WO2023143435A1 - 表达tpk的重组病毒及其治疗阿尔茨海默病的用途 - Google Patents

表达tpk的重组病毒及其治疗阿尔茨海默病的用途 Download PDF

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WO2023143435A1
WO2023143435A1 PCT/CN2023/073317 CN2023073317W WO2023143435A1 WO 2023143435 A1 WO2023143435 A1 WO 2023143435A1 CN 2023073317 W CN2023073317 W CN 2023073317W WO 2023143435 A1 WO2023143435 A1 WO 2023143435A1
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tpk
seq
amino acid
polypeptide
raav
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PCT/CN2023/073317
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French (fr)
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王晶晶
桑绍明
张寰
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上海日馨医药科技股份有限公司
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • 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
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus

Definitions

  • the present invention relates to the field of gene therapy. Specifically, the present invention relates to a diseased recombinant adeno-associated virus (rAAV) or a recombinant lentivirus comprising a polynucleotide encoding thiamine pyrophosphate kinase (TPK), which can be used for Treat Alzheimer's.
  • rAAV diseased recombinant adeno-associated virus
  • TPK recombinant lentivirus comprising a polynucleotide encoding thiamine pyrophosphate kinase
  • AD Alzheimer's disease
  • a ⁇ ⁇ -amyloid cascade hypothesis
  • Tau protein hypothesis the phosphorylated Tau protein hypothesis
  • neuroinflammatory response hypothesis the oxidative stress hypothesis
  • the brain accounts for about 2% of the body weight, but consumes about 20% of the total energy consumed by the human body, which shows that glucose metabolism is very important for the normal function of the brain.
  • regulation of gene expression in cells can be achieved by methods such as calcium transfer, liposome transfection, electroporation and transgenic virus infection.
  • Adeno-associated virus is a single-stranded DNA virus that widely exists in humans. Its natural hosts are humans and other primates. There is no report that AAV can cause disease.
  • Recombinant adeno-associated virus rAAV, which is engineered on the basis of AAV, is widely used as a genetic Treatment vector.
  • Luxturna was jointly developed by Spark Therapeutics and Children's Hospital of Florida for the treatment of Burger's congenital amaurosis.
  • Luxturna uses AAV2 as a vector carrying human RPE65cDNA, and the administration method is subretinal injection.
  • Zolgensma is being developed by Novartis for the treatment of pediatric spinal muscular atrophy.
  • Zolgensma uses AAV9 as a vector to carry the SMN1 gene necessary for the survival of motor neurons, and is administered intravenously.
  • Vitamin B1 also known as thiamine (TM) is converted in the body to the biologically active thiamine diphosphate (TDP) by thiamine pyrophosphate kinase (TPK).
  • TDP is an essential coenzyme for transketolase, pyruvate dehydrogenase, and ⁇ -ketoglutarate dehydrogenase in glucose metabolism.
  • Studies have shown that the activity of transketolase in the brain tissue of AD patients decreased by more than 45%, and the activity of ⁇ -ketoglutarate dehydrogenase decreased by more than 75%; the TDP level in the whole blood of AD patients was significantly lower than the normal level, and the TDP in the whole blood The level is highly correlated with the decline of brain glucose metabolism in AD patients.
  • TPK mRNA and protein in the brain tissue of AD patients were significantly lower than those of age-sex-matched normal human brain tissue. Ratings were positively correlated with the Braak score and negatively correlated in AD patients. The results indicated that TPK expression may be a protective factor to delay the onset and progression of AD disease.
  • Conditional knockout of the TPK gene in mouse brain excitatory neurons significantly leads to abnormal brain glucose metabolism and cognitive impairment, brain atrophy due to synapse and neuron loss, A ⁇ deposition and plaque formation, and abnormal phosphorylation of Tau Neurofibrillary tangle formation, microglial and astrocyte activation and neuroinflammation, impaired microvasculogenesis, and dysregulation of peripheral glucose metabolism are all important multipathophysiological features of human AD disease (see, e.g., Sang et al ., Thiamine pyrophosphokinase deficiency induces Alzheimer's pathology, Biorxiv, 2020; and WO2021023069A1).
  • the present invention provides a recombinant adeno-associated virus (rAAV) or recombinant lentivirus comprising in its genome an expression cassette comprising a thiamine pyrophosphokinase ( TPK) polynucleotide.
  • rAAV adeno-associated virus
  • TPK thiamine pyrophosphokinase
  • the TPK comprises the amino acid sequence of SEQ ID NO: 1.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO:2.
  • the promoter is a neuron-specific promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:3.
  • the expression cassette further comprises a WPRE element.
  • the WPRE element comprises the nucleotide sequence of SEQ ID NO:4.
  • the promoter is a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the promoter comprises the nucleotide sequence of SEQ ID NO:7.
  • the rAAV is rAAV of serotype AAV_PHP eB.
  • the present invention provides a modified thiamine pyrophosphate kinase (TPK) polypeptide comprising, compared to the starting TPK polypeptide, a group selected from positions 13, 30, 31, 37, 85, 129, 158, Amino acid substitutions at one or more of positions 181, 11, and 35, where the amino acid substitution at position 13 is P, the amino acid substitution at position 30 is A, the amino acid substitution at position 31 is R, the amino acid substitution at position 37 is K, and the amino acid substitution at position 85
  • the amino acid at position 129 is replaced by K, the amino acid at position 129 is replaced by G, the amino acid at position 158 is replaced by K, the amino acid at position 181 is replaced by S, the amino acid at position 11 is replaced by W, and the amino acid at position 35 is replaced by W, where the same as the starting Compared with the TPK polypeptide, the modified TPK polypeptide has an improved activity of catalyzing the conversion of thiamine (TM) into thiamine diphosphate (TDP)
  • the starting TPK polypeptide is a wild-type TPK polypeptide.
