WO2024074143A1 - Construction pour améliorer l'expression génique - Google Patents

Construction pour améliorer l'expression génique Download PDF

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WO2024074143A1
WO2024074143A1 PCT/CN2023/123282 CN2023123282W WO2024074143A1 WO 2024074143 A1 WO2024074143 A1 WO 2024074143A1 CN 2023123282 W CN2023123282 W CN 2023123282W WO 2024074143 A1 WO2024074143 A1 WO 2024074143A1
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aav
sequence
seq
nos
expression construct
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PCT/CN2023/123282
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Qing Lin
Yixiong CHEN
Xiaojing SHENG
Tianqi JIANG
Qiang Wu
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Lingyi Biotech Co., Ltd.
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    • 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
    • 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
    • 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
    • C12N15/861—Adenoviral 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C12N2830/008—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
    • 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
    • C12N2830/42—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
    • 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)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase

Definitions

  • the present disclosure relates to a nucleotide construct.
  • the invention further relates to said nucleotide construct, an expression vector, and its use.
  • the present disclosure an expression construct comprising a transcription regulatory element operably linked to a polynucleotide sequence of interest, wherein the expression construct comprises a promoter, and an enhancer, the enhancer is upstream of the promoter,
  • Enhance 3 is optional
  • Enhance 1 comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 13 and 15, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 13 and 15, the all or the portion of the sequence retains the functionality of enhancer, and
  • the Enhance 2 comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 8-11 and 13, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 8-11 and 13, the all or the portion of the sequence retains the functionality of enhancer.
  • the Enhance 3 comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 8-11, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 8-11, the all or the portion of the sequence retains the functionality of enhancer.
  • the promoter comprises all or a portion of a sequence selected from the sequence consisting of SEQ ID NOs: 2-6, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 2-6, the all or the portion of the sequence retains the functionality of promoter;
  • the promoter comprises all or a portion of a sequence selected from the sequence consisting of SEQ ID NOs: 3-6, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 3-6, the all or the portion of the sequence retains the functionality of promoter.
  • the Enhance 1 comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 13 and 15, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 13 and 15, the all or the portion of the sequence retains the functionality of enhancer;
  • the Enhance 2 comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 8-9, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 8-9, the all or the portion of the sequence retains the functionality of enhancer.
  • the Enhance 1 comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 13 and 15, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 13 and 15, the all or the portion of the sequence retains the functionality of enhancer;
  • the Enhance 2 comprises all or a portion of a sequence comprising SEQ ID NO: 8, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 8, the all or the portion of the sequence retains the functionality of enhancer.
  • the expression construct further comprises an untranslated intron region.
  • the untranslated intron region comprises all or a portion of a sequence selected from the sequence consisting of SEQ ID NOs: 24-43, and at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%identical to SEQ ID NOs: 24-43.
  • the untranslated intron region is operably linked to 5’ terminal of the polynucleotide sequence of interest.
  • the untranslated intron region is located between 5’ and 3’ terminal of the polynucleotide sequence of interest.
  • the disclosure provides a vector comprising the expression construct of present disclosure.
  • the vector is viral vector, preferably AAV vector.
  • the vector further comprises two adeno-associated virus inverted terminal repeats (ITR) sequences flanking the expression construct, preferably further comprises a poly A sequence.
  • ITR adeno-associated virus inverted terminal repeats
  • the disclosure provides an adeno-associated virus (AAV) comprising the vector of present disclosure and capsid protein.
  • AAV adeno-associated virus
  • the AAV is selected from the group consisting of: serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVhu37 or any one of the AAV serotypes isolated from human and nonhuman mammalians or variant thereof.
  • the disclosure provides a composition comprising the expression construct, vector, or AAV of present disclosure and a pharmaceutically acceptable excipient.
  • the disclosure provides the expression construct, vector, or AAV of present disclosure for use in a method of treatment.
  • the disclosure provides use of the expression construct, vector, or AAV of present disclosure in the preparation of a medication for treatment of a disease or condition in a subject.
  • the disclosure provides the expression construct, vector, or AAV of present disclosure for use in a method for treating a disease or condition in a subject.
