspect or the viral particle of the eighth aspect, and a pharmaceutically acceptable excipient.
  • the present application provides a method for treating a lysosomal storage disease, especially Fabry disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the rAAV vector of the seventh aspect, the rAAV particle of the eighth aspect or the pharmaceutical composition of the ninth aspect.
  • the present application provides the use of the rAAV vector of the seventh aspect, the rAAV particle of the eighth aspect or the pharmaceutical composition of the ninth aspect in treating a lysosomal storage disease, especially Fabry disease.
  • the present application provides the use of the rAAV vector of the seventh aspect, or the rAAV particle of the eighth aspect in the manufacture of a medicament in treating a lysosomal storage disease, especially Fabry disease.
  • FIG. 1 illustrates the schematic diagram of the GLA-expressing vector for the evaluation of the optimized coding sequence (CDS) .
  • FIG. 2A shows the concentrations of ⁇ -Gal A protein expressed by different coding sequences in the supernatants of Hep3B cells.
  • FIG. 2B shows the concentrations of ⁇ -Gal A protein expressed by different coding sequences in the supernatants of Huh7 cells.
  • FIG. 3 illustrates the schematic diagram of the GLA-expressing vector for the evaluation of the ⁇ -Gal A mutants.
  • FIG. 4A shows the ⁇ -Gal A activities of ⁇ -Gal A mutant expression evaluated in HepG2 cells.
  • FIG. 4B shows the ⁇ -Gal A activities of ⁇ -Gal A mutant expression evaluated in Hep3B cells.
  • FIG. 4C shows the ⁇ -Gal A activities of ⁇ -Gal A mutant expression evaluated in Huh7 cells.
  • FIG. 5 illustrates the schematic diagrams of the two GLA-expressing vectors for the evaluation of the ⁇ -Gal A mutants in in vivo mouse studies.
  • FIGS. 6A, 6B and 6C show the results of in vivo evaluation of the ⁇ -Gal A mutant M7 as compared to the wild-type M0.
  • FIG. 6A shows the time course of the plasma ⁇ -Gal A activities.
  • FIG. 6B shows ⁇ -Gal A activities in the liver, heart and kidney at day 21.
  • FIG. 6C shows the time course of the plasma lyso-Gb3 concentrations after administration of the rAAVs at the indicated doses.
  • FIG. 7 illustrates the schematic diagrams of the Luciferase expression vectors for in vitro evaluation of promoters P0-P13.
  • FIG. 8A shows the results of the luciferase assay for evaluation of expression driven by promoters P0-P13 in HepG2 cells.
  • FIG. 8B shows the results of the luciferase assay for evaluation of expression driven by promoters P0-P13 in Hep3B cells.
  • FIG. 8C shows the results of the luciferase assay for evaluation of expression driven by promoters P0-P13 in Huh7 cells.
  • FIG. 9 shows the bioluminescence imaging results comparing the expression efficiency and biodistribution of luciferase after systematic delivery of the rAAVs expressing luciferase driven by four different promoters, P0, P4, P7 and P10.
  • H heart; Li: liver; S: spleen; Lu: lung; S. i. : small intestine; K: kidney; B: brain; S. m. : skeletal muscle.
  • FIG. 10 are bar graphs showing luciferase activities in protein extracts of the indicated tissues (H: heart; Li: liver; S: spleen; Lu: lung; S. i. : small intestine; K: kidney; B: brain; S. m. : skeletal muscle) from mice that received AAV8-P0, AAV8-P4, AAV8-P7 or AAV8-P10 injection.
  • FIG. 14 are bar graphs showing luciferase activities in protein extracts of the indicated tissues (H: heart; Li: liver; S: spleen; Lu: lung; S. i. : small intestine; K: kidney; B: brain; S. m. : skeletal muscle) from mice that received AAV8-P14, AAV8-P16, AAV8-P18 or AAV8-P21 injection.
  • FIG. 15 illustrates the schematic diagrams of the ⁇ -Gal A expression vectors for in vivo efficacy study of Example 4.
  • FIG. 16 shows the plasma ⁇ -Gal A activities in mice that received AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 or AAV8-P21-M7 at the indicated doses before and after (day 7, day 14 and day 28) the rAAV treatment.
  • FIG. 20A shows the time courses of the plasma ⁇ -Gal A activities in mice that received AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7 or AAV9-P14-M7 at the indicated doses before and after (day 7, day 14, day 28, day 56, day 84 and day 112) the rAAV treatment.
  • FIG. 20B shows the time courses of the plasma ⁇ -Gal A activities in mice that received AAV5-P18-M7 or AAV6-P18-M7 at the indicated doses before and after (day 7, day 14, day 28, day 56, day 84 and day 112) the rAAV treatment.
