[go: up one dir, main page]

WO2025026198A1 - Polynucléotides pour le traitement de la maladie de wilson - Google Patents

Polynucléotides pour le traitement de la maladie de wilson Download PDF

Info

Publication number
WO2025026198A1
WO2025026198A1 PCT/CN2024/107705 CN2024107705W WO2025026198A1 WO 2025026198 A1 WO2025026198 A1 WO 2025026198A1 CN 2024107705 W CN2024107705 W CN 2024107705W WO 2025026198 A1 WO2025026198 A1 WO 2025026198A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polynucleotide
variant
nos
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/107705
Other languages
English (en)
Inventor
Qing Lin
Yixiong CHEN
Qiang Wu
Yuzhe Peng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lingyi Biotech Co Ltd
Original Assignee
Lingyi Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lingyi Biotech Co Ltd filed Critical Lingyi Biotech Co Ltd
Priority to CN202480018081.XA priority Critical patent/CN120882865A/zh
Publication of WO2025026198A1 publication Critical patent/WO2025026198A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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
    • 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
    • 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
    • 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
    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/007—Vectors comprising a special origin of replication system tissue or cell-specific

Definitions

  • the present disclosure relates to polynucleotides that comprise a nucleotide sequence encoding ATP7B, and also includes the design of expression constructs that contain these polynucleotides, as well as recombinant AAV vectors and viral particles that carry the polynucleotides.
  • the use of these polynucleotides and constructs has potential applications in gene therapy of diseases associated with ATP7B dysfunction.
  • the invention provides a method for enhancing the expression of ATP7B in various host cells and tissues, which may lead to the treatment of genetic disorders such as Wilson's disease, and the development of new therapeutic strategies for other diseases.
  • Wilson disease is a rare autosomal recessive disorder of copper metabolism that primarily affects the liver and subsequent neurological system and other tissues, caused by mutations of the ATP7B gene located on chromosome 13.
  • ATP7B encodes a P-type copper-transporting ATPase, which is expressed mainly in hepatocytes and functions in the transmembrane transport of copper.
  • Dysfunction of ATP7B protein leads to decreased hepatocellular excretion of copper into bile, and causes copper accumulation in the liver and subsequent tissues. Without normal copper metabolism, ceruloplasmin lacks incorporation of copper and results in additional hemolytic anemia, etc.
  • ATP7B has a basic P-type ATPase architecture that includes a large, cytosolic N-terminal tail with six 70-aa long independently folded Cu (II) responsive elements (CREs) and a transmembrane part with eight transmembrane domains that form an intramembranous Cu channel.
  • II copper
  • CREs Cu responsive elements
  • transmembrane part with eight transmembrane domains that form an intramembranous Cu channel.
  • the full-length ATP7B gene is about 4.4 kb in size, which is oversized for packaging into adeno-associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • a truncated form of ATP7B with comparable copper transportation capacity to wild-type ATP7B was developed for use in gene therapy vectors to treat Wilson disease.
  • This invention provides a potential solution to the challenge of delivering large genes to target tissues, which has the potential to revolutionize the treatment of genetic disorders.
  • a polynucleotide encoding an ATP7B that has been codon-optimized for expression comprising a coding region of the ATP7B, wherein the coding region of the ATP7B comprises a sequence selected from the group consisting of (a) a sequence of SEQ ID NO: 28-35, (b) a sequence that is 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 NO: 28-35, and (c) a functional fragment of (a) or (b) that retains the functionality of ATP7B.
  • the ATP7B comprises an amino acid sequence of SEQ ID NO: 36.
  • the polynucleotide further comprises an untranslated intron region, preferably, the untranslated intron region comprises all or a portion of a sequence selected from the sequence consisting of SEQ ID NOs: 37-56, and sequences that are 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: 37-56.
  • the untranslated intron region is located between the 51-52bp, 1285-1286bp, 1543-1544bp, 1707-1708bp, 1869-1870bp, 1946-1947bp, 2121-2122bp, 2355-2356bp, 2447-2448bp, 2575-2576bp, 2730-2731bp, 2865-2866bp, 3060-3061bp, 3243-3244bp, 3412-3413bp, 3556-3557bp, 3699-3700bp, 3903-3904bp, 4021-4022bp, 4124-4125bp of SEQ ID NOs: 28-35.
  • polynucleotide comprising:
  • the disclosure provides a polynucleotide encoding a truncated ATP7B that has been codon-optimized for expression, comprising a coding region of the truncated ATP7B, the truncated ATP7B comprises one or more sequences selected from CRE5, CRE6, and variants thereof, and one or more sequences selected from CRE1, CRE2, and variants thereof.
  • the coding region of CRE1 comprises a sequence selected from the group consisting of SEQ ID NO: 1, and sequences that are 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 NO: 1;
  • the coding region of CRE2 comprises a sequence selected from the group consisting of SEQ ID NO: 2, and sequences that are 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 NO: 2;
  • the coding region of CRE5 comprises a sequence selected from the group consisting of SEQ ID NO: 5, and sequences that are 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 NO: 5;
  • the coding region of CRE6 comprises a sequence selected from the group consisting of SEQ ID NO: 6, and sequences that are 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 NO: 6.
  • truncated ATP7B comprises a flexible linker connecting CREs.
  • the truncated ATP7B comprises a structure as shown in Formula Ia from N-terminus to C-terminus:
  • A, B, C, or D is selected from CRE1, CRE2, CRE5, CRE6 and its variants thereof; A, B, C, and D are different, respectively; A and B are present, C and/or D are optionally present; and
  • L is none or a flexible linker.
  • the truncated ATP7B further comprises LP and/or CTR, where LP is a leader peptide, and CTR is a C-terminal region.
  • the truncated ATP7B has a structure as shown in Formula Ib from N-terminus to C-terminus:
  • A, B, C, or D is selected from CRE1, CRE2, CRE5, CRE6 and its variants thereof; A, B, C, and D are different, respectively; A and B are present, C and/or D are optionally present; and L is none or a flexible linker, LP is a leader peptide, and CTR is a C-terminal region.
  • truncated ATP7B comprises a flexible linker connecting CREs.
  • the flexible linker comprises a nucleotide sequence encoding any amino acid sequences selected from SEQ ID NOs: 22-26, and sequences 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: 22-26; preferably, the flexible linker comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 17-21, and sequences that are 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: 17-21.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which 1) CRE5 or variant thereof and/or 2) CRE6 or variant thereof is present; and 3) CRE1 or variant thereof and/or 4) CRE2 or variant thereof is present;
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof and CRE5 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof and CRE6 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE5 or variant thereof, and CRE6 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE2 or variant thereof and CRE5 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE2 or variant thereof and CRE6 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE2 or variant thereof, CRE5 or variant thereof, and CRE6 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE2 or variant thereof, and CRE5 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE2 or variant thereof, and CRE6 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE2 or variant thereof, CRE5 or variant thereof, and CRE6 or variant thereof are present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE4 or variant thereof is present.
  • the present disclosure provides a polynucleotide encoding a functional ATP7B protein comprises one or more sequences selected from SEQ ID NO: 145-158, and sequences 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 NO: 145-158, preferably the truncated ATP7B comprises one or more nucleotide sequences selected from SEQ ID NOs: 107-120, and sequences that are 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: 107-120.
  • the truncated ATP7B comprises a CREs region selected from SEQ ID NOs: 1-6 and sequences 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: 1-6.
  • the CREs region has one or more sequences selected from SEQ ID NOs: 1, 2, 5, and 6, and sequences 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: 1, 2, 5, and 6.
  • the polynucleotide 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: 37-56, and sequences that are 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: 37-56.
  • the truncated ATP7B comprises one or more polynucleotide encodes a functional ATP7B protein comprises one or more sequences selected from SEQ ID NOs: 141-144, and sequences that are 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: 141-144;
  • the polynucleotide comprises one or more sequences selected from SEQ ID NOs: 121-124, and sequences that are 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: 121-124.
  • the present disclosure provides a polynucleotide encodes a functional ATP7B protein comprises one or more sequences selected from SEQ ID NOs: 141-158, and sequences that are 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: 141-158.
  • this disclosure provides an expression construct comprising a transcription regulatory element operably linked to the said polynucleotide sequence of the present disclosure, wherein the transcription regulatory element comprises a promoter, and/or an enhancer, and/or a metal responsive element (MRE) , preferably the enhancer is upstream of the promoter, more preferably, the MRE is upstream of the enhancer.
  • the transcription regulatory element comprises a promoter, and/or an enhancer, and/or a metal responsive element (MRE) , preferably the enhancer is upstream of the promoter, more preferably, the MRE is upstream of the enhancer.
  • MRE metal responsive element
  • the promoter comprises all or a portion of a sequence selected from the sequence consisting of SEQ ID NOs: 80-83, and sequences that are 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: 80-83, the all or the portion of the sequence retains the functionality of promoter.
  • the enhancer comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 77-79, and sequences that are 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: 77-79, the all or the portion of the sequence retains the functionality of enhancer.
  • the metal responsive element comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 96-103, and 105, and sequences that are 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: 96-103, and 105, the all or the portion of the sequence retains the functionality of metal responsive element.
  • the promoter, enhancer or metal responsive element is single-copy or multi-copy sequence.
  • the expression construct comprises one or more sequences selected from SEQ ID NOs: 125-140, and sequences 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: 125-140.
  • the expression construct further comprises a 5’ -inverted terminal repeat (ITR) sequence, a polyA sequence; and a 3’ -ITR sequence.
  • ITR inverted terminal repeat
  • this disclosure provides a vector comprising the said polynucleotide of the present disclosure, or the said expression construct of the present disclosure.
  • the vector is a virus vector.
  • virus vector is AAV vector.
  • this disclosure provides a recombinant adeno-associated virus (rAAV) comprising the vector of the present disclosure and capsid protein.
  • rAAV adeno-associated virus
  • the AAV is selected from the group consisting of: serotype 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10, or hu37 as well as any one of the AAV serotypes isolated from human and nonhuman mammalians or variant thereof.
  • this disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the said polynucleotide, the said expression construct, the said vector, or the said rAAV, and a pharmaceutically acceptable carrier.
  • this disclosure provides a method for treating a disease in the subjects, comprising administrating the effective amount of the said polynucleotide, the said expression construct, the said vector, the said rAAV, or the said pharmaceutical composition.
  • the disease is an ATP7B related disease.
  • the disease is Wilson disease.