  • the disclosure provides a method for treating a disease or condition in a subject, comprising administering an effective amount of the expression construct, vector, or AAV of present disclosure to the patient.
  • the disclosure provides the expression construct, vector, or AAV of present disclosure for use in a method of expressing the nucleotide sequence of interest in a subject.
  • the disclosure provides the expression construct, vector, or AAV of present disclosure for use in a method of expressing the nucleotide sequence of interest in a subject.
  • FIG. 1 showed luciferase and GCase activity in HepG2 Cells with chimeric HSRE constructs.
  • A Luciferase activity comparisons for different CHSREs based on promoter HSRE002.
  • B Luciferase activity comparisons for different CHSREs based on promoter HSRE005.
  • FIG. 2 showed luciferase and GCase activities in HSRE012 constructs in HepG2.
  • A Luciferase activity in constructs with various HSRE012 locations flanked by promoter HSRE002. Significant differences observed: PG133 vs. PG131 (P ⁇ 0.01) , PG134 vs. PG132 (P ⁇ 0.01) .
  • (D) GCase enzyme activity comparisons for various HSRE012 copy numbers flanked by promoter HSRE002. No significant differences observed: PG055 vs. PG025, PG056 vs. PG026, PG057 vs. PG027 (Values: mean ⁇ SEM, N 3 per group) .
  • FIG. 3 showed GCase Enzyme activities in constructs with exogenous introns flanked by HSREs in vitro.
  • A GCase enzyme activities of construct with endogenous intron in Huh7 cells. P ⁇ 0.05 for Group PG059 compared with PG058.
  • B GCase enzyme activities of different constructs in HepG2 cells in vitro. P ⁇ 0.01 for Group PG076, PG078 and PG081 compared with PG074. P ⁇ 0.01 for Group PG090 compared with PG082 and P ⁇ 0.05 for Group PG091 compared with PG082.
  • FIG. 4 showed GCase enzyme activities in constructs with various introns flanked by Cchimeric HSREs in vitro.
  • A GCase enzyme activities of different constructs in HepG2 cells. P ⁇ 0.05 for Group PG107 and PG108 compared with PG168. P ⁇ 0.01 for Group PG114 and PG115 compared with PG169.
  • FIG. 5 showed GCase enzyme activities in serum and tissue lysates 2 weeks post-injection of AAV8 vectors at a dose of 2E12 vg/kg in wild-type mice.
  • A GCase enzyme activities were measured in serum. P ⁇ 0.05 for Group PG026, PG037, PG051 and PG105 and P ⁇ 0.01 for Group PG103, PG104 and PG105 compared with PG127. P ⁇ 0.01 for Group PG103 and PG104 compared with PG102. P ⁇ 0.01 for Group PG103 and PG104 compared with PG026.
  • C GCase enzyme activity measured in lung lysates.
  • FIG. 6 showed markable efficacy of AAV8 gene therapy candidates regulated by chimeric HSREs in Gaucher mice.
  • A GCase enzyme activities were measured in serum 8 weeks post-injection of AAV8 candidates at dose of 2E12 vg/kg.
  • Substrate accumulation was measured in serum 8 weeks post-injection of AAV8 candidates at dose of 2E12 vg/kg.
  • FIG. 7 showed GCase enzyme activities in tissue lysates from Gaucher mice 12 weeks post-injection of AAV8 candidates at a dose of 2E12 vg/kg.
  • A GCase enzyme activities were measured in liver lysates. P ⁇ 0.01 for Group PG118 -PG122 compared with Buffer control or Cerezyme groups. P ⁇ 0.05 for Group PG120 and PG122 and P ⁇ 0.01 for Group PG118 and PG119 compared with PG127.
  • B GCase enzyme activities measured in lung lysates. P ⁇ 0.05 for Group PG118 -PG122 compared with Buffer control or Cerezyme groups.
  • FIG. 8 showed substrate accumulation of glucosylsphingosine in tissue lysates 12 weeks post-injection of AAV8 candidates at a dose of 2E12 vg/kg in Gaucher mice.
  • A Substrate accumulation was measured in liver lysates. P ⁇ 0.01 for Group PG001, PG011, PG119 and PG120 compared with Buffer control or Cerezyme groups. P ⁇ 0.01 for groups of PG119 and PG120 compared with PG127.