  • FIG. 21 shows the ⁇ -Gal A activities in the plasma, heart, liver and kidney of mice that received AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 or AAV6-P18-M7 at 16 weeks after administration of the respective rAAV at the indicated doses.
  • FIG. 22A shows the time courses of the plasma lyso-Gb3 concentrations in mice that received AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7 or AAV9-P14-M7 at the indicated doses before and after (day 7, day 14, day 28, day 56, day 84 and day 112) the rAAV treatment.
  • FIG. 22B shows the time courses of the plasma lyso-Gb3 concentrations in mice that received AAV5-P18-M7 or AAV6-P18-M7 at the indicated doses before and after (day 7, day 14, day 28, day 56, day 84 and day 112) the rAAV treatment.
  • FIG. 23 shows the lyso-Gb3 concentrations in the plasma, heart, liver and kidney of mice that received AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 or AAV6-P18-M7 at the indicated doses at 16 weeks after administration of the respective rAAV.
  • FIG. 24 shows the one-year time courses of the plasma ⁇ -Gal A activities and lyso-Gb3 concentrations in mice that received 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 vg/kg AAV8-P18-M7.
  • the wording “comprise” and variations thereof such as “comprises” and “comprising” will be understood to imply the inclusion of a stated element, e.g., an amino acid sequence, a nucleotide sequence, a property, a step or a group thereof, but not the exclusion of any other elements, e.g., amino acid sequences, nucleotide sequences, properties and steps.
  • the term “comprise” or any variation thereof can be substituted with the term “contain” , “include” or sometimes “have” or equivalent variation thereof.
  • the wording “comprise” also includes the scenario of “consisting of” .
  • ⁇ -galactosidase A or “ ⁇ -Gal A” refers to the enzyme of EC 3.2.1.22, which hydrolyzes terminal, non-reducing alpha-D-galactose residues in alpha-D-galactosides.
  • ⁇ -Gal A is encoded by GLA gene in human.
  • the amino acid sequence of the wild-type human ⁇ -Gal A is shown as SEQ ID NO: 1.
  • isolated nucleic acid means a DNA or RNA which is removed from all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature.
  • An isolated nucleic acid molecule “comprising” a specific nucleotide sequence may include, in addition to the specified sequence, operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences. Due to the codon degeneracy, one skilled in the art understands that any specific amino acid sequence can be coded by several different nucleotide sequences.
  • codon-optimized coding sequence refers to a nucleotide sequence coding for a polypeptide of interest, e.g., human ⁇ -Gal A polypeptide, modified from their wild-type coding sequence accommodating codon bias. Optimization may be achieved by reducing sequence complexity, adjusting GC content, adjusting codon usage and/or avoiding rare codons.
  • the coding sequence which has been codon optimized usually shows an increased translational efficiency of the gene of interest (GOI) , leading to a higher protein expression.
  • GOI gene of interest
  • promoter refers to a DNA sequence that enables initiation of transcription of a downstream gene under the control of the said promoter. Promoters include but not limited to constitutive promoters, cell type-specific promoters, tissue-specific promoters, development stage-specific promoters. Promoter can be a naturally occurring promoter of a gene, a modified version of a naturally occurring promoter or a synthetic promoter.
  • the term “enhancer” is a regulatory DNA sequence which can enhance the transcription of the GOI in AAV together with the promoter.
  • chimeric promoter refers to a promoter element that drives the expression of a gene that is operatively linked to it.
  • the chimeric promoter of the present application may be consisted of a conventional promoter, such as a minimal promoter, and optionally also an enhancer and/or cis-regulatory element.
  • a minimal promoter is the minimal portion of the promoter required to properly initiate transcription.
  • a cis-regulatory element or a cis-regulatory replication element (CRE) is a nucleotide fragment derived from a non-coding region which has regulatory function on the transcription of a neighboring gene.
  • operatively linked means that the promoter or chimeric promoter is in a functionally appropriate location and/or orientation in relation to the coding sequence so as to control the transcription of the coding sequence.
  • expression cassette herein refers to a DNA component included in a vector (e.g., rAAV vector) and consisted of a gene (e.g., human GLA gene) to be expressed in host cells transfected by the vector and regulatory sequence (s) .
  • a vector e.g., rAAV vector
  • a gene e.g., human GLA gene
  • N-linked glycosylation refers to a type of modification in which oligosaccharide is attached to the amino group of an asparagine (N) side chain. N-linked glycosylation predominantly occurs in N-X-S/T peptide sequences, and rarely in N-X-C, in which N is asparagine, S is serine, T is threonine, C is cysteine, and X can be any amino acid other than proline. Accordingly, in the context of the present application, the term “N-linked glycosylation site” refers to any of the aforesaid three-amino-acid peptide stretch selected from N-X-S, N-X-T and N-X-C.