  • the subject is mammalian, preferably human.
  • Figure 1 showed codon-optimized versions of ATP7B led to a significant increase in ATP7B protein expression.
  • the LYM3P086 version exhibited the highest level of protein expression among all the tested versions.
  • Figure 2 showed the introduction of certain designed endogenous introns of ATP7B resulted in a significant increase in ATP7B protein expression. Particularly, designed endogenous intron-1, intron-5 and intron-14 of ATP7B were found to achieve higher protein expression levels.
  • Figure 3 showed the evaluation of the copper-transporting capacity of codon-optimized ATP7B expressed by various hepatic-specific chimeric elements (CHSRE) with Dual Luciferase Assay. A higher relative Fluci/Rluci indicated a lower copper-transporting capacity.
  • Figure 4 showed the copper-dependent transcriptional activity of metal responsive elements (MREs) from ATP7B endogenous promoter.
  • Figure 5 showed the copper-dependent transcriptional activity of the hepatic-specific regulatory element (CHSRE) with MREs.
  • Figure 6 showed the copper-transporting capacity of codon-optimized ATP7B with specific combinations of CREs.
  • Figure 7 showed the copper-transporting capacity of different AAV candidates, which carried codon-optimized truncated ATP7B with/without designed endogenous intron-1 of ATP7B driven by CHSRE.
  • Figure 8 showed the liver copper level 4-week after a single dose (2.0 ⁇ 10 12 vg/kg) of AAV candidates, which carried codon-optimized truncated ATP7B with/without designed endogenous intron-1 of ATP7B driven by CHSRE.
  • Figure 9 showed schematic representation of the nucleic acid constructs of pCMV vector carrying ATP7B transgenes (a) ; AAV-ITR vector carrying ATP7B transgenes (b) ; vector carrying MREs-E1b TATA box-Luciferase (c) and vector carrying MREs-CHSREs-Luciferase (d) .
  • the present disclosure relates to a polynucleotide that encodes an optimized version of ATP7B for efficient expression, comprising a coding region of the ATP7B, wherein the coding region of the ATP7B comprises a sequence selected from the group consisting of (a) a sequence of SEQ ID NOs: 28-35, (b) a sequence that is 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: 28-35, and (c) a functional fragment of (a) or (b) that retains the functionality of ATP7B.
  • the ATP7B comprises an amino acid sequence comprising SEQ ID NO: 36.
  • the polynucleotide further comprises an untranslated intron region, preferably, the untranslated intron region comprises all or a portion of a sequence selected from the sequence consisting of SEQ ID NOs: 37-56, and sequences that are 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: 37-56.
  • the untranslated intron region is located between the 51-52bp, 1285-1286bp, 1543-1544bp, 1707-1708bp, 1869-1870bp, 1946-1947bp, 2121-2122bp, 2355-2356bp, 2447-2448bp, 2575-2576bp, 2730-2731bp, 2865-2866bp, 3060-3061bp, 3243-3244bp, 3412-3413bp, 3556-3557bp, 3699-3700bp, 3903-3904bp, 4021-4022bp, 4124-4125bp of SEQ ID NOs: 28-35.
  • a polynucleotide comprising: (a) a sequence 100%identical to a nucleotide sequence of any one of SEQ ID NOs: 57-76; (b) a sequence that is 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 a nucleotide sequence of any one of SEQ ID NOs: 57-76, or (c) a functional fragment of (a) or (b) that retains the functionality of human ATP7B.
  • the ATP7B comprises an amino acid sequence comprising SEQ ID NO: 36.
  • the disclosure provides a polynucleotide encoding a truncated ATP7B that has been codon-optimized for expression, comprising a coding region of the truncated ATP7B, the truncated ATP7B comprises one or more sequences selected from CRE5, CRE6, and variants thereof, and one or more sequences selected from CRE1, CRE2, and variants thereof., wherein
  • the coding region of CRE1 comprises a sequence selected from the group consisting of SEQ ID NO: 1, and sequences that are 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 NO: 1;
  • the coding region of CRE2 comprises a sequence selected from the group consisting of SEQ ID NO: 2, and sequences that are 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 NO: 2;
  • the coding region of CRE5 comprises a sequence selected from the group consisting of SEQ ID NO: 5, and sequences that are 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 NO: 5;
  • the coding region of CRE6 comprises a sequence selected from the group consisting of SEQ ID NO: 6, and sequences that are 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 NO: 6;
  • the truncated ATP7B comprises a structure as shown in Formula Ia from N-terminus to C-terminus:
  • A, B, C, or D is selected from CRE1, CRE2, CRE5, CRE6 and its variants thereof; A, B, C, and D are different, respectively; A and B are present, C and/or D are optionally present; and L is none or a flexible linker.
  • the truncated ATP7B further comprises LP and/or CTR.
  • the truncated ATP7B has a structure as shown in Formula Ib from N-terminus to C-terminus:
  • A, B, C, or D is selected from CRE1, CRE2, CRE5, CRE6 and its variants thereof; A, B, C, and D are different, respectively; A and B are present, C and/or D are optionally present; and L is none or a flexible linker, LP is a leader peptide, and CTR is a C-terminal region.
  • truncated ATP7B comprises a flexible linker connecting CREs.
  • the flexible linker comprises a nucleotide sequence encoding any amino acid sequences selected from SEQ ID NOs: 22-26, and sequences 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: 22-26; preferably, the flexible linker comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 17-21, and sequences that are 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: 17-21.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which 1) CRE5 or variant thereof and/or 2) CRE6 or variant thereof is present; and 3) CRE1 or variant thereof and/or 4) CRE2 or variant thereof is present.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof and CRE5 or variant thereof are present; preferably, wherein the CRE1 comprises an amino acid sequence of SEQ ID NO: 9, the CRE5 comprises an amino acid sequence of SEQ ID NO: 13; more preferably, wherein the CRE1 comprises a nucleotide sequence of SEQ ID NO: 1, the CRE5 comprises a nucleotide sequence of SEQ ID NO: 5.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof and CRE6 or variant thereof are present; preferably, wherein the CRE1 comprises an amino acid sequence of SEQ ID NO: 9, the CRE6 comprises an amino acid sequence of SEQ ID NO: 14; more preferably, wherein the CRE1 comprises a nucleotide sequence of SEQ ID NO: 1, the CRE6 comprises a nucleotide sequence of SEQ ID NO: 6.