  • the starting TPK polypeptide is a human TPK polypeptide, and wherein the modified TPK polypeptide comprises one or more positions selected from positions 13, 30, 31, 37, 85, 129, 158, and 181 position, preferably all, of amino acid substitutions.
  • the starting TPK polypeptide is a mouse TPK polypeptide, and wherein the modified TPK polypeptide comprises an amino acid substitution at positions 11 and/or 35.
  • the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11.
  • the present invention also provides a polynucleotide encoding the modified TPK polypeptide of the present invention.
  • the present invention also provides an expression cassette comprising a polynucleotide of the present invention operably linked to a promoter.
  • the promoter is a neuron-specific promoter.
  • the promoter comprises the nucleotide sequence of SEQ ID NO:3.
  • the expression cassette further comprises a WPRE element.
  • the WPRE element comprises the nucleotide sequence of SEQ ID NO:4.
  • the present invention also provides rAAV or recombinant lentivirus, which contains the expression cassette provided in the third aspect of the present invention in its genome.
  • the rAAV is rAAV of serotype AAV_PHP eB.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the modified TPK polypeptide, polynucleotide, rAAV or recombinant lentivirus of the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for intravenous, intracerebral, or intrathecal administration.
  • the present invention provides a method for treating or preventing Alzheimer's disease, comprising administering the modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition of the present invention to a subject in need .
  • the modified TPK polypeptide, polynucleoside is administered intravenously, intracerebrally, or intrathecally. acid, rAAV, recombinant lentivirus or pharmaceutical composition.
  • the present invention also provides the use of the modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition of the present invention in the preparation of medicines for treating or preventing Alzheimer's disease.
  • the drug is administered intravenously, intracerebrally, or intrathecally.
  • the present invention also provides the modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition of the present invention, which is used for treating or preventing Alzheimer's disease.
  • the rAAV, recombinant lentivirus, or pharmaceutical composition is administered intravenously, intracerebrally, or intrathecally.
  • Figure 1 shows the plasmid map of the genome comprising AAV-TPK ( Figure 1A) and the genome of the TPK lentivirus ( Figure 1B).
  • Figure 2 shows fluorescence microscopy images of neuronal cells infected with lentivirus.
  • Figure 3 shows TPK expression (Figure 3A, parental represents cells not infected with virus), TDP content (Figure 3B) and TM content (Figure 3C) in neuronal cells infected with lentivirus.
  • Figure 4 shows the body weight (left and middle panels) and food intake (right panel) of mice injected with rAAV through the tail vein.
  • FIG. 5 shows TDP (left panel) and TM (right panel) levels in the blood of mice injected with rAAV through the tail vein.
  • Figure 6 shows images of immunofluorescence of sections from various regions in the brain of mice injected with rAAV through the tail vein.
  • FIG. 7 shows TPK expression (left panel) and TDP and TM contents (right panel) in the brain of mice injected with rAAV through the tail vein.
  • FIG. 8 shows TDP content (left panel) and TM content (right panel) in the liver of mice injected with rAAV through the tail vein.
  • Figure 9 shows body weight (left and middle panels) and food intake (right panel) of mice injected intracerebroventricularly with rAAV.
  • FIG 10 shows the TDP (left panel) and TM (right panel) levels in the blood of mice injected with rAAV intracerebroventricularly.
  • Figure 11 shows images of immunofluorescence of sections from various regions in the brain of mice injected with rAAV intracerebroventricularly.
  • Figure 12 shows TPK expression (left panel) and TDP and TM content (right panel) in the brain of mice injected with rAAV intracerebroventricularly.
  • FIG. 13 shows TDP content (left panel) and TM content (right panel) in the liver of rAAV-injected mice.
  • Figure 14 shows the body weight changes within 2 weeks after weaning of mice developed from rAAV-injected embryos on day 21 of birth.
  • Figure 15 shows the TDP (left panel) and TM (right panel) contents in the blood of mice developed from rAAV-injected embryos 21 days after birth.
  • Fig. 16 shows immunofluorescence images of sections of brains of mice developed from rAAV-injected embryos 21 days after birth.
  • FIG 17 shows the expression of TPK (left panel) and the contents of TDP and TM (right panel) in the brain of mice developed from rAAV-injected embryos 21 days after birth.
  • Figure 18 shows the TDP content (left panel) and TM content (right panel) in the liver of mice developed from rAAV-injected embryos 21 days after birth.
  • Figure 19 shows the body weight changes within 2 weeks after neonatal mice were weaned after intracerebroventricular injection of rAAV for 21 days.
  • Figure 20 shows the TDP (left panel) and TM (right panel) contents in the blood of neonatal mice injected with rAAV 21 days after the lateral ventricle.
  • Figure 21 shows images of immunofluorescence of sections from various regions in the brain of neonatal mice 21 days after intracerebroventricular injection of rAAV.
  • Figure 22 shows the expression of TPK (left panel) and the content of TDP and TM (right panel) in the brain of neonatal mice after 21 days of intracerebroventricular injection of rAAV.
  • Figure 23 shows the body weight changes within 2 weeks after neonatal mice were injected with rAAV in the hippocampus for 21 days after weaning.
  • Figure 24 shows the TDP (left panel) and TM (right panel) contents in the blood of neonatal mice injected with rAAV 21 days after hippocampus.
  • Figure 25 shows images of immunofluorescence of sections of brains of neonatal mice 21 days after hippocampus injection of rAAV.
  • Figure 26 shows the expression of TPK in the brain of neonatal mice injected with rAAV 21 days after hippocampus.
  • Figure 27 shows TDP content (left panel) and TM content (right panel) in the liver of neonatal mice 21 days after intracerebroventricular injection of rAAV.