  • the number of “N-linked glycosylation site” in a certain polypeptide is calculated by adding the numbers of N-X-S, N-X-T and N-X-C occurred in the said polypeptide.
  • composition refers to a composition suitable for delivering to a subject.
  • administration when applied to a subject, e.g., an animal, including human, or to cell, tissue, organ, or biological fluid, means contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid.
  • Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid, where the fluid is in contact with the cell.
  • administration and treatment also include in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
  • polypeptide variants of the human ⁇ -Gal A with increased enzyme activity of lysosomal ⁇ -galactosidase A. This is mainly achieved by creating new N-linked glycosylation sites in the amino acid sequence of the enzyme variant.
  • the polypeptide variant of the human ⁇ -Gal A comprises at least one amino acid substitution selected from a group consisting of Q422N, E418N, D233N, E398N, and D244N, as compared to the wild-type human ⁇ -Gal A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.
  • the polypeptide variant of human ⁇ -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 as shown in any one of SEQ ID NOs: 24, and 26-29.
  • the polypeptide variant of the human ⁇ -Gal A comprises or consists of the amino acid sequence as shown in SEQ ID NO: 24.
  • the variant of the present application can achieve an ⁇ -Gal A activity of 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%higher than that of the wild-type human ⁇ -Gal A, or as high as at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold of that of the wild-type human ⁇ -Gal A, when being expressed by a rAAV vector in the same cell type (e.g., liver cells or liver cancer cells) or in the same tissue (liver, heart, kidney, or blood) .
  • the enzyme activity can be determined by any conventional means known in the art.
  • the present application also provides codon-optimized coding sequences for the wild-type human ⁇ -Gal A and polypeptide variants of the human ⁇ -Gal A of the present application.
  • the preferred codon-optimized coding sequences of the present application are designed based on a codon-optimized coding sequence for wild-type human ⁇ -Gal A as shown in SEQ ID NO: 5. Therefore, the coding sequence of a variant of the present application only differs from the nucleotide of SEQ ID NO: 5 in the codon (s) corresponding to the amino acid substitution (s) .
  • the codon for replacement can be determined based on the codon usage of the intended host cells.
  • the nucleotide sequence encoding for the human ⁇ -Gal A variant comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 37 and 39-42.
  • the nucleotide sequence encoding for the polypeptide variant of the human ⁇ -Gal A comprises or consists of a nucleotide sequence as shown in SEQ ID NOs: 37.
  • chimeric promoters comprising the cis-regulatory element, enhancer and (minimal) promoter in specific orders can lead to significantly increased expression of the GOI.
  • the cis-regulatory element is arranged in a position 5’ upstream of the enhancer and promoter, instead of between the enhancer and promoter, or any other position.
  • the chimeric promoter of the present application from 5’ to 3’ comprises or is consisted of (a) - (c) :
  • the cis-regulatory element of the present application can be one derived from the non-coding region, e.g., the intron region, of a gene highly expressed in the target tissue, e.g., liver.
  • the preferred cis-regulatory element of the present application has a nucleotide sequence as shown in any one of SEQ ID NOs: 44, and 47-56.
  • the promoter or minimal promoter in the chimeric promoter of the present application can be one that drives higher expression of the gene of interest in liver and preferably also in heart as compared to in other tissues.
  • the promoter or minimal promoter in the chimeric promoter of the present application can be a TTR promoter, e.g., having a nucleotide sequence of SEQ ID NO: 13.
  • the enhancer in the chimeric promoter of the present application can be one that locates upstream of the promoter in nature.
  • the enhancer in the chimeric promoter of the present application can be a TTR enhancer when the promoter is a TTR promoter.
  • the TTR enhancer can have a nucleotide sequence of SEQ ID NO: 12.
  • the expression cassette can further comprise elements such as KOZAK sequence, polyadenylation sequence and translation termination signal.
  • SV40 polyA sequence can be included after the coding sequence of the GOI.
  • the SV40 polyA sequence can have a nucleotide sequence of SEQ ID NO: 16.
  • the coding sequence of ⁇ -Gal A or the expression cassette of the present application can be constructed into a recombinant AAV (rAAV) vector, to obtain rAAV particles for delivery into a subject in need thereof.
  • rAAV recombinant AAV
  • the rAAV vectors can be a self-complementary AAV (scAAV) vector or a single- stranded AAV (ssAAV) vector.
  • the rAAV vector is comprised of two inverted terminal repeat (ITR) sequences at both ends of the inserted expression cassette.