  • B Substrate accumulation measured in lung lysate. P ⁇ 0.05 for Group PG001 and P ⁇ 0.01 for Group PG011, PG119 and PG120 compared with Buffer control or Cerezyme groups.
  • FIG. 9 showed GCase enzyme activities in serum and tissue lysates 2 weeks post-injection of AAV9 candidates at a dose of 2E12 vg/kg in wild-type mice.
  • A GCase enzyme activities were measured in serum. P ⁇ 0.05 for Group PG124 and P ⁇ 0.01 for Group PG123, PG125 and PG126 compared with Buffer control group. P ⁇ 0.05 for Group PG124 and PG125 and P ⁇ 0.01 for Group PG123 and PG126 compared with PG001. P ⁇ 0.05 for Group PG125 and P ⁇ 0.01 for Group PG123 and PG126 compared with PG128.
  • B GCase enzyme activities were measured in liver lysates.
  • P ⁇ 0.01 for Group PG123 -PG126 compared with Buffer control group P ⁇ 0.05 for Group PG124 and PG125 and P ⁇ 0.01 for Group PG123 and PG126 compared with PG001.
  • C GCase enzyme activities were measured in lung lysates.
  • compositions and methods are intended to mean that the compositions and methods include the recited elements, but do not exclude others.
  • nucleotide and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other natural, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases.
  • expression refers to the two-step process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • encodes or "encoding” as it is applied to polynucleotides refers to a polynucleotide which is said to "encode” a polypeptide if it can be transcribed to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • promoter means a control sequence that is a region of a polynucleotide sequence at winch the initiation and rate of transcription of a coding sequence, such as a gene or a transgene, are controlled. Promoters may be constitutive, inducible, repressible, or tissue-specific. In embodiments, the promoter is used together with an enhancer to increase the transcription efficiency. An enhancer is a regulatory element that increases the expression of a target sequence.
  • protein protein
  • peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds.
  • the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least, two amino acids and no limitation is placed on the maximum number of amino acids which may comprise, consist essentially of or consist of a protein's or peptide's sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • Identity refers to sequence similarity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between sequences is a function of the number of matching positions shared by the sequences.
  • vector refers to a nucleic acid comprising, consisting essentially of, or consisting of an intact replicon such that the vector may be replicated when placed within a cell, for example by a process of transfection, infection, or transformation. It is understood in the art that once inside a cell, a vector may replicate as an extrachromosomal (episome) element or may be integrated into a host cell chromosome.
  • Vectors may include nucleic acids derived from retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papovaviruses, AAV viral vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, e.g., Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying, et al. (1999) Nat. Med. 5 (7) : 823-827.
  • Adeno-associated virus refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae.
  • Adeno-associated virus is a single-stranded DNA virus that grows only in cells in which certain functions are provided by a co-infecting helper virus. All AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins. At least 13 sequentially numbered naturally-occurring AAV serotypes are known in the art.
  • Non-limiting exemplary serotypes useful in the methods disclosed herein include any of those 13 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ and AAV PHP.
  • the AAV particle comprises, consists essentially of, or consists of three major viral proteins: VP1, VP2 and VP3.
  • the AAV refers to the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.
  • the AAV particle comprises an AAV capsid protein selected from the group consisting of AAVPHP.
  • the AAV refers to the serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, AAVrh10, AAVhu37 or any one of the AAV serotypes isolated from human and nonhuman mammalians or variant thereof.
  • the AAV particle comprises an AAV capsid protein selected from the group consisting of: AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15,
  • AAV1-8/rh. 49 AAV2-15/rh. 62, AAV2-3/rh. 61, AAV2-4/rh. 50, AAV2-5/rh. 51, AAV3.1/hu. 6, AAV3.1/hu. 9, AAV3-9/rh. 52, AAV3-11/rh. 53, AAV4-8/r11.64, AAV4-9/rh. 54, AAV4-19/rh. 55, AAV5-3/rh. 57, AAV5-22/rh. 58, AAV7.3/hu. 7, AAV16.8/hu. 10, AAV16.12/hu. 11, AAV29.3/bb. 1, AAV29.5/bb. 2, AAV106.1/hu.