  • Figure 28 shows the activity of modified human TPK compared to wild type human TPK and mouse TPK.
  • Figure 29 shows the activity of modified mouse TPK compared to wild-type mouse TPK.
  • FIG. 30 shows the Western Blot results of TPK protein in brain tissue.
  • Figure 31 shows the HPLC results of the content of TDP in brain tissue.
  • Figure 32 shows the curves of body weight (A), food intake (B) and blood glucose (C) of mice injected with AAV-hTPK.
  • Figure 33 shows the results of Western Blot of TPK protein in the brain tissue and liver of APP/PS1 mice injected with AAV-TPK.
  • Figure 34 shows the contents of TDP and TM in brain tissue, liver and blood of APP/PS1 mice injected with AAV-TPK.
  • Figure 35 shows the Western Blot results of p-GSK3 ⁇ and GSK3 ⁇ in the brain tissue of APP/PS1 mice injected with AAV-TPK intracerebroventricularly.
  • Figure 36 shows the ratio of p-GSK3 ⁇ /GSK3 ⁇ in the brain tissue of APP/PS1 mice injected intracerebroventricularly with AAV-TPK.
  • Thiamine pyrophosphate kinase or “TPK” is an enzyme that catalyzes the conversion of thiamine (TM) to thiamine diphosphate (TDP).
  • AAV Adeno-Associated Virus
  • rAAV refers to recombinant adeno-associated virus, also known as a recombinant AAV vector (or "rAAV vector”).
  • AAV includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and Sheep AAV.
  • Primary AAV refers to AAV that infects primates
  • non-primate AAV refers to AAV that infects non-primate mammals
  • bovine AAV refers to AAV that infects bovine mammals, and the like.
  • AAV genome sequences of the different subtypes of AAV are known in the art, as well as the sequences of the native terminal repeat (TR), Rep protein and capsid subunits. Such sequences can be found in the literature or in public databases such as GenBank. See, e.g., Genbank Accession Nos.
  • NC_002077 AAV-1
  • AF063497 AAV-1
  • NC_001401 AAV-2
  • AF043303 AAV-2
  • NC_001729 AAV-3
  • NC_001829 AAV-4
  • U89790 AAV-4
  • NC_006152 AAV-5
  • AF513851 AAV-7
  • AF513852 AAV-8
  • NC_006261 AAV-8
  • rAAV vector refers to an AAV vector comprising a polynucleotide sequence of non-AAV origin (ie, a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of cells. Generally, the heterologous polynucleotide is flanked by at least one, and often two, AAV inverted terminal repeats (ITRs).
  • ITRs AAV inverted terminal repeats
  • rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be single-stranded (ssAAV) or self-complementary (scAAV).
  • Packaging refers to the series of intracellular events leading to AAV particle assembly and encapsidation.
  • AAV rep and cap genes refer to the polynucleotide sequences encoding the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are also called AAV "packaging genes”.
  • helper virus for AAV refers to a virus that allows mammalian cells to replicate and package AAV (eg, wild-type AAV).
  • helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses (eg, vaccinia).
  • Adenoviruses encompass many different subclasses, although adenovirus type 5 of subgroup C is most commonly used.
  • Many adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC.
  • Viruses of the Herpesviridae family include, for example, herpes simplex virus (HSV) and Epstein-Barr viruses (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); also available from depositories such as ATCC.
  • HSV herpes simplex virus
  • EBV Epstein-Barr viruses
  • CMV cytomegalovirus
  • PRV pseudorabies virus
  • Helper virus function refers to the functions encoded in the genome of the helper virus that allow AAV replication and packaging (along with other requirements for replication and packaging as described herein).
  • helper virus function can be provided in a variety of ways, including by providing a helper virus or providing a producer cell with, for example, a polynucleotide sequence encoding the necessary function in trans.
  • a plasmid also known as a helper plasmid
  • other expression vector comprising a nucleotide sequence encoding one or more adenoviral proteins is transfected into producer cells along with the rAAV vector.
  • Lentiviral vectors are viral vectors derived from HIV-1.
  • "Recombinant lentiviral vector” or “recombinant lentiviral vector particle” refers to recombinant virus particles and recombinant virus-like particles produced in host cells or production cells after transfection with plasmid vectors selected by transfer vectors, encoding
  • the envelope vector consists of an envelope protein and a packaging vector providing lentiviral proteins in trans, such as lentiviral GAG and POL proteins, especially mutated POL proteins to prevent integration, according to methods well known in the art.
  • Virus-like particles result from incomplete assembly of proteins presented for encapsidation of recombinant lentiviral genomes in a manner that does not allow the formation of true virus particles.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure, if present, can be imparted either before or after polymer assembly.
  • the term polynucleotide refers alternately to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the invention described herein as a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • Nucleic acid hybridization reactions can be performed under conditions of varying "stringency”. Conditions that increase the stringency of hybridization reactions are well known and disclosed in the art. See, e.g., Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.
  • a polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, that percentage of the bases or amino acids are the same when comparing the two sequences.
  • Sequence similarity can be determined in a number of different ways. For determining sequence identity, available methods and computer programs for aligning sequences include, for example, BLAST, available at ncbi.nlm.nih.gov/BLAST/, and FASTA, available in the Genetics Computing Group (GCG) package , from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Gene refers to a polynucleotide containing at least one open reading frame capable of encoding a specific protein after transcription and translation.
  • recombinant means that polynucleotides are the product of various combinations of cloning, restriction or ligation steps and other procedures that produce constructs other than polynucleotides found in nature.
  • a recombinant virus is a viral particle comprising a recombinant polynucleotide. The term includes copies of the progeny of the propolynucleotide construct and the proviral construct, respectively.