  • ITR inverted terminal repeat
  • the ITR of the present disclosure can be ITR derived from any AAV serotypes. When reference is made to serotype of AAV ITR, the phrase “derived from” means that the ITR can be the ITR of a certain serotype or a variant derived therefrom with modification (s) .
  • the rAAV vector comprises two ITRs derived from AAV2.
  • the scrAAV vector comprises two AAV2 ITRs, or comprises a wild-type AAV2 ITR and a truncated version of AAV2 ITR lacking the terminal resolution site (trs) and region D.
  • the ssrAAV vector comprises two AAV2 ITRs, or comprises a wild-type AAV2 ITR and a truncated version of AAV2 ITR lacking the region C or region C’ .
  • the wild-type AAV2 ITR locates in the 5’ of the inserted nucleotide sequence, while the AAV2 ITR variant locates in the 3’ of the inserted nucleotide sequence; or vice versa.
  • the rAAV is scAAV, and the two ITRs comprised in the rAAV have nucleotide sequences as shown in SEQ ID NO: 63 (5’ ITR) and SEQ ID NO: 64 (3’ ITR) , respectively.
  • the rAAV is ssAAV, and the two ITRs comprised in the rAAV have nucleotide sequences as shown in SEQ ID NO: 10 (5’ ITR) and SEQ ID NO: 17 (3’ ITR) , respectively.
  • AAV capsid is composed of three viral proteins, namely VP1, VP2 and VP3.
  • the capsid of the present application can be derived from any AAV serotype known in the art or characterized in the future. By “derived from” , it means that the capsid can be the wild-type capsid of a certain AAV serotype or a variant thereof, e.g. a variant comprising one or more amino acid modifications in one or more of VP1, VP2 or VP3.
  • the capsid of the present application can be a chimeric AAV capsid that comprises capsid proteins with amino acid fragments, regions, or domains derived from two or more of AAV serotypes.
  • the capsid and ITRs of the present application can be derived from the same serotype of AAV or from different serotypes of AAV.
  • the capsid can be a capsid suitable for intravenous (IV) delivery (e.g., IV injection) to the peripheral tissues.
  • IV intravenous
  • peripheral tissue in the context of the present application refers to any tissue that is not a part of the brain or spinal cord.
  • the AAV vector comprises a capsid of 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 serotype, a variant thereof, or a chimeric AAV capsid.
  • the capsid is the AAV5, AAV6, AAV8 or AAV9 capsid, a variant thereof, or a chimeric capsid derived from two or more of AAV5, AAV6, AAV8 and AAV9.
  • the capsid is the AAV5 or AAV8 capsid, a variant thereof, or a chimeric capsid derived from AAV5 or AAV8.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated nucleic acid comprising the coding sequence, the rAAV vector or the viral particle of the present application, along with a pharmaceutically acceptable excipient.
  • Conventional pharmaceutically acceptable excipients are known in the art and can be solid or liquid excipients.
  • the pharmaceutical composition can be a liquid for injection.
  • the rAAV vector of the present application can be administered into a subject via systemic delivery or local delivery.
  • the rAAV vector of the present application can be delivered to peripheral tissues or organs, e.g., into peripheral blood, via any parental or enteral route.
  • the rAAV vector of the present application can be administered by intravenous (IV) , intramuscular (IM) , subcutaneous (SC) , intra-arterial, intraperitoneal (IP) , intradermal, transdermal, oral, nasal or rectal route.
  • IV intravenous
  • IM intramuscular
  • SC subcutaneous
  • IP intraperitoneal
  • the rAAV can be delivered by injection.
  • the rAAV vector can be delivered by a combined administration via more than one delivery route.
  • the rAAV vector can be administered via a single dose or multiple doses. In a specific embodiment, the rAAV vector is administered via a single injection.
  • the rAAV vector or the pharmaceutical composition can be used to treat a disease or condition related to or caused by deficiency or absence of lysosomal ⁇ -galactosidase A, e.g., a lysosomal storage disease such as Fabry disease.
  • a disease or condition related to or caused by deficiency or absence of lysosomal ⁇ -galactosidase A e.g., a lysosomal storage disease such as Fabry disease.
  • the therapeutically effective dosage of the rAAV vector can range from about 1 x 10 11 vg/kg to about 5 x 10 14 vg/kg.
  • the rAAV vector may be administered at a dosage between about 1 x 10 12 and about 5 x 10 14 vg/kg.
  • the rAAV vector may be administered at a dosage of at least about 1 x 10 11 , at least about 5 x 10 11 , at least about 1 x 10 12 , at least about 5 x 10 12 , at least about 1 x 10 13 , at least about 5 x 10 13 , at least about 1 x 10 14 vg/kg, or at least about 5 x 10 14 vg/kg.