  • AAV114.3/hu. 40 AAV127.2/hu. 41, AAV127.5/hu. 42, AAV128.3/hu. 44, AAV130.4/hu. 48, AAV145.1/hu. 53, AAV145.5/hu. 54, AAV145.6/hu. 55, AAV161.10/hu. 60, AAV161.6/hu. 61, AAV33.12/hu. 17, AAV33.4/hu. 15, AAV33.8/hu. 16, AAV52/hu. 19, AAV52.1/hu. 20, AAV58.2/hu.
  • AAVhu. 45 AAVhu. 46, AAVhu. 47, AAVhu. 48, AAVhu. 48R1, AAVhu. 48R2, AAVhu. 48R3, AAVhu. 49, AAVhu. 51, AAVhu. 52, AAVhu. 54, AAVhu. 55, AAVhu. 56, AAVhu. 57, AAVhu. 58, AAVhu. 60, AAVhu. 61, AAVhu. 63, AAVhu. 64, AAVhu. 66, AAVhu. 67, AAVhu. 14/9, AAVhu. t 19, AAVrh. 2, AAVrh.
  • AAV true type AAV
  • UPENN AAV 10 Japanese AAV 10 serotypes
  • AAV CBr-7.1, AAV CBr-7.10 AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1,
  • AAV vector refers to a vector comprising one or more heterologous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeat sequences (ITRs) .
  • HNA heterologous nucleic acid
  • ITRs AAV inverted terminal repeat sequences
  • AAV vectors can be replicated in a host cell that provides the functionality of rep and cap gene products, and allow the ITRs and the nucleic acid between the ITRs to be packaged into infectious viral particles.
  • AAV vectors comprise a promoter, at least one nucleic acid that may encode at least one protein or RNA, and/or an enhancer and/or a terminator within the flanking ITRs that is packaged into the infectious AAV particle.
  • the ITRs and the nucleic acid between the ITRs may be encapsulated into the AAV capsid, and this encapsidated nucleic acid may be referred to as the “AAV vector genome. ”
  • AAV vectors may contain elements in addition to the encapsidated portion, for example, antibiotic resistance genes or other elements known in the art included in the plasmid for manufacturing purposes but not packaged into the AAV particle.
  • viral capsid refers to the proteinaceous shell or coat of a viral particle. Capsids function to encapsidate, protect, transport, and/or release into the host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of protein ( “capsid proteins” ) .
  • the viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3.
  • AAV virion or “AAV viral particle” or “AAV particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide from an AAV vector referred to herein as the AAV vector genome.
  • a "subject" of diagnosis or treatment is an animal such as a mammal, or a human.
  • a subject is not limited to a specific species and includes non-human animals subject to diagnosis or treatment and those subject to infections or animal models, including, without limitation, simian, murine, rat, canine, or leporid species, as well as other livestock, sport animals, or pets.
  • the subject is a human.
  • treating or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease) , stabilized (i.e., not worsening) state of a condition (including disease) , delay or slowing of condition (including disease) progression, amelioration or palliation of the condition (including disease) states and remission (whether partial or total) , whether detectable or undetectable.
  • an effective amount intends to mean a quantity sufficient to achieve a desired effect.
  • the effective amount may depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions.
  • an effective amount is an amount sufficient to result in gaining partial or full function of a gene that is deficient in a subject.
  • the effective amount of an AAV viral particle is the amount sufficient to result in expression of a gene in a subject. The skilled artisan will be able to determine appropriate amounts depending on these and other factors.
  • the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target subject and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations.
  • the effective amount may comprise, consist essentially of, or consist of one or more administrations of a composition depending on the embodiment.
  • administering intends to mean delivery' of a substance to a subject such as an animal or human. Administration can be affected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and wall vary with the composition used for therapy, the purpose of the therapy, as well as the age, health or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and other animals, treating veterinarian.
  • AAV8 and AAV9 particles were produced by transient triple-transfection of HEK293T cells or suspension HEK293 cells with plasmids encoding the AAV Rep and Cap proteins, as well as the adenoviral helper genes, as well the recombinant genome containing the GBA1 construct.