  • control element or "control sequence” is a nucleotide sequence that participates in molecular interactions that contribute to the regulation of the function of a polynucleotide, including the replication, repetition, transcription, splicing, translation, or degradation of the polynucleotide. Modulation can affect the frequency, speed, or specificity of the process, and can be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a region of DNA that is capable, under certain conditions, of binding RNA polymerase and initiating transcription of a coding region that is usually located downstream (3' direction) from the promoter.
  • “Operably linked” refers to the juxtaposition of genetic elements, wherein the elements are in a relationship permitting their operation in an intended manner.
  • a promoter is operably linked to a coding region if the promoter facilitates the initiation of transcription of the coding sequence. Intervening residues are possible between the promoter and coding region as long as this functional relationship is maintained.
  • an "expression vector” is a vector that contains a region encoding a polypeptide of interest, and is used to effect expression of a protein in a predetermined target cell.
  • Expression vectors also contain control genes operably linked to the coding region to facilitate expression of the protein in the target. system components. The combination of control elements and genes operably linked thereto for expression is sometimes referred to as an "expression cassette,” many of which are known in the art and are available or can be readily constructed from components available in the art.
  • Heterologous refers to an entity that is derived from and is genotyped different from the rest of the entities to which it is compared.
  • polynucleotides introduced into plasmids or vectors derived from different species by genetic engineering techniques are heterologous polynucleotides.
  • a promoter that is taken from its native coding sequence and operably linked to a coding sequence with which it is not found naturally linked is a heterologous promoter.
  • an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes nucleic acid not normally included in naturally occurring wild-type AAV, and the encoded heterologous gene product is a wild-type AAV that does not typically occur in nature encoded gene product.
  • polypeptide polypeptide
  • peptide protein
  • modified amino acid polymers for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or association with a labeling component.
  • the modifications also include modifications to the polypeptide sequence, including, but not limited to, substitutions, deletions, insertions and/or additions of one or more amino acids.
  • substitution also referred to as substitution with a "homologous" amino acid residue, refers to a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain, for example, an amino acid with a basic side chain (e.g., lysine , arginine and histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chain amino acids (such as glycine, asparagine, glutamine, serine, threonine acid, tyrosine, cysteine), nonpolar side chain amino acids (e.g.
  • a basic side chain e.g., lysine , arginine and histidine
  • amino acids with acidic side chains such as aspartic acid, glutamic acid
  • uncharged polar side chain amino acids such as glycine, asparagine, glutamine, serine, threonine acid, tyrosine, cysteine
  • Conservative amino acid substitutions generally have minimal effect on the activity of the resulting protein. Such substitutions are described below.
  • a conservative substitution is the replacement of an amino acid with an amino acid of similar size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, etc. Such substitutions are generally conservative when one wishes to fine-tune the properties of a protein.
  • homologous amino acid residues refer to amino acid residues that have similar chemical properties relating to hydrophobicity, charge, polarity, steric character, aromatic character, and the like.
  • amino acids that are homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine , aromatic phenylalanine, tyrosine, tryptophan, serine and threonine with chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.
  • amino acid conservative substitutions in proteins include: Ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Met, Leu or Tyr for Phe, Thr for Ser, Ser for Thr, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu replaces Val.
  • isolated plasmid, nucleic acid, vector, virus, virion, host cell, or other material refers to a preparation of material that is free of at least some of the other components that may be present when the material or a similar material exists in nature or when it was originally prepared. Thus, for example, purification counts can be used to prepare isolated substances to be enriched from a source mixture.
  • treatment refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms and/or may be therapeutic in terms of partial or complete cure of the disease and/or side effects attributable to the disease.
  • treatment includes any treatment of a disease in a mammal, especially a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible or at risk of the disease but has not yet been diagnosed with the disease ; (b) inhibiting the disease, ie arresting its development; and (c) ameliorating the disease, ie causing the disease to regress.
  • mammals including including, but not limited to, humans and non-human primates, including apes and humans; mammalian sport animals (e.g., horses); mammalian livestock (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (eg, mice, rats, etc.).
  • mammalian sport animals e.g., horses
  • mammalian livestock e.g., sheep, goats, etc.
  • mammalian pets e.g., dogs, cats, etc.
  • rodents eg, mice, rats, etc.
  • mouse thiamine pyrophosphate kinase is more active in catalyzing the conversion of TM to TDP than human TPK (hTPK).
  • the inventors modified the sequences of hTPK and mTPK to improve the activity of catalyzing the conversion of TM into TDP. For example, the surface charge properties of hTPK were murinized by replacing residue Xh in hTPK with Xm when the charge properties of residue Xh in hTPK were different from residue Xm at the matching position in mTPK.
  • the present invention provides a modified thiamine pyrophosphate kinase (TPK) polypeptide comprising, compared to the starting TPK polypeptide, a Amino acid substitutions at one or more positions of and 35, wherein compared with the starting TPK polypeptide, the modified TPK polypeptide has an improved activity of catalyzing the conversion of TM into TDP, and wherein the positions refer to SEQ ID NO: 1 or 8 numbers.
  • TPK modified thiamine pyrophosphate kinase
  • the amino acid substitution at position 13 is P.
  • the amino acid substitution at position 30 is A.
  • the amino acid substitution at position 31 is R.
  • the amino acid substitution at position 37 is K.
  • the amino acid substitution at position 85 is K.
  • the amino acid substitution at position 129 is G.
  • the amino acid substitution at position 158 is a K.
  • the amino acid substitution at position 181 is S.
  • the amino substitution at position 11 is W.
  • the amino acid substitution at position 35 is W.
  • the TPK polypeptide based on which the amino acid modification is carried out is referred to as the starting TPK polypeptide.