  • the rAAV vector may be administered at a dosage of about 1 x 10 11 , about 5 x 10 11 , about 1 x 10 12 , about 5 x 10 12 , about 1 x 10 13 , about 5 x 10 13 , or about 1 x 10 14 vg/kg, or about 5 x 10 14 vg/kg.
  • rAAV By administration of the rAAV of the present application, one or more of the following therapeutic effects can be achieved: (1) increase of ⁇ -Gal A level; (2) increase of ⁇ -Gal A activity; (3) decrease or elimination of glycosphingolipids, in particular globotriaosylsphingosine (lyso-Gb3) , globotriaosylceramide (Gb3 or GL-3) or galactosylceramide.
  • the increase or decrease is determined by comparing to the level or activity of the same subject before receiving the rAAV administration.
  • the increase of ⁇ -Gal A level, the increase of ⁇ -Gal A activity and/or the decrease or elimination of glycosphingolipids such as lyso-Gb3 is observed in one or more of the tissues selected from plasma, heart, liver, and kidney of the subject.
  • the ⁇ -Gal A amount and/or activity increased by at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 1,000 times, at least about 2,000 times, at least about 5,000 times, at least about 10,000 times, at least about 20,000 times, at least about 50,000 times, or even more as compared to the ⁇ -Gal A amount and/or activity of the same subject before receiving the rAAV administration.
  • the level of glycosphingolipids in particular globotriaosylsphingosine (lyso-Gb3) or globotriaosylceramide (Gb3 or GL-3) can be 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%, as compared to the level of the same subject before receiving the rAAV administration.
  • the glycosphingolipids in particular globotriaosylsphingosine (lyso-Gb3) or globotriaosylceramide (Gb3 or GL-3) can be substantially eliminated in the plasma of the subject after the rAAV administration.
  • one or more of the therapeutic effects can be observed immediately after administration of the rAAV. In some embodiments, one or more of the 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 after the administration of the rAAV, or in an even longer period of time.
  • HepG2 cells used in the examples were purchased from ATCC. All the other types of cells were purchased from Procell Life Science &Technology Co., Ltd..
  • rAAVs were prepared into scAAVs for in vitro coding sequence evaluation and ssAAVs for in vivo and in vitro ⁇ -Gal A mutant, chimeric promoter and final construct evaluation.
  • CDS optimized coding sequences of GLA
  • C0 refers to the wild type GLA CDS
  • C1-C7 are the optimized CDS.
  • the optimized coding sequences were built into a plasmid vector having the structure as shown in Figure 1 to evaluate the GOI expression.
  • the plasmid vector comprised the following elements between the two ITRs: 3x HS-CRM (SEQ ID NO: 11) , TTR enhancer (SEQ ID NO: 12) , TTR promoter (SEQ ID NO: 13) , a chimeric intron (SEQ ID NO: 14) , the 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) .
  • the sequence information of the various elements is provided in Table 2 below.
  • Hep3B and Huh7 cells were maintained in DMEM + 10%FBS and passaged every 3 days. The day before transfection, cells were inoculated into 96-well plates at a density of 1.7 ⁇ 10 4 cells/well (Hep3B cells) or 1.48 ⁇ 10 4 cells/well (Huh7 cells) . The cells were transfected by the plasmids comprising C0-C7 with Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000008) following the user’s guide. 72 hours after transfection, supernatants were collected and the ⁇ -Gal A concentration was detected by ELISA (Sino Biological, SEK12078) . ELISA results were shown in Figure 2 and Table 3.
  • C3 gave the highest GLA expression.
  • C3 sequence has the lowest CpG number while the highest percentage GC content.
  • CpG in the AAV vectors has the potential to cause immunoreaction and result in exogenous gene silencing [3, 4] , lower CpG number would be favored for clinical use. Therefore, C3 is a promising candidate to provide more endurable and stronger GLA expression.
  • a limitation for Fabry Disease treatment is the poor cross uptake efficiency of the ⁇ -Gal A protein by the relevant tissues, e.g., heart and kidney.
  • the expressed ⁇ -Gal A enters the recipient cells via the mannose-6-phosphate (M6P) receptor (M6PR) [5] .
  • M6PR mannose-6-phosphate
  • M6PR mannose-6-phosphate receptor
  • M6PR is widely distributed in a variety of tissues and cells, the M6PR expression is limited, which restricts the amount of ⁇ -Gal A entering into heart or kidney, rendering insufficient therapeutic effect.