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE5 or variant thereof, and CRE6 or variant thereof are present; preferably, wherein the CRE1 comprises an amino acid sequence of SEQ ID NO: 9, the CRE5 comprises an amino acid sequence of SEQ ID NO: 13, the CRE6 comprises an amino acid sequence of SEQ ID NO: 14; preferably, wherein the CRE1 comprises a nucleotide sequence of SEQ ID NO: 1, the CRE5 comprises a nucleotide sequence of SEQ ID NO: 5, the CRE6 comprises a nucleotide sequence of SEQ ID NO: 6.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE2 or variant thereof and CRE5 or variant thereof are present; preferably, wherein the CRE2 comprises an amino acid sequence of SEQ ID NO: 10, the CRE5 comprises an amino acid sequence of SEQ ID NO: 13; preferably, wherein the CRE2 comprises a nucleotide sequence of SEQ ID NO: 2, the CRE5 comprises a nucleotide sequence of SEQ ID NO: 5.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE2 or variant thereof and CRE6 or variant thereof are present; preferably, wherein the CRE2 comprises an amino acid sequence of SEQ ID NO: 10, the CRE6 comprises an amino acid sequence of SEQ ID NO: 14; preferably, wherein the CRE2 comprises a nucleotide sequence of SEQ ID NO: 2, the CRE6 comprises a nucleotide sequence of SEQ ID NO: 6.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE2 or variant thereof, CRE5 or variant thereof, and CRE6 or variant thereof are present; preferably, wherein the CRE2 comprises an amino acid sequence of SEQ ID NO: 10, the CRE5 comprises an amino acid sequence of SEQ ID NO: 13, the CRE6 comprises an amino acid sequence of SEQ ID NO: 14; preferably, wherein the CRE2 comprises a nucleotide sequence of SEQ ID NO: 2, the CRE5 comprises a nucleotide sequence of SEQ ID NO: 5, the CRE6 comprises a nucleotide sequence of SEQ ID NO: 6.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE2 or variant thereof, and CRE5 or variant thereof are present; preferably, wherein the CRE1 comprises an amino acid sequence of SEQ ID NO: 9, the CRE2 comprises an amino acid sequence of SEQ ID NO: 10, the CRE5 comprises an amino acid sequence of SEQ ID NO: 13; preferably, wherein the CRE1 comprises a nucleotide sequence of SEQ ID NO: 1, the CRE2 comprises a nucleotide sequence of SEQ ID NO: 2, the CRE5 comprises a nucleotide sequence of SEQ ID NO: 5.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE2 or variant thereof, and CRE6 or variant thereof are present; preferably, wherein the CRE1 comprises an amino acid sequence of SEQ ID NO: 9, the CRE2 comprises an amino acid sequence of SEQ ID NO: 10, the CRE6 comprises an amino acid sequence of SEQ ID NO: 14; preferably, wherein the CRE1 comprises a nucleotide sequence of SEQ ID NO: 1, the CRE2 comprises a nucleotide sequence of SEQ ID NO: 2, the CRE6 comprises a nucleotide sequence of SEQ ID NO: 6.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE1 or variant thereof, CRE2 or variant thereof, CRE5 or variant thereof, and CRE6 or variant thereof are present; preferably, the CRE1 comprises an amino acid sequence of SEQ ID NO: 9, the CRE2 comprises an amino acid sequence of SEQ ID NO: 10, the CRE5 comprises an amino acid sequence of SEQ ID NO: 13, the CRE6 comprises an amino acid sequence of SEQ ID NO: 14; preferably, the CRE1 comprises a nucleotide sequence of SEQ ID NO: 1, the CRE2 comprises a nucleotide sequence of SEQ ID NO: 2, the CRE5 comprises a nucleotide sequence of SEQ ID NO: 5, the CRE6 comprises a nucleotide sequence of SEQ ID NO: 6.
  • this disclosure provides a polynucleotide encoding a truncated ATP7B in which CRE4 or variant thereof is present.
  • the truncated ATP7B comprises a flexible linker connecting CREs.
  • the flexible linker comprises a nucleotide sequence encoding any amino acid sequences selected from SEQ ID NOs: 22-26, and sequences 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: 22-26; preferably, the flexible linker comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 17-21, and sequences that are 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: 17-21.
  • the truncated ATP7B comprises one or more nucleotide sequences selected from SEQ ID NO: 107-120, and sequences 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 NO: 107-120.
  • the truncated ATP7B comprises one or more sequences selected from SEQ ID NOs: 107, 108, 110, 111, 114, 116, 117, and 120, and sequences 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: 107, 108, 110, 111, 114, 116, 117, and 120.
  • the present disclosure provides a polynucleotide encoding a functional ATP7B protein comprises one or more sequences selected from SEQ ID NO: 145-158, and sequences 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 NO: 145-158.
  • the truncated ATP7B comprises a CREs region selected from SEQ ID NOs: 1-6 and sequences 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: 1-6.
  • the CREs region has one or more sequences selected from SEQ ID NOs: 1, 2, 5, and 6, and sequences 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: 1, 2, 5, and 6.
  • the polynucleotide 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: 37-56, and sequences that are 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: 37-56.
  • the truncated ATP7B comprises one or more polynucleotide encodes a functional ATP7B protein comprises one or more sequences selected from SEQ ID NOs: 141-144, and sequences that are 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: 141-144;
  • the polynucleotide comprises one or more sequences selected from SEQ ID NOs: 121-124, and sequences that are 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: 121-124.
  • the present disclosure provides a polynucleotide encodes a functional ATP7B protein comprises one or more sequences selected from SEQ ID NOs: 141-158, and sequences that are 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: 141-158.
  • this disclosure provides an expression construct comprising a transcription regulatory element operably linked to the said polynucleotide sequence of the present disclosure, wherein the transcription regulatory element comprises a promoter, and/or an enhancer, and/or a metal responsive element (MRE) , preferably the enhancer is upstream of the promoter, more preferably, the MRE is upstream of the enhancer.