  • rAAV particles were purified using iodixanol based density gradient ultracentrifugation method. Subsequently, rAAV was then quantified by probe-based ddPCR (Biorad) assay and characterized by silver staining.
  • HEK293T or the liver hepatocyte cell line HepG2 and Huh7 were plated in a 24-well plate at a cell density of 1.5E5 cells/well. Each well received 500 ⁇ l of complete cell culture medium.
  • a PEI-based transfection reagent was used for transfection. Specifically, 0.15 ⁇ g of plasmids containing transgene sequence, as well as 0.15 ⁇ g of plasmids containing Luciferase reporter gene, was co-transfected into each well. 48h after transfection, 300 ⁇ l of fresh complete cell culture medium was added to each well, and cells were incubated for additional 24 hours.
  • GCase activity assay cell culture supernatants were proceeded to enzyme activity assay according to the method described below, cells were lysed by using cell lysis buffer (Promega) and proceeded to Luciferase detection by using Steady-Glo Luciferase detection system (Promega) . The 96-well plates containing cell lysate and detection reagent were read on Varioskan LUX reader (ThermoFisher) . All enzyme activity results were first normalized to corresponding Firefly Luciferase intensity. The results were then normalized to the control group. rAAV biopotency assay was performed by cell transduction using HEK293T, Huh7 or HepG2 cells.
  • rAAV transduction was performed at defined multiplicity of infection (MOI) of 1E5 or 1E6.48h after infection, 300 ⁇ l of fresh complete cell culture medium was added to each well, and cells were incubated for another 24 hours. 72h after infection, cell culture supernatant was proceeded to enzyme activity test according to the method described below.
  • MOI multiplicity of infection
  • AAV vector containing the GBA1 transgene were administered through tail vein injection of wild type (C57BL/6) male mice at age of 8-9 weeks.
  • AAV dose was 1E12 vg/ml.
  • serum GCase levels were measured at various time intervals (1, 2, or 4 weeks) post injection. Mice were followed up to 4 weeks post AAV treatment and sacrificed for biochemical and pathological analysis.
  • AAV vector containing the GBA1 transgene were administered through tail vein injection of Gaucher (a combination of two different types of GBA1 mutations) mice at age of 7-12 weeks old. All mice were maintained under a special pathogen-free environment and in individually ventilated cages. All cages, cob bedding, and water were sterilized before use. The cages, cob bedding, food and water will be changed twice a week.
  • Gaucher a combination of two different types of GBA1 mutations
  • the AAV dose was ranged from 2E11 to 5E13 vg/ml.
  • serum GCase levels and substrate accumulation levels were measured at various time intervals post injection. Mice were followed up to the end point of study and sacrificed for biochemical and pathological analysis.
  • Cerezyme was resuspended according to the manufacturer's instructions and aliquoted (40IU/ml) and stored at -80°C. Before injection, the aliquot was thawed on ice and diluted, and gently but thoroughly mixed.
  • Serum was separated from fresh blood without anticoagulation within 0.5 hour at 4°C by centrifugation at 12,000 rpm for 15 mins. Serum was stored at -80°C. For the Cerezyme group, serum was collected 1.5 hours after injection.
  • mice were anesthetized and euthanized. Tissues were collected from the mice after perfusion with saline and stored at -80°C. For the Cerezyme group, tissue samples were collected 1.5 hours after injection. Tissue samples were divided into 4 parts, with 3 parts frozen in individual tubes and stored at -80°C for GCase activity assay, glucosylsphingosine analysis and mRNA analysis. The remaining part was fixed in 10%neutral buffered formalin solution (NBF, pH 7.4) for about 24-48 h at room temperature for histology analysis. Bone marrow were collected from femurs and tibias of both legs of the mice.
  • NAF 10%neutral buffered formalin solution
  • Serum samples were obtained from mouse blood and stored at -80°C. Tissues were lysed in tissue lysis buffer (Citrate-phosphate buffer, pH5.0, 0.25%Sodium taurocholic, 1%TX-100 with Proteinase inhibitor cocktail) using a homogenizer (Shanghai jingxin) at specific program (50 Hz, working 30s and cooling down 30s, 4 min totally) .