  • M6P is involved in phosphorylation of the protein glycosylation sites, with each glycosylation site having multiple M6Ps. So ⁇ -Gal A mutants with increased N-linked glycosylation sites were designed to increase the total numbers of M6P in the expressed ⁇ -Gal A protein. Thirteen ⁇ -Gal A mutants each with a single amino acid substitution were designed and the sequence information is described in Table 4, including the amino acid sequences and the corresponding nucleotide sequences. The nucleotide sequence selected as the template for the mutants was C3 (SEQ ID NO: 5) as shown in Example 1.
  • Recombinant AAV vectors were constructed for each of the mutants following the construct diagram as showed in Figure 3. Specifically, the vector comprised the following elements between the two ITRs: 3x HS-CRM (SEQ ID NO: 11) , TTR enhancer (SEQ ID NO: 12) , TTR promoter (SEQ ID NO: 13) , coding sequence of the wild-type protein or any one of the mutants (see Table 4) , and SV40 poly A (SEQ ID NO: 16) .
  • Table 5 The sequence information of the regulatory motifs of the GLA expression vectors for the evaluation of ⁇ -Gal A mutants.
  • HepG2, Hep3B, and Huh7 cells were maintained in DMEM + 10%FBS and passaged every 3 days. The day before transfection, cells were inoculated into 96-well plates in 1.48 ⁇ 10 4 cells/well (HepG2 cells or Huh7 cells) or 1.7 ⁇ 10 4 cells/well (Hep3B cells) . Plasmids were transfected by Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000008) following the user’s guide.
  • assay buffer 0.1 M sodium citrate, 0.05 M Na 2 HPO 4 and 100 mM N-acetyl-D-galactosamine
  • the vector comprised the following elements between the two ITRs: CRE I (SEQ ID NO: 44) , 3x HS-CRM (SEQ ID NO: 11) , TTR enhancer (SEQ ID NO: 12) , TTR promoter (SEQ ID NO: 13) , coding sequence of the wild-type protein (SEQ ID NO: 5) or the mutant (SEQ ID NO: 37) , and SV40 poly A (SEQ ID NO: 16) .
  • the sequence information is also shown in Table 7.
  • Table 7 The sequence information of the regulatory motifs of the GLA expression vectors for in vivo evaluation of the ⁇ -Gal A mutant M7.
  • Table 9 The ⁇ -Gal A activities in the heart, liver and kidney of mice receiving AAV8-P10-M0 or AAV8-P10-M7 at 4 weeks after rAAV administration.
  • Plasma lyso-Gb3 is considered as a promising biomarker for monitoring Fabry disease in the clinic.
  • the plasma lyso-Gb3 concentrations were evaluated by liquid chromatography–mass spectrometry (LC-MS) before and after (days 7, 14, 21 and 28) administration of the rAAVs. Results are shown in Figure 6C and Table 10.
  • M7 achieved higher ⁇ -Gal A enzyme activities in the liver and heart, and as a result, M7 also achieved better lyso-Gb3 lowering in the plasma.
  • Chimeric promoters based on the TTR promoter were designed to improve GLA liver expression.
  • Cis-regulatory elements (CREs) from genes with high expression in the human liver were selected [6] .
  • the selected CREs were inserted in different locations of P1 to generate P2-P13.
  • a liver-specific promoter, P0 was used as a control. Illustrations of the luciferase expressing constructs comprising P0-P13 are shown in Figure 7.
  • the sequence information is provided in Table 11.
  • HepG2, Hep3B, and Huh7 cells were maintained in DMEM + 10%FBS and passaged every 3 days. The day before transfection, cells were inoculated into 96-well plates in 1.48 ⁇ 10 4 cell/well (HepG2 or Huh7 cells) or 1.7 ⁇ 10 4 cell/well (Hep3B cells) . Plasmids were transfected by Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000008) following the user’s guide.
  • bioluminescence imaging was used to evaluate luciferase activities in different tissues ( Figure 9) .
  • P10 mediated the strongest luciferase expression.
  • the luciferase activities in protein extracts from various tissues were evaluated as RLU per 20 ⁇ g total protein and the results are shown in Figure 10 and Table 15.
  • Table 16 The sequence information of the regulatory motifs of the luciferase expression vectors for in vitro evaluation of P14-P21.
  • the HepG2, Hep3B, Huh7, AC16, IHC-SV40 (IHC) , H9C2 and 769P cells were passaged every 3 days. The day before transfection, cells were inoculated into 96-well plates in 1.48 ⁇ 10 4 cell/well (HepG2 or Huh7 cells) or 1.7 ⁇ 10 4 cell/well (Hep3B, AC16, IHC, H9C2 or 769P cells) . Plasmids were transfected by Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000008) following the user’s guide.
  • Table 17 The relative light unit (RLU) of cell lines transfected with P14-P21, P0 and cTNT.