  • the transcription regulatory element comprises a promoter, and/or an enhancer, and/or a metal responsive element (MRE) , preferably the enhancer is upstream of the promoter, more preferably, the MRE is upstream of the enhancer.
  • MRE metal responsive element
  • the promoter comprises all or a portion of a sequence selected from the sequence consisting of SEQ ID NOs: 80-83, and sequences that are 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: 80-83, the all or the portion of the sequence retains the functionality of promoter.
  • the enhancer comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 77-79, and sequences that are 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: 77-79, the all or the portion of the sequence retains the functionality of enhancer.
  • the metal responsive element comprises all or a portion of a sequence comprising at least one selected from the sequence consisting of SEQ ID NOs: 96-103, and 105, and sequences that are 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: 96-103, and 105, the all or the portion of the sequence retains the functionality of metal responsive element.
  • the promoter, enhancer or metal responsive element is a single-copy or multi-copy sequence.
  • the expression construct comprises one or more sequences selected from SEQ ID NOs: 125-140, and sequences 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: 125-140.
  • the expression construct further comprises a 5’ -inverted terminal repeat (ITR) sequence, a polyA sequence; and a 3’ -ITR sequence.
  • ITR inverted terminal repeat
  • this disclosure provides a vector comprising the said polynucleotide of the present disclosure, or the said expression construct of the present disclosure.
  • the vector is a virus vector.
  • virus vector is AAV vector.
  • this disclosure provides a recombinant adeno-associated virus (rAAV) comprising the vector of the present disclosure and capsid protein.
  • rAAV adeno-associated virus
  • the AAV is selected from the group consisting of: serotype 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10, or hu37 as well as any one of the AAV serotypes isolated from human and nonhuman mammalians or variant thereof.
  • this disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the said polynucleotide, the said expression construct, the said vector, or the said rAAV, and a pharmaceutically acceptable carrier.
  • this disclosure provides a method for treating a disease in the subjects, comprising administrating the effective amount of the said polynucleotide, the said expression construct, the said vector, the said rAAV, or the said pharmaceutical composition.
  • the disease is an ATP7B related disease.
  • the disease is Wilson disease.
  • the subject is mammalian, preferably human.
  • 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.
  • a polynucleotide may be DNA or RNA.
  • the disclosure provides, in some aspects, an isolated polynucleotide comprising an expression construct encoding ATP7B or a portion thereof.
  • the isolated polynucleotide comprises an ATP7B-encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, for example human cells) , such as the sequence set forth in SEQ ID NOs: 28-35 or a sequence 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 a nucleotide sequence of any one of SEQ ID NOs: 28-35.
  • the polynucleotide further comprises an untranslated intron region.
  • 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.
  • 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, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, AAVrh10, or AAVhu37 as well as 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 the infectious viral particles.
  • AAV vectors comprise a promoter, at least one nucleic acid sequence 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.
  • AAV5, 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, adenoviral helper genes, and the recombinant genome containing the ATP7B construct.
  • the resulting rAAV particles were purified using an iodixanol-based density gradient ultracentrifugation method.
  • the rAAV particles were then quantified using a probe-based ddPCR assay (Biorad) and characterized by silver staining.
  • rAAV biopotency assay involved transduction by Huh-7 cell lines, and was assessed using a reporter plasmid (pGL4 ⁇ MRE-LUC) with an internal control plasmid (pCMV-RL) .
  • pGL4 ⁇ MRE-LUC reporter plasmid
  • pCMV-RL internal control plasmid
  • MREs metal response elements
  • Cells were first plated at a density of 8.5 ⁇ 10 6 cells/plate in a 15 cm 2 plate. After 24 hours, the cells were transfected with pGL4 ⁇ MRE-LUC and the pCMV-RL plasmid. Following another 48 hours, the cells were trypsinized and seeded in 96-well plates at a cell density of 1.5 ⁇ 10 4 cells/well 24 hours before transduction. The rAAV particles were transduced at a defined multiplicity of infection (MOI) of 3 ⁇ 10 6 . After 48 hours of transduction, 150 ⁇ M CuSO 4 was added to the cells and incubated for 24 hours before collection.
  • MOI multiplicity of infection
  • the firefly and Renilla luciferase activity were measured with the Double-Luciferase Reporter Assay Kit from Transgen Biotech.
  • the baseline control has no copper overload, and the relative luminescence intensity units were calculated by normalizing firefly luciferase activity with Renilla luciferase activity.
  • MREs metal response elements
  • the reporter plasmid is responsive to bioavailable cytosolic copper by activating luciferase expression.
  • HEK293T cells were transfected with pGLN ⁇ MRE-LUC or the empty pcDNA 3.1 plasmid as a control. The cells were then incubated with CuSO 4 for 24 hours, while baseline control have no copper overload. The firefly luciferase activity was measured with the Single-Luciferase (Firefly) Reporter Assay Kit from Transgen Biotech.
  • the copper-transporting capacity of truncated ATP7B in pCMV vectors were assessed using the reporter plasmid (pGL4 ⁇ MRE-LUC) .
  • HEK293T cells were co-transfected with pGL4 ⁇ MRE-LUC and a plasmid expressing truncated ATP7B under the control of the CMV promoter, the empty pcDNA 3.1 plasmid was used as a control. All cells were co-transfected with the pCMV-RL plasmid for internal control. After transfection, cells were incubated with 75 ⁇ M CuSO 4 for 24 hours, while the absence of copper overload was considered as the basal state.
  • the firefly and Renilla luciferase activity were measured with the Double-Luciferase Reporter Assay Kit from Transgen Biotech.
  • the relative luminescence intensity units were calculated by normalizing firefly luciferase activity with Renilla Luciferase activity.