  • tissue lysis buffer Concentrate-phosphate buffer, pH5.0, 0.25%Sodium taurocholic, 1%TX-100 with Proteinase inhibitor cocktail
  • homogenizer Shanghai jingxin
  • specific program 50 Hz, working 30s and cooling down 30s, 4 min totally
  • 4-Methylumbelliferyl ⁇ -D-glucopyranoside (4MU-Glc, Carbosynth) served as the substrate for enzyme GCase.
  • serum was diluted 1: 100 using enzyme assay buffer (Citrate-phosphate buffer, pH5.0, 0.25%Sodium taurocholic, 0.25%TX-100) .
  • Tissue lysate was diluted 1: 40 using lysis buffer (Citrate-phosphate buffer, pH5.0, 0.25%Sodium taurocholic, 1%TX-100 with Proteinase inhibitor cocktail) .
  • Fluorescence levels were then converted to nmol/h/ml (serum) or nmol/h/mg of total protein (liver, spleen, lung, bone marrow and brain) based on a 4-Methylumbelliferone (4-MU, Sigma-Aldrich) standard curve.
  • RNeasy Kit QIAGEN
  • TAKARA Primescript RT master mix
  • Rabbit anti-mouse CD68 antibody (1: 25 Abcam AB53444) was used to visualize mouse macrophage.
  • the formalin-fixed mouse tissues were deparaffined with xylene and graded ethanol washes, followed by antigen retrieval using pepsin according to product use recommendations. Sections were counterstained with haematoxylin. Biotin labeled secondary antibody was used for detecting.
  • the signals were visualized by using Streptavidin-HRP and Tyramide signal amplification kit according to recommendations.
  • Tissue sections were stained with hematoxylin and eosin (H&E) .
  • the stained tissues were scanned with Aperio AT2 (Leica, 40X) .
  • the tissue images were processed with Aperio ImageScope (V12.4.3.5008) . All Gaucher cells on a whole tissue section for both liver and lung per mouse are counted manually. Gaucher cell counts from the whole section was normalized to the tissue slice area (square centimeter) for data graph.
  • Tissue homogenate was prepared by homogenizing with 9 volumes (w: v) of PBS buffer. Aliquots (10 pL) of tissue lysates or serum sample were subjected to LC/MS analysis. The quantitated tissue Lyso_GL1 were normalized by tissue weight, and substrate level in serum was normalized by serum volume. Values bellowed the lower quantitation limit of Lyso_GL1 (LLOQ) of 10 ng/g for tissues and 1 ng/ml for serum or plasma will be labelled as BQL in the corresponding figures.
  • LLOQ lower quantitation limit of Lyso_GL1
  • this disclosure outlines an integrated approach for designing and screening novel expression cassettes that efficiently and selectively express therapeutic GCase in the liver.
  • the first step involves cloning the nucleotide sequences of Firefly Luciferase gene and GBA1 gene into target vectors.
  • the nucleotide sequences of Firefly Luciferase gene and GBA1 gene were driven by a series of chimeric hepatic specific regulatory elements (CHSREs) that were rationally designed with the combination of different promoters and various regulatory elements.
  • CHSREs chimeric hepatic specific regulatory elements
  • the regulatory elements described herein are HSRE006, HSRE007, HSRE008, HSRE009, HSRE010, HSRE011, HSRE012, HSRE013 and HSRE014. Introns were also employed in some embodiments to further increase the GCase expression.
  • the different promoters and various regulatory elements ID as well as their nucleotide sequences used in the present discourse are shown in Table 1. Codon-optimized GBA1 gene with K321N mutation is an Exemplary single nucleotide sequence of interest.
  • Luciferase or GCase To confine the expression of the Luciferase or GCase in the liver, a series of chimeric hepatic specific regulatory elements (CHSREs) comprising core promoter and one, two, three or more regulatory elements were well designed and underwent in vitro or in vivo screening by comparing the Luciferase or GCase expression efficiency in HepG2 cells. Firefly Luciferase or mGBA-C110 (a codon-optimized GBA1 version with K321N mutation) were expressed under the control of various CHSREs.