  • rAAVs of the AAV8 capsid were generated comprising these four chimeric promoters (AAV8-P14, AAV8-P16, AAV8-P18 and AAV8-P21) for in vivo evaluation.
  • bioluminescence imaging was used to evaluate the luciferase activities in different tissues ( Figure 13) .
  • the luciferase activity in protein extracts from various tissues were evaluated as RLU per 20 ⁇ g total protein ( Figure 14 and Table 18) .
  • Table 18 The relative light unit (RLU) of tissues from mice receiving AAV8-P14, AAV8-P16, AAV8-P18 or AAV8-P21 injection.
  • the GLA -/Y mice receiving PBS and WT mice were used as controls.
  • the ⁇ -Gal A activities in plasma were evaluated and results are shown in Figure 16 and Table 20.
  • the ⁇ -Gal A activities increased 38,149.0, 261,649.1, 24,461.2, 515,638.0 and 30,157.0 times for AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 and AAV8-P21-M7, separately, which were 8,882.9, 60,924.5, 5,695.7, 120,065.4 and 7,022.0 folds higher than that of the WT.
  • Lyso-Gb3 concentrations in the plasma, heart, liver and kidney were assessed. Results are shown in Figures 18-19 and Tables 22-23.4 weeks after 1 ⁇ 10 13 vg/kg AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 and AAV8-P21-M7 administrations, the plasma lyso-Gb3 decreased 98.7%, 98.9%, 98.6%, 99.1%and 98.8%, respectively. Accordingly, the heart lyso-Gb3 decreased 99.1%, 99.2%, 98.8%, 99.4%and 99.1%.
  • the liver lyso-Gb3 decreased 99.5%, 99.5%, 99.2%, 99.6%and 99.5%.
  • the kidney lyso-Gb3 decreased 98.6%, 99.0%, 97.7%, 99.5%and 97.9%. The above results demonstrated the dramatic therapeutic effects four weeks after the rAAV administrations.
  • mice receiving AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 or AAV8-P21-M7 were treated with AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 or AAV8-P21-M7.
  • mice receiving AAV8-P10-M7, AAV8-P14-M7, AAV8-P16-M7, AAV8-P18-M7 or AAV8-P21-M7 at 4 weeks after the rAAV administrations.
  • AAV5-P14-M7 and AAV5-P18-M7 were dosed at the concentrations of 1 ⁇ 10 12 vg/kg (low dose) , 1 ⁇ 10 13 vg/kg (middle dose) and 5 ⁇ 10 13 vg/kg (high dose) .
  • AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7 and AAV6-P18-M7 were dosed at the concentrations of 1 ⁇ 10 11 vg/kg (low dose) , 1 ⁇ 10 12 vg/kg (middle dose) and 1 ⁇ 10 13 vg/kg (high dose) .
  • the GLA -/Y mice receiving PBS and WT mice were used as controls.
  • the plasma ⁇ -Gal A activities were tracked till 16 weeks after dosing. The results are shown in Figures 20A, 20B, 21 and Table 24. The plasma ⁇ -Gal A activities increased since the AAV administration and reached its plateau on about 14 days after dosing.
  • the ⁇ -Gal A activities increased 38434.3, 203.5, 303784.6, 183069.0, 106195.3 and 176325.0 times for AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 and AAV6-P18-M7, respectively, which were 9709.7, 51.4, 76745.6, 46249.0, 26828.3 and 46401.3 folds higher than that of the WT.
  • mice receiving AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 or AAV6-P18-M7.
  • the ⁇ -Gal A activities in the peripheral tissues reached 1.1, 1.1, 23.1, 44.0, 30.1 and 15.4 times of that of the WT for GLA -/Y mice receiving AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 and AAV6-P18-M7, respectively.
  • the ⁇ -Gal A activities in the liver reached 107.7, 1.0, 230.5, 110.3, 258.2 and 657.7 times of that of the WT.
  • the ⁇ -Gal A activities in the kidney reached 18.8, 1.0, 70.1, 22.1, 21.3 and 20.1 times of that of the WT.
  • mice receiving AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 or AAV6-P18-M7 at 16 weeks after rAAV administrations.
  • the plasma lyso-Gb3 decreased 98.5%, 55.5%, 98.6%, 96.8%, 98.2%and 89.0%, respectively, as compared to the level before dosing.
  • the heart lyso-Gb3 decreased 98.9%, 59.2%, 99.3%, 98.9%, 99.4%and 92.3%, respectively.
  • the liver lyso-Gb3 decreased 99.4%, 82.0%, 99.6%, 99.5%, 99.6%and 97.9%, respectively.