  • the starting TPK polypeptide can be a wild-type TPK polypeptide, or a variant of the wild-type TPK polypeptide.
  • the polypeptide of SEQ ID NO: 1 is the "starting TPK polypeptide" with respect to the modified TPK polypeptide; and if the variant polypeptide of SEQ ID NO: 1
  • the variant polypeptide is the "starting TPK polypeptide" relative to the modified TPK polypeptide when the modification is initiated.
  • the starting TPK polypeptide is a wild-type TPK polypeptide, such as a human TPK polypeptide and a mouse TPK polypeptide.
  • the starting TPK polypeptide is a human TPK polypeptide, and wherein the modified TPK polypeptide comprises a position selected from positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35 Amino acid substitutions for amino acid substitutions at one or more positions.
  • the starting TPK polypeptide is a human TPK polypeptide, and wherein the modified TPK polypeptide comprises one or more positions selected from positions 13, 30, 31, 37, 85, 129, 158, and 181 Amino acid substitutions for amino acid substitutions at positions.
  • said starting TPK polypeptide is a human TPK polypeptide, and wherein said modified TPK polypeptide comprises amino acid substitutions of amino acid substitutions at positions 13, 30, 31, 37, 85, 129, 158 and 181.
  • the starting TPK polypeptide is a mouse TPK polypeptide, and wherein the modified TPK polypeptide comprises an amino acid substitution at positions 11 and/or 35.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, Amino acid sequences with 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity, wherein compared to the starting TPK polypeptide, the modified TPK polypeptide comprises a position selected from One or more of positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35, such as positions 13, 30, 31, 37, 85, 129, 158, and 181, positions 13, 30, 31 , 37, 85, 129, 158, 181, and 11, positions 13, 30, 31, 37, 85, 129, 158, 181, and 35, or positions 13, 30, 31, 37, 85, 129, 158, 181, Amino acid substitutions at 11 and 35.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, An amino acid sequence having a sequence identity of 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99%, wherein said modified TPK polypeptide comprises positions 11W and and/or amino acid substitutions of 35W amino acid substitutions.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, An amino acid sequence having a sequence identity of 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99%, wherein said modified TPK polypeptide comprises a position 11W compared to said starting TPK polypeptide Amino Acid Substitutions Amino Acid Substitutions.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity amino acid sequence, wherein compared with the starting TPK polypeptide, the modified TPK polypeptide comprises a Amino Acid Substitutions Amino Acid Substitutions.
  • said modified TPK polypeptide comprises the following amino acid substitutions or combinations of amino acid substitutions:
  • the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11 or has at least 65%, preferably at least 70%, 75% or Amino acid sequences having a sequence identity of 80%, more preferably at least 85%, 90% or 95%, especially preferably at least 96%, 97%, 98% or 99%.
  • the modified TPK polypeptide comprises one or more amino acid substitutions compared to SEQ ID NO: 9, 10 or 11.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 9, except for positions 13, 30, 31, 37, 85, 129, 158 and 181 and, preferably, between 241
  • the outer positions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 10, except for positions 11 and 241 and, preferably, 13, 30, 31, 37, 85, 129, 158 and Positions other than 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 11, except for positions 35 and 241 and, preferably, 13, 30, 31, 37, 85, 129, 158 and Positions other than 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the activity of the modified TPK polypeptide to catalyze the conversion of TM to TDP is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or higher.
  • the present invention provides an isolated polynucleotide encoding a polynucleotide for thiamine pyrophosphate kinase (TPK).
  • TPK thiamine pyrophosphate kinase
  • the TPK may be a TPK from a human or a non-human animal, such as a non-human mammal.
  • TPKs from non-human mammals include, but are not limited to, TPKs from non-human primates such as monkeys, horses, cows, sheep, goats, pigs, dogs, cats, mice, rats.
  • the TPK is wild-type TPK. In some embodiments, the TPK is mouse TPK or human TPK.
  • the TPK comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the TPK comprises the nucleotide sequence of SEQ ID NO:2. In some embodiments, the TPK consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the TPK consists of the nucleotide sequence of SEQ ID NO:2.
  • the TPK is a modified TPK that, compared to the starting TPK, comprises one or more amino acid substitution, insertion, deletion and/or addition, wherein the activity of the modified TPK is at least 10%, 20%, 30%, 40%, 50%, 60%, 70% of the starting TPK , 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or higher.
  • the modified TPK comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, insertions, deletions compared to the starting TPK and/or add.
  • the modified TPK is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the starting TPK.
  • the starting TPK may be a wild-type TPK or a modified TPK.
  • the starting TPK is wild-type mouse TPK or wild-type human TPK.
  • the starting TPK comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the starting TPK consists of the amino acid sequence of SEQ ID NO: 1.
  • the modified TPK polypeptide comprises one or more positions selected from positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35 compared to the starting TPK polypeptide Amino acid substitutions, wherein compared with the starting TPK polypeptide, the modified TPK polypeptide has an improved activity of catalyzing the conversion of TM into TDP, and wherein the position is numbered with reference to SEQ ID NO: 1 or 8.
  • the amino acid substitution at position 13 is P.
  • the amino acid substitution at position 30 is A.
  • the amino acid substitution at position 31 is R.
  • the amino acid substitution at position 37 is K.
  • the amino acid substitution at position 85 is K.
  • the amino acid substitution at position 129 is G.
  • the amino acid substitution at position 158 is a K.
  • the amino acid substitution at position 181 is S.
  • the amino substitution at position 11 is W.
  • the amino acid substitution at position 35 is W.
  • the starting TPK polypeptide is a wild-type TPK polypeptide, such as a human TPK polypeptide and a mouse TPK polypeptide.