  • AAV vector containing the truncated ATP7B transgene were administered via tail vein injection of Atp7b -/- mice at age of 8-20 weeks. All mice were maintained in a special pathogen-free environment and in individually ventilated cages, with all cages, cob bedding, and water were sterilized before use. The cages, cob bedding, food and water were changed twice a week.
  • AAV dose was optimized to be 2.0 ⁇ 10 12 vg/kg.
  • serum ceruloplasmin activity, alanine aminotransferase level and the metabolic copper contents were measured at various time intervals post injection. The mice were followed up to the end point of study and then sacrificed for tissue copper accumulation, biochemical, and pathological analysis.
  • Serum was obtained from fresh blood collected within 30 minutes of collection and stored at ⁇ -60 °Cafter centrifugation at 12,000 rpm for 15 minutes at 4°C. Metabolic cages were used to collect 24-hours urine and feces for metabolic copper content analysis.
  • the ceruloplasmin activity assay kit (Nanjing Jiancheng Bioengineering Institute, China) was utilized to determine ceruloplasmin activity in serum using o-dianisidine dihydrochloride as a substrate (Stepien and Guy 2018) , some modifications were made according to the manufacturer's instructions. In brief, 5 ⁇ L of diluted serum was added to 96-well plates, followed by addition of 80 ⁇ L reagent I and 20 ⁇ L reagent II. The samples were mixed thoroughly and incubated at 37 °C for 140 minutes.
  • reagent III was added after incubation for termination, and the absorbance at 540 nm was measured spectrophotometrically (Varioskan TM LUX, ThermoFisher) .
  • the serum ALT activity was determined using the IFCC alanine aminotransferase assay kit (Nanjing Jiancheng Bioengineering Institute, China) with modifications. Firstly, 10 ⁇ L diluted serum was pipetted into each well of a 96-well plate, followed by the addition of 200 ⁇ L of reagent R1.The plate was then incubated at 37 °C for 10 minutes. Subsequently, 50 ⁇ L reagent R2 was added to each well and mixed well.
  • the plate was then read spectrophotometrically (Varioskan TM LUX, ThermoFisher) at 340 nm every 2 minutes.
  • Solid samples (feces or tissues) were weighed, homogenized and dried to constant weight. The dried samples were then digested in a nitric acid solution overnight. For urine samples, centrifugation was performed to remove insoluble or suspended particles before 100 times dilution with 1%nitric acid and 0.5%hydrochloric acid. Similarly, solid samples were diluted with 10%nitric acid and 1%hydrogen peroxide before analysis. Copper content was determined using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) , which was calibrated using working aqueous standards.
  • ICP-MS Inductively Coupled Plasma Mass Spectrometry
  • TAKARA Primescript RT master mix
  • anti-mouse IgG, HRP-linked antibody (1: 1000, 7076S, CST) or anti-rabbit IgG, HRP-linked antibody (1: 1000, 7074S, CST) was used as the secondary antibody.
  • the signals were visualized using Pierce Western Blot Signal Enhancer (ThermoFisher) according to the manufacturer's instructions.
  • Rabbit anti-human ATP7B antibody (1: 100, ThermoFisher, PA5-102826) was used to visualize hATP7B expression in mice tissue samples.
  • the formalin-fixed mouse tissues were subjected to deparaffinization and followed by antigen retrieval using pepsin according to manufacturer's recommendations.
  • the tissue sections were counterstained with haematoxylin-eosin and a biotin-labeled secondary antibody was used for detection.
  • the signals were visualized using the Streptavidin-HRP and Tyramide signal amplification kit as per the manufacturer's instructions.
  • Timm s sulfide silver staining
  • Timm sulfide silver staining kit (80115.1, Genmed Scientifics Inc. ) was used to visualize copper accumulation in various tissues. Mice were firstly deeply anesthetized and perfused via the ascending aorta with 100 mL 0.9%saline. Tissues were collected and postfixed in Reagent A for 45 minutes, followed by Reagent B for 16-24 hours at room temperature. Fixed tissues were then transferred into Reagent C for another 16-24 hours at room temperature, and processed for paraffin embedding. Sections were cut at a thickness of 4 ⁇ M and stained according to manufacturer's recommendations. Subsequently, signals representing copper accumulation were visible under a microscope.
  • Example 1 Codon optimization of ATP7B significantly increased copper-transporting capacity
  • Codon optimization is widely used to enhance translational efficiency by adapting the codon usage bias of the host organism, thereby increasing gene expression without altering the protein sequence.
  • codon frequency in homo sapiens
  • eight versions of codon-optimized ATP7B transgenes were designed, synthesized and cloned into the pCMV vectors ( Figure 9a) , the ATP7B sequences of which were listed in Table 1.
  • the vectors were then transfected into HEK293T cells, and the resulting cell lysates were analyzed by western blotting to measure the protein expression level of ATP7B.
  • LYM3P082 (SEQ ID NO. 30) , LYM3P085 (SEQ ID NO. 33) , LYM3P086 (SEQ ID NO. 34) and LYM3P087 (SEQ ID NO. 35) significantly increased the ATP7B expression, while the LYM3P085 (SEQ ID NO. 33) and LYM3P086 (SEQ ID NO. 34) were proven to exhibit the highest expression level (Fig. 1) .
  • Example 2 Designed ATP7B endogenous introns increased the ATP7B copper-transporting capacity and protein expression
  • Introns have been known to play crucial roles in regulating alternative splicing, enhancing gene expression, and controlling mRNA transport or chromatin assembly, etc. Some exogenous introns, primarily sourced from virus genomes, have been utilized in gene therapy to increase gene expression. However, considering the safety and the immunogenicity concerns associated with interspecies sequence, endogenous introns of ATP7B were prioritized.
  • the genomic length of ATP7B is approximately 80 kb, which includes 20 introns varying in size from 82 bp to 36 kb.
  • introns that are longer than 300 bp were truncated to a mini-version while preserving the full length of other introns that are less than 300 bp (Table 2) .
  • the codon-optimized ATP7B (SEQ ID NO: 34) was inserted with designed ATP7B endogenous introns at their native location as in the wild type ATP7B of NC_000013.11 (51932669.. 52012132, complement) .
  • the nucleotides encoding the adjacent 3 amino acids of ATP7B that flanking the intron were reverted to their wild-type sequence to ensure compatibility of inserted introns.
  • These ATP7B transgenes with designed endogenous introns (Table 3) , driven by TTRm, were then cloned into AAV-ITR vector ( Figure 9b) .
  • One reference product of LYM3P021 was constructed with a truncated ATP7B driven by a hepatic-specific promoter as described in SEQ ID NO. 8 from WO2016097219A1.
  • the two constructs of LYM3P181 (ATP7B transgene is wild-type ATP7B, SEQ ID NO. 27) and LYM3P283 (ATP7B transgene is ATP7B-C07, SEQ ID NO. 34) were also included as non-intron controls.
  • codon-optimized ATP7B with introns inserted including LYM3P285-ATP7Bi1-C07, LYM3P288-ATP7Bi4-C07, LYM3P289-ATP7Bi5-C07, LYM3P293-ATP7Bi9-C07 and LYM3P298-ATP7Bi14-C07, all exhibited significant increase in protein expression compared to the non-intron controls of LYM3P181-wtATP7B and LYM3P283-ATP7B-C07 (Fig. 2) .
  • Example 3 In vitro screening of chimeric hepatic specific promoters for ATP7B expression
  • CHSREs chimeric hepatic-specific regulatory elements consisting of enhancers (CHSRE01, CHSRE02, CHSRE03) and core promoters (CHSRE04, CHSRE05, CHSRE06, CHSRE07) were combined and permuted as CHSRE08–19 (Table 4) .
  • the CHSREs were fused with codon-optimized ATP7B (SEQ ID NO: 34) and cloned into AAV-ITR vector as listed in Table 4, the constructs of which were then transfected into Huh-7 cells to measure the relative copper-transporting capacity via Dual Luciferase Assay to screen out the vectors with higher copper-transporting potency.
  • Enhancers of CHSRE01, CHSRE02 and CHSRE03 were found to increase transcription efficiency of promotes of CHSRE04, CHSRE06 and CHSRE07, which were evidenced in Fig. 3 that LYM3P341-CHSRE08, LYM3P342-CHSRE09, LYM3P343-CHSRE10, LYM3P347-CHSRE14, LYM3P348-CHSRE15, LYM3P349-CHSRE16, LYM3P350-CHSRE17, LYM3P351-CHSRE18 and LYM3P352-CHSRE19 were found to have higher transcription efficiency compared to corresponding single-promoter control constructs of LYM3P283-CHSRE04, LYM3P339-CHSRE06 and LYM3P340-CHSRE07.
  • LYM3P345-CHSRE12 which had the combination of enhancer CHSRE02 and the promoter CHSRE05, possessed higher copper-transporting capacity than the corresponding single-promoter control of LYM3P338-CHSRE05 (Fig. 3) .
  • Example 4 Further combined with metal responsive elements (MREs) on CHSRE exhibited copper-dependent gene transcription
  • MREs Metal responsive elements
  • TGCRCNC The core consensus sequence TGCRCNC of MREs was analyzed in the human ATP7B endogenous promoter (GRCh38/hg38 chr13: 52011451-52014450) to identify MREs that are suitable for inducing potent copper-dependent ATP7B expression, which are listed in Table 5.
  • MREe4 or MREe7 4 or 7 tandem repeats of MREe (MREe4 or MREe7) were cloned upstream of the hepatic-specific promoter CHSRE05, as listed in Table 6, to drive the expression of luciferase transgene ( Figure 9d) .
  • the constructs were transfected into Huh-7 cells to quantify the copper-dependent transcriptional activity.
  • the results showed that MREe7 significantly increased the transcription efficiency of CHSRE05 promoter in a copper-dependent manner (Fig. 5) .
  • ATP7B is a multi-domain protein comprising several independently folded Cu (II) responsive elements (CREs) situated between the leader peptide (LP) and C-terminal region (CTR) .
  • CREs Cu (II) responsive elements
  • LP leader peptide
  • CTR C-terminal region
  • Table 7 lists the codon-optimized sequence of leader peptide (LP) , CRE1 -6, C-terminal region (CTR) and linkers that connect two adjacent CREs.
  • the CREs listed herein can be arranged in any order, combined directly without any sequences or with any flexible sequences, including endogenous linkers between CREs from ATP7B, to constitute a functional ATP7B.
  • Full-length ATP7B which is approximately 4.4 kb, can be oversized for packaging into adeno-associated virus (AAV) , making it necessary to obtain a truncated form with comparable copper transportation capacity to the wild type ATP7B for developing treatments with gene therapy vectors against Wilson disease. Due to the limitations in AAV packaging, three or fewer combinations of CREs were preferable. As illustrated in Table 8, truncated codon-optimized ATP7B with specific combinations of CREs, driven by TTRm promoter, were cloned into AAV-ITR vectors (Fig. 9b) together with the constant LP and CTR domains to express functional ATP7B proteins.
  • Example 6 In vivo study of therapeutic potential of AAV candidates against Wilson disease
  • MREe7 SEQ ID NO: 105 was cloned upstream of the chimeric hepatic-specific elements of CHSRE08 or CHSRE18 in constructs of LYM3P465, LYM3P466, LYM3P467, LYM3P468, LYM3P469, LYM3P470, LYM3P471 and LYM3P472 as listed in Table 10.
  • the therapeutic AAV constructs with CHSRE-driven truncated codon-optimized ATP7B with or without insertion of intron were described in Table 10.
  • AAV candidates 2.0 ⁇ 10 12 vg/kg
  • LYM3P452, LYM3P453, LYM3P455, LYM3P456, LYM3P469, LYM3P470 and LYM3P471, formulation buffer control, or the reference product of LYM3P021 were administrated through a tail vein injection to 12-week-age Atp7b -/- mice, while C57BL/6J mice were used as wild-type control with a formulation buffer administration. After the administration, serum samples were collected at week 1 post-administration to assess the activity of ceruloplasmin (Cp) .
  • Cp ceruloplasmin
  • mice Four weeks later, the mice were sacrificed to determine the liver copper accumulation.
  • the results showed that the selected AAV candidates produced with the constructs from Table 10 more effectively eliminated liver copper accumulation (Fig. 8) than the reference of LYM3P021 in the Atp7b -/- Wilson disease mouse model.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente divulgation concerne des polynucléotides à codons optimisés d'ATP7B et d'ATP7B tronqué. La présente divulgation concerne en outre des constructions d'expression, des vecteurs, des particules virales ou des compositions comprenant le polynucléotide de la divulgation, ainsi que des méthodes et des utilisations des polynucléotides, de la construction d'expression, des vecteurs, des particules virales ou des compositions en thérapie génique pour le traitement de la maladie de Wilson (WD).
PCT/CN2024/107705 2023-07-28 2024-07-26 Polynucléotides pour le traitement de la maladie de wilson Pending WO2025026198A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202480018081.XA CN120882865A (zh) 2023-07-28 2024-07-26 用于治疗威尔逊病的多核苷酸