  • CHSREs hepatic specific regulatory elements
  • Chimeric hepatic regulatory element comprising of one, two, three or multiple copy of enhancers selected from HSRE010, HSRE014, HSRE012, HSRE006, HSRE009 are then constructed with the combination of 4 promoters of HSRE002, HSRE004, HSRE003 or HSRE005 to drive the expression of Luciferase gene or GBA1 gene as the constructs of PG130 -PG165 and PG023, PG024, PG025, PG026, PG027, PG028, PG029, PG030, PG033, PG034, PG035, PG036, PG037, PG038, PG040, PG041, PG042, PG043, PG044, PG045, PG047, PG048, PG049, PG050, PG051, PG052 for further examination of Luciferase or GCase expression in HepG2 cells.
  • CHSRE
  • PG026-CHSRE005) have no effect on the expression of GCase (Fig. 1E) .
  • Further tandem adding HSRE010 to CHSRE005 as CHSRE009 in PG143 significantly augmented Luciferase expression compared to PG132-CHSRE005 (Fig. 1A) significantly augmented Luciferase expression compared to PG132-CHSRE005 (Fig. 1A) , however, no effect on the GCase expression with CHSRE009 in PG030 was observed compared to the corresponding control of PG026-CHSRE005 (Fig. 1E) .
  • introns selected from human GBA1 derived endogenous introns of Int001 inserted between 27-28bp of SEQ ID NO: 21) , Int002 (inserted between 115-116bp of SEQ ID NO: 21) , Int003 (inserted between 307-308bp of SEQ ID NO: 21) , Int004 (inserted between 454-455bp of SEQ ID NO: 21) , Int005 (inserted between 588-589bp of SEQ ID NO: 21) , Int006 (inserted between 761-762bp of SEQ ID NO: 21) , Int007 (inserted between 999-1000bp of SEQ ID NO: 21) , Int008 (inserted between 1224-1225bp of SEQ ID NO: 21) , Int009 (inserted between 1388-1389bp of SEQ ID NO: 21) and Int0010 (inserted between 1505-1506bp of SEQ ID NO: 21) were
  • Endogenous introns selected from the group of Int002, Int003, Int005 and exogenous intron selected from Int011 and Int020 were cloned with mGBA-C110 under the promoters of HSRE002 as the constructs of PG069-PG073.
  • the above constructs along with the PG058 and PG059 were transfected into Huh7 cells for the examination of GCase activity in cell culture supernatant.
  • the PG059 constructs with Int001 inserted were proven to have higher GCase expression than the control without intron. (Fig. 3C) .
  • exogenous intron selected from group of Int012, Int013, Int014, Int015, Int016, Int017, Int018, Int019 and Int011 were cloned with mGBA-C110 under the hepatic specific promoters of HSRE001, HSRE015 and HSRE016 respectively as the constructs of PG074-101.
  • These constructs with exogenous intron inserted upstream of GBA1 coding sequence were transfected in HepG2 cells and HEK293T cells for the examination of GCase activity in cell culture supernatant. The results showed that all constructs expressed GCase efficiently in HepG2 cells but very low in HEK293T cells (Fig. 3A and 3B) .
  • Constructs with exogenous chimeric intron inserted between chimeric HSREs and GBA1 coding sequence are PG110, PG111, PG112 and PG113, PG169, PG114 and PG115.
  • Transfections were conducted on HepG2 and HEK293T cells, the culture supernatants were harvested for measuring GCase activity. The results showed that all these constructs with the combination of chimeric HSREs and both of endogenous intron 1 and exogenous chimeric intron expressed GCase efficiently in HepG2 cells GBA1 (Fig. 4A) .
  • constructs of PG102, PG026, PG103, PG104, PG037, PG107, PG108, PG051, PG105 and PG106 were selected and packaged with AAV8 for further study in wild type mice.
  • AAV8 products along with reference product of PG127, which is constructed with the combination of sequence of SEQ ID NO: 14, SEQ ID NO: 5, and SEQ ID NO: 23 as described in patent WO2020161483A1, were injected into wild type mice at the dose of 2E12 vg/kg (Fig. 5) .
  • Buffer control group and enzyme replacement therapy (Cerezyme) group were also included in this study.
  • constructs of PG011, PG103, PG104, PG107 and PG105 described above all carried the Ampicillin resistance, which were all replaced by Kanamycin and renamed as PG117, PG118, PG119, PG120, PG121 and PG122.
  • constructs with CHSRE-driven mGBAi1-C110 of PG119, PG120, PG121 and PG122 constructs with CHSRE-driven GBAi1-C110 of PG118, and also the CRE001-driven mGBA-C110 of PG117, were packaged as AAV8.
  • AAV8 candidates along with wild type GBA1 control of PG001 and PG127 as the reference product were administered into Gaucher mice at dose of 2E12 vg/kg by tail vein injection and underwent a 12-weeks therapeutic study.
  • Some control groups including wild type control (Naive) , buffer control and enzyme replacement therapy (Cerezyme) group were also included in this study. Serum enzyme activity and glucosylsphingosine accumulation were monitored at interval time point (week 1, 2, 4, 6, 8, and 12) post injection. According to the results, all AAV8 injected group showed high and stable GCase activity in serum.
  • the AAV8 candidates of PG117, PG118, PG119, PG120, PG121 and PG122 showed higher GCase activity than the buffer control group, Cerezyme group and reference product of PG127 (Fig. 6A) .
  • the glucosylsphingosine level in serum decreased quickly after the injection of AAV8 products.
  • AAV8 candidates of PG117, PG118, PG119, PG120, PG121 and PG122 decreased the glucosylsphingosine close to the level of naive wild type mice after 8 weeks post injection (Fig. 6B) .
  • tissue samples were collected at the end of the study for GCase enzyme activity and glucosylsphingosine analysis.
  • the GCase enzyme activity were increased in liver, lung, and spleen for all AAV injected groups. Consistent with the results in serum described above, groups of AAV8 products of PG117, PG118, PG119, PG120, PG121 and PG122 showed higher GCase activity and lower glucosylsphingosine accumulation in all tested tissues than groups of Cerezyme and reference product of PG127 (Fig. 7 and Fig. 8) .
  • AAV8 products of PG117, PG118, PG119, PG120, PG121 and PG122 were proven to be promising therapeutic candidates against Gaucher disease, and performed superior than the existing therapies of Cerezyme.
  • endogenous GBA1 introns of Int001, Int002, Int005 or a combination of Int001 and Int005 were inserted into the expression cassette of mGBA-C110 under the universal promoter CRE001 as mGBAi1-C110 (PG123) , mGBAi2-C110 (PG124) , mGBAi5-C110 (PG125) and mGBAi1i5-C110 (PG126) .
  • Constructs of PG001, PG123, PG124, PG125 and PG126 were packaged as AAV9 and proceeded for in vivo evaluation in wild type mice.
  • AAV9 products along with reference product of PG128, which was constructed with the sequence of SEQ ID NO: 1 (149bp-3806bp) as described in patent US10837028B2, were injected into wild type mice at the dosage of 2E12 vg/kg by tail vein injection. Buffer control group and enzyme replacement therapy (Cerezyme) group were also included in this study.
  • AAV9 products of PG123, PG124, PG125 and PG126 showed higher GCase activity in both serum and tissue lysates compared with buffer control, PG001 and reference product group of PG128. GCase activity in all groups from serum and different tissue lysates were highly consistent (Fig. 9) .
  • novel designed candidates for AAV9 product could increase GCase activity in both serum and tissues, which suggest their superior therapeutic potential for Gaucher disease, andalso the Parkinson’s disease and Alzheimer's disease.
  • AAV9 could cross the blood-brain-barrier and deliver GBA1 gene to the CNS efficiently to enable the expression of GCase, which have the potential to alleviate the neurological symptoms and benefit type II and III Gaucher disease, as well as Parkinson’s disease and Alzheimer's disease.

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Abstract

L'invention concerne une construction d'expression, des vecteurs, des particules virales ou des compositions. La construction d'expression comprend un élément régulateur de transcription fonctionnellement lié à une séquence polynucléotidique d'intérêt, la construction d'expression comprenant un promoteur, un activateur et un intron facultatif, l'activateur étant en amont du promoteur. L'invention concerne également des utilisations de la construction d'expression, des vecteurs, des particules virales ou des compositions.
PCT/CN2023/123282 2022-10-08 2023-10-07 Construction pour améliorer l'expression génique WO2024074143A1 (fr)

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