  • the starting TPK polypeptide is a human TPK polypeptide, and wherein the modified TPK polypeptide comprises a position selected from positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35 Amino acid substitutions for amino acid substitutions at one or more positions.
  • the starting TPK polypeptide is a human TPK polypeptide, and wherein the modified TPK polypeptide comprises one or more positions selected from positions 13, 30, 31, 37, 85, 129, 158, and 181 Amino acid substitutions for amino acid substitutions at positions.
  • said starting TPK polypeptide is a human TPK polypeptide, and wherein said modified TPK polypeptide comprises amino acid substitutions of amino acid substitutions at positions 13, 30, 31, 37, 85, 129, 158 and 181.
  • the starting TPK polypeptide is a mouse TPK polypeptide, and wherein the modified TPK polypeptide comprises an amino acid substitution at positions 11 and/or 35.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO:8 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, Amino acid sequences with 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity, wherein compared to the starting TPK polypeptide, the modified TPK polypeptide comprises a position selected from One or more of positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35, such as positions 13, 30, 31, 37, 85, 129, 158, and 181, positions 13, 30, 31 , 37, 85, 129, 158, 181, and 11, positions 13, 30, 31, 37, 85, 129, 158, 181, and 35, or positions 13, 30, 31, 37, 85, 129, 158, 181, Amino acid substitutions at 11 and 35.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, An amino acid sequence having a sequence identity of 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99%, wherein said modified TPK polypeptide comprises positions 11W and and/or amino acid substitutions of 35W amino acid substitutions.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, Amino acid sequences having a sequence identity of 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99%, wherein the starting The modified TPK polypeptide comprises an amino acid substitution of an amino acid substitution at position 11W compared to a TPK polypeptide.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity amino acid sequence, wherein compared with the starting TPK polypeptide, the modified TPK polypeptide comprises a Amino Acid Substitutions Amino Acid Substitutions.
  • said modified TPK polypeptide comprises the following amino acid substitutions or combinations of amino acid substitutions:
  • the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11 or has at least 65%, preferably at least 70%, 75% or Amino acid sequences having a sequence identity of 80%, more preferably at least 85%, 90% or 95%, especially preferably at least 96%, 97%, 98% or 99%.
  • the modified TPK polypeptide comprises one or more amino acid substitutions compared to SEQ ID NO: 9, 10 or 11.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 9, except for positions 13, 30, 31, 37, 85, 129, 158 and 181 and, preferably, between 241
  • the outer positions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 10, except for positions 11 and 241 and, preferably, 13, 30, 31, 37, 85, 129, 158 and Positions other than 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 11, except for positions 35 and 241 and, preferably, 13, 30, 31, 37, 85, 129, 158 and Positions other than 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the activity of the modified TPK polypeptide to catalyze the conversion of TM to TDP is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or higher.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15, 16 or 17, or at least 70%, 75%, 80%, 85% with SEQ ID NO: 15, 16 or 17 %, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity of nucleotide sequences.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15, or has at least 70%, 75%, 80%, 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98% or 99% sequence identity.
  • the polynucleotide comprises SEQ ID NO: 15, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% of SEQ ID NO: 15 , 98% or 99% sequence identity of the nucleotide sequence, and said nucleotide sequence encoding compared with SEQ ID NO: 9, except at positions 13, 30, 31, 37, 85, 129, 158 and 181 and, preferably, positions other than 241 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservatively substituted amino acid sequences.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16, or has at least 70%, 75%, 80%, 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98% or 99% sequence identity.
  • the polynucleotide comprises, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% of SEQ ID NO: 16 , 98% or 99% sequence identity of the nucleotide sequence, and said nucleotide sequence encoding compared with SEQ ID NO: 10, except positions 11 and 241 and, preferably, 13, 30, 31, Positions other than 37, 85, 129, 158, and 181 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservatively substituted amino acid sequences.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17, or has at least 70%, 75%, 80%, 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98% or 99% sequence identity.
  • the polynucleotide comprises, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% of SEQ ID NO: 17 , 98% or 99% sequence identity of the nucleotide sequence, and said nucleotide sequence encoding compared with SEQ ID NO: 11, except at positions 35 and 241 and, preferably, 13, 30, 31, Positions other than 37, 85, 129, 158 and 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservatively substituted amino acid sequences.
  • the polynucleotide is DNA or RNA, such as mRNA.
  • the present invention also provides an expression cassette comprising a polynucleotide of the present invention operably linked to a promoter.
  • the promoter is a neuron-specific promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the promoter consists of the nucleotide sequence of SEQ ID NO:3. In some embodiments, the promoter comprises nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:3 acid sequence. In some embodiments, the promoter is composed of nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:3 acid sequences.
  • the promoter is a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter.
  • the promoter comprises the nucleotide sequence of SEQ ID NO:7.
  • the promoter consists of the nucleotide sequence of SEQ ID NO:7.
  • the promoter comprises nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7 acid sequence.
  • the promoter is composed of nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7 acid sequences.
  • the expression cassette may also contain additional control sequences, such as enhancers, introns, mRNA stabilizing sequences, and polyadenylation signal sequences.
  • the expression cassette further comprises an mRNA stabilizing sequence, such as a WPRE element.
  • the WPRE element comprises the nucleotide sequence of SEQ ID NO:4.
  • the WPRE element consists of the nucleotide sequence of SEQ ID NO:4.
  • the WPRE element comprises a nucleoside having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:4 acid sequence.
  • the WPRE element is composed of nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:4 acid sequences.
  • the WPRE element is downstream of a polynucleotide encoding a TPK.
  • the expression cassette further comprises a polyadenylation signal sequence, such as, but not limited to, the polyadenylation signal sequence of the human growth factor gene and the polyadenylation signal sequence of the rabbit globulin gene. In some embodiments, the expression cassette further comprises a polyadenylation signal sequence of a human growth factor gene. In some embodiments, the polyadenylation signal sequence comprises the nucleotide sequence of SEQ ID NO:5. In some embodiments, the polyadenylation signal sequence consists of the nucleotide sequence of SEQ ID NO:5.