  • the kidney lyso-Gb3 decreased 98.0%, 65.7%, 99.2%, 97.3%, 98.5%and 92.4%, respectively.
  • the above results demonstrated the dramatic therapeutic effects at 16 weeks after the rAAV administrations.
  • mice receiving AAV5-P14-M7, AAV6-P14-M7, AAV8-P14-M7, AAV9-P14-M7, AAV5-P18-M7 or AAV6-P18-M7.
  • Table 27 The lyso-Gb3 concentrations in 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 at 16 weeks after the rAAV administrations.
  • AAV5-P14-M7 and AAV5-P18-M7 were dosed at the concentration of 1 ⁇ 10 13 vg/kg.
  • AAV8-P14-M7 and AAV8-P18-M7 were dosed at the concentration of 1 ⁇ 10 12 vg/kg.
  • the GLA -/Y mice receiving PBS and WT mice were used as controls.
  • the plasma ⁇ -Gal A activities and lyso-Gb3 concentrations were tracked as long as 52 weeks after dosing. The results are shown in Figure 24 and Tables 28, 29.
  • the plasma ⁇ -Gal A activities increased soon after the AAV administration and maintained as a high-level plateau to more than 52 weeks.
  • the ⁇ -Gal A activities kept at high levels of 125.8, 2514.6, 591.6 and 2299.7 times of that of the WT for AAV5-P14-M7, AAV5-P18-M7, AAV8-P14-M7 and AAV8-P18-M7, respectively.
  • nearly complete elimination of plasma lyso-Gb3 was sustained in all four groups receiving rAAV treatment.
  • SEQ ID NO: 1 wild-type GLA amino acid sequence
  • SEQ ID Nos: 2-9, 11-16 are listed above.
  • SEQ ID NO: 40 nucleotide sequence of M10
  • SEQ ID NO: 46 nucleotide sequence of Luciferase

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  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des variants polypeptidiques de l'α-Gal A humaine, la séquence codante optimisée desdits variants polypeptidiques et de la protéine de type sauvage, ainsi que des vecteurs viraux adéno-associés recombinés (rAAV) comportant ladite séquence codante. La présente invention concerne également des promoteurs chimériques induisant une forte expression dans le foie et le cœur, ce qui convient aux rAAV de la présente invention.
PCT/CN2024/129227 2023-11-03 2024-11-01 Vecteurs viraux adéno-associés recombinés pour le traitement de la maladie de fabry Pending WO2025092951A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105916984A (zh) * 2013-10-23 2016-08-31 建新公司 重组糖蛋白及其用途
CN107208081A (zh) * 2015-01-22 2017-09-26 Jcr制药股份有限公司 用于从包含污染宿主细胞蛋白质的材料纯化重组人α‑半乳糖苷酶A的方法
WO2019161059A1 (fr) * 2018-02-14 2019-08-22 Generation Bio Co. Vecteurs d'adn non viraux et utilisations associées pour la production d'anticorps et de protéines de fusion
US20210015898A1 (en) * 2018-04-09 2021-01-21 Allen Institute Rescuing voltage-gated sodium channel function in inhibitory neurons
CN112912112A (zh) * 2018-10-26 2021-06-04 Vrije布鲁塞尔大学 肝特异性核酸调节元件以及其方法及用途
CN113423434A (zh) * 2018-12-05 2021-09-21 阿贝奥纳治疗有限公司 用于基因递送的重组腺相关病毒载体
CN113631182A (zh) * 2018-10-10 2021-11-09 阿米库斯治疗学公司 二硫键稳定的多肽组合物和使用方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105916984A (zh) * 2013-10-23 2016-08-31 建新公司 重组糖蛋白及其用途
CN107208081A (zh) * 2015-01-22 2017-09-26 Jcr制药股份有限公司 用于从包含污染宿主细胞蛋白质的材料纯化重组人α‑半乳糖苷酶A的方法
WO2019161059A1 (fr) * 2018-02-14 2019-08-22 Generation Bio Co. Vecteurs d'adn non viraux et utilisations associées pour la production d'anticorps et de protéines de fusion
US20210015898A1 (en) * 2018-04-09 2021-01-21 Allen Institute Rescuing voltage-gated sodium channel function in inhibitory neurons
CN113631182A (zh) * 2018-10-10 2021-11-09 阿米库斯治疗学公司 二硫键稳定的多肽组合物和使用方法
CN112912112A (zh) * 2018-10-26 2021-06-04 Vrije布鲁塞尔大学 肝特异性核酸调节元件以及其方法及用途
CN113423434A (zh) * 2018-12-05 2021-09-21 阿贝奥纳治疗有限公司 用于基因递送的重组腺相关病毒载体

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