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2023110008 2023-07-28
CNPCT/CN2023/110008 2023-07-28

Publications (1)

Publication Number Publication Date
WO2025026198A1 true WO2025026198A1 (fr) 2025-02-06

Family

ID=94394228

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/107705 Pending WO2025026198A1 (fr) 2023-07-28 2024-07-26 Polynucléotides pour le traitement de la maladie de wilson

Country Status (2)

Country Link
CN (1) CN120882865A (fr)
WO (1) WO2025026198A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (fr) * 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de produits biologiques et de protéines associées à une maladie humaine
WO2017103624A1 (fr) * 2015-12-18 2017-06-22 Ucl Business Plc Thérapie génique de la maladie de wilson
WO2018126116A1 (fr) * 2016-12-30 2018-07-05 The Trustees Of The University Of Pennsylvania Thérapie génique pour le traitement de la maladie de wilson
CN111088285A (zh) * 2019-08-15 2020-05-01 北京锦篮基因科技有限公司 携带atp7b基因表达框及变异体的aav载体及应用
WO2020102723A1 (fr) * 2018-11-16 2020-05-22 Encoded Therapeutics, Inc. Compositions et méthodes pour le traitement de la maladie de wilson
WO2020142653A1 (fr) * 2019-01-04 2020-07-09 Ultragenyx Pharmaceutical Inc. Constructions de thérapie génique pour le traitement de la maladie de wilson
WO2021209574A1 (fr) * 2020-04-15 2021-10-21 Fondazione Telethon Constructions comprenant des intéines

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (fr) * 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de produits biologiques et de protéines associées à une maladie humaine
WO2017103624A1 (fr) * 2015-12-18 2017-06-22 Ucl Business Plc Thérapie génique de la maladie de wilson
WO2018126116A1 (fr) * 2016-12-30 2018-07-05 The Trustees Of The University Of Pennsylvania Thérapie génique pour le traitement de la maladie de wilson
WO2020102723A1 (fr) * 2018-11-16 2020-05-22 Encoded Therapeutics, Inc. Compositions et méthodes pour le traitement de la maladie de wilson
WO2020142653A1 (fr) * 2019-01-04 2020-07-09 Ultragenyx Pharmaceutical Inc. Constructions de thérapie génique pour le traitement de la maladie de wilson
CN111088285A (zh) * 2019-08-15 2020-05-01 北京锦篮基因科技有限公司 携带atp7b基因表达框及变异体的aav载体及应用
WO2021209574A1 (fr) * 2020-04-15 2021-10-21 Fondazione Telethon Constructions comprenant des intéines

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PADULA, A. ET AL.: "Full-length ATP7B reconstituted through protein trans-splicing corrects Wilson disease in mice", MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT, vol. 36, 30 September 2022 (2022-09-30), pages 495 - 504, XP093078404, DOI: 10.1016/j.omtm.2022.08.004 *

Also Published As

Publication number Publication date
CN120882865A (zh) 2025-10-31

Similar Documents

Publication Publication Date Title
KR102743219B1 (ko) 아데노-관련 바이러스 변이체 캡시드 및 그 용도
RU2705249C2 (ru) Вектор aav и анализ на нейтрализующие антитела против aav (аденоассоциированного вируса)
JP2022071049A (ja) 臨床使用に適した無血清懸濁細胞培養システムにおいて組換えアデノ随伴ウイルス（ａａｖ）ベクターを産生するスケーラブルな方法
BR112018072849B1 (pt) proteína de capsídeo variante de vírus adeno-associado, ácido nucleico isolado, vírion de raav infeccioso e respectivo uso e composição farmacêutica
KR20210009317A (ko) 미토콘드리아 dna 고갈 증후군을 포함하는 불균형 뉴클레오타이드 풀에 의해 초래된 질환에 대한 유전자 요법
WO2024079665A1 (fr) Éléments régulateurs d'acide nucléique pour l'expression génique constitutive et méthodes d'utilisation
CN118086341B (zh) 在肝脏中高表达人源葡萄糖脑苷脂酶基因的表达框
CN117925624B (zh) 一种金属响应调控元件
WO2024074142A1 (fr) Polynucléotides pour le traitement de maladies associées à une déficience en gcase
JP2021534766A (ja) ガラクトース血症の処置のための遺伝子治療
KR20240162557A (ko) 증가된 심장 풍부화를 갖는 아데노-연관 바이러스 조성물
WO2025026198A1 (fr) Polynucléotides pour le traitement de la maladie de wilson
CN118064436B (zh) 一种增强基因表达的嵌合启动子
WO2021129624A1 (fr) Molécule d'acide nucléique isolée et application associée
TW202208407A (zh) 使用質膜修復蛋白(dysferlin)雙載體之基因療法
WO2024143440A1 (fr) Procédé de production de virus adéno-associé, et ensemble de vecteurs
WO2024074143A1 (fr) Construction pour améliorer l'expression génique
WO2024183769A1 (fr) Gène atp7b tronqué et son utilisation
WO2025157262A1 (fr) Acides nucléiques codant pour la follistatine et leurs utilisations
CN114269935A (zh) 用于治疗婴儿贝敦氏病的鞘内和静脉内组合基因疗法
CN119876192B (zh) 用于治疗苯丙酮尿症的多核苷酸
JP2025533550A (ja) Ａａｖ遺伝子治療ベクターによる心筋症の治療
CN118620930A (zh) 唾液酸尿症动物模型的构建方法及其用途
WO2024179585A1 (fr) Méthode de traitement ou d'amélioration de la cachexie
WO2024125494A1 (fr) Procédé de régulation génique et utilisation associée

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24848155

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202480018081.X

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 202480018081.X

Country of ref document: CN