  • the polyadenylation signal sequence comprises a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:5.
  • sexual nucleotide sequence In some embodiments, the polyadenylation signal sequence is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:5 Sexual nucleotide sequence composition.
  • the polyadenylation signal sequence is downstream of the WPRE element.
  • the expression cassette may be part of the genome of rAAV.
  • the expression cassette is flanked by the ITRs of AAV.
  • ITRs include those derived from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, Avian AAV, Bovine AAV, Canine AAV, Equine AAV, Primate AAV, Non-primate AAV, and Ovine AAV ITR.
  • the ITR is an ITR of AAV-2.
  • the 5'ITR and 3'ITR are the same. In some embodiments, the 5'ITR and 3'ITR are different.
  • both the 5'ITR and the 3'ITR are ITR130, e.g., comprising the nucleotide sequence of SEQ ID NO:6.
  • the 5'ITR and 3'ITR are each composed of the nucleotide sequence of SEQ ID NO:6.
  • the 5'ITR and 3'ITR respectively comprise at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:6 Sexual nucleotide sequence.
  • the 5'ITR and 3'ITR are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:6, respectively.
  • Sexual nucleotide sequence composition is provided.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:4, SEQ ID NO:5.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:4, SEQ ID NO:5.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:5.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:4, SEQ ID NO:5.
  • the invention also provides a vector comprising a polynucleotide or expression cassette of the invention.
  • the vector is an expression vector.
  • the vector is a eukaryotic expression vector.
  • the vector is a vector that provides the rAAV genome in rAAV packaging, ie, a transgenic plasmid.
  • the vector is one that provides the lentiviral genome in lentiviral packaging, i.e., a transgenic Because of the plasmid.
  • the present invention provides a recombinant adeno-associated virus (rAAV) or a recombinant lentivirus, which contains an expression cassette in its genome, and the expression cassette contains a polynucleotide encoding TPK operably linked to a promoter.
  • rAAV adeno-associated virus
  • lentivirus which contains an expression cassette in its genome, and the expression cassette contains a polynucleotide encoding TPK operably linked to a promoter.
  • the TPK may be a TPK from a human or a non-human animal, such as a non-human mammal.
  • TPKs from non-human mammals include, but are not limited to, TPKs from non-human primates such as monkeys, horses, cows, sheep, goats, pigs, dogs, cats, mice, rats.
  • the TPK is wild-type TPK. In some embodiments, the TPK is mouse TPK or human TPK.
  • the TPK comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the TPK comprises the nucleotide sequence of SEQ ID NO:2. In some embodiments, the TPK consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the TPK consists of the nucleotide sequence of SEQ ID NO:2.
  • the TPK is a modified TPK comprising one or more amino acid substitutions, insertions, deletions and/or additions compared to the starting TPK, wherein the modified TPK has an activity of At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150% of the starting TPK %, 200%, 250%, 300% or higher.
  • the modified TPK comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, insertions, deletions compared to the starting TPK and/or add.
  • the modified TPK is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the starting TPK.
  • the starting TPK may be a wild-type TPK or a modified TPK.
  • the starting TPK is wild-type mouse TPK or wild-type human TPK.
  • the starting TPK comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the starting TPK consists of the amino acid sequence of SEQ ID NO: 1.
  • the modified TPK polypeptide comprises one or more positions selected from positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35 compared to the starting TPK polypeptide Amino acid substitutions, wherein compared with the starting TPK polypeptide, the modified TPK polypeptide has an improved activity of catalyzing the conversion of TM into TDP, and wherein the position is numbered with reference to SEQ ID NO: 1 or 8.
  • the amino acid substitution at position 13 is P.
  • the amino acid substitution at position 30 is A.
  • the amino acid substitution at position 31 is R.
  • the amino acid substitution at position 37 is K.
  • the amino acid substitution at position 85 is K.
  • the amino acid substitution at position 129 is G.
  • the amino acid substitution at position 158 is a K.
  • the amino acid substitution at position 181 is S.
  • the amino substitution at position 11 is W.
  • the amino acid substitution at position 35 is W.
  • the starting TPK polypeptide is a wild-type TPK polypeptide, such as a human TPK polypeptide and a mouse TPK polypeptide.
  • the starting TPK polypeptide is a human TPK polypeptide, and wherein the modified TPK polypeptide comprises a position selected from positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35 Amino acid substitutions for amino acid substitutions at one or more positions.
  • the starting TPK polypeptide is a human TPK polypeptide, and wherein the modified TPK polypeptide comprises one or more positions selected from positions 13, 30, 31, 37, 85, 129, 158, and 181 Amino acid substitutions for amino acid substitutions at positions.
  • said starting TPK polypeptide is a human TPK polypeptide, and wherein said modified TPK polypeptide comprises amino acid substitutions of amino acid substitutions at positions 13, 30, 31, 37, 85, 129, 158 and 181.
  • the starting TPK polypeptide is a mouse TPK polypeptide, and wherein the modified TPK polypeptide comprises an amino acid substitution at positions 11 and/or 35.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, Amino acid sequences with 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity, wherein compared to the starting TPK polypeptide, the modified TPK polypeptide comprises a position selected from One or more of positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35, such as positions 13, 30, 31, 37, 85, 129, 158, and 181, positions 13, 30, 31 , 37, 85, 129, 158, 181, and 11, positions 13, 30, 31, 37, 85, 129, 158, 181, and 35, or positions 13, 30, 31, 37, 85, 129, 158, 181, Amino acid substitutions at 11 and 35.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, An amino acid sequence having a sequence identity of 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99%, wherein said modified TPK polypeptide comprises positions 11W and and/or amino acid substitutions of 35W amino acid substitutions.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, An amino acid sequence having a sequence identity of 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99%, wherein said modified TPK polypeptide comprises a position 11W compared to said starting TPK polypeptide Amino Acid Substitutions Amino Acid Substitutions.
  • the starting TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity amino acid sequence, wherein compared with the starting TPK polypeptide, the modified TPK polypeptide comprises a Amino Acid Substitutions Amino Acid Substitutions.
  • said modified TPK polypeptide comprises the following amino acid substitutions or combinations of amino acid substitutions:
  • the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11 or has at least 65%, preferably at least 70%, 75% or Amino acid sequences having a sequence identity of 80%, more preferably at least 85%, 90% or 95%, especially preferably at least 96%, 97%, 98% or 99%.
  • the modified TPK polypeptide comprises one or more amino acid substitutions compared to SEQ ID NO: 9, 10 or 11.
  • the modified TPK polypeptide comprises 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 10, except for positions 11 and 241 and, preferably, 13, 30, 31, 37, 85, 129, 158 and Positions other than 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the modified TPK polypeptide is compared with the amino acid sequence of SEQ ID NO: 11, except for positions 35 and 241 and, preferably, 13, 30, 31, 37, 85, 129, 158 and Positions other than 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions.
  • the activity of the modified TPK polypeptide to catalyze the conversion of TM to TDP is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or higher.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15, 16 or 17, Or a nucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, 16 or 17 sequence.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15, or has at least 70%, 75%, 80%, 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98% or 99% sequence identity.
  • the polynucleotide comprises SEQ ID NO: 15, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% of SEQ ID NO: 15 , 98% or 99% sequence identity of the nucleotide sequence, and said nucleotide sequence encoding compared with SEQ ID NO: 9, except at positions 13, 30, 31, 37, 85, 129, 158 and Positions other than 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservatively substituted amino acid sequences.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16, or has at least 70%, 75%, 80%, 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98% or 99% sequence identity.
  • the polynucleotide comprises SEQ ID NO: 16, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% of SEQ ID NO: 16 , 98% or 99% sequence identity of the nucleotide sequence, and said nucleotide sequence encoding compared with SEQ ID NO: 10, except at positions 11 and 241 and, preferably, 13, 30, 31, Positions other than 37, 85, 129, 158 and 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservatively substituted amino acid sequences.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17, or has at least 70%, 75%, 80%, 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98% or 99% sequence identity.
  • the polynucleotide comprises, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% of SEQ ID NO: 17 , 98% or 99% sequence identity of the nucleotide sequence, and said nucleotide sequence encoding compared with SEQ ID NO: 11, except at positions 35 and 241 and, preferably, 13, 30, 31, Positions other than 37, 85, 129, 158 and 181 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservatively substituted amino acid sequences.
  • the promoter is a neuron-specific promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the promoter consists of the nucleotide sequence of SEQ ID NO:3. In some embodiments, the promoter comprises nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:3 acid sequence. In some embodiments, the promoter is composed of nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:3 acid sequences.
  • the promoter is a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter.
  • the promoter comprises the nucleotide sequence of SEQ ID NO:7.
  • the promoter consists of the nucleotide sequence of SEQ ID NO:7.
  • the promoter comprises nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7 acid sequence.
  • the promoter is composed of nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7 acid sequences.
  • the expression cassette may also contain additional control sequences, such as enhancers, introns, mRNA stabilizing sequences, and polyadenylation signal sequences.
  • the expression cassette further comprises an mRNA stabilizing sequence, such as a WPRE element.
  • the WPRE element comprises the nucleotide sequence of SEQ ID NO:4.
  • the WPRE element consists of the nucleotide sequence of SEQ ID NO:4.
  • the WPRE element comprises a nucleoside having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:4 acid sequence.
  • the WPRE element is composed of nucleosides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:4 acid sequences.
  • the WPRE element is downstream of a polynucleotide encoding a TPK.
  • the expression cassette further comprises a polyadenylation signal sequence, such as, but not limited to, the polyadenylation signal sequence of the human growth factor gene and the polyadenylation signal sequence of the rabbit globulin gene.
  • the expression cassette further comprises a polyadenylation signal sequence of a human growth factor gene.
  • the polyadenylation signal sequence of the human growth factor gene comprises the nucleotide sequence of SEQ ID NO:5.
  • the polyadenylation signal sequence of the human growth factor gene consists of the nucleotide sequence of SEQ ID NO:5.
  • the polyadenylation signal sequence of the human growth factor gene comprises at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO:5. Nucleotide sequences with % or greater identity. In some embodiments, the polyadenylation signal sequence of the human growth factor gene has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO:5. nucleotide sequence composition with % or greater identity.
  • the polyadenylation signal sequence is downstream of the WPRE element.
  • ITRs in the genome of rAAV include those derived from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV- 9.
  • ITRs for avian AAV, bovine AAV canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV.
  • the ITR is an ITR of AAV-2.
  • the ITR is an ITR of AAV-2.
  • the 5'ITR and 3'ITR are the same. In some embodiments, the 5'ITR and 3'ITR are different.
  • both the 5'ITR and the 3'ITR are ITR130, e.g., comprising the nucleotide sequence of SEQ ID NO:6.
  • the 5'ITR and 3'ITR are each composed of the nucleotide sequence of SEQ ID NO:6.
  • the 5'ITR and 3'ITR respectively comprise at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:6 Sexual nucleotide sequence.
  • the 5'ITR and 3'ITR are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:6, respectively.
  • Sexual nucleotide sequence composition is provided.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5. In some embodiments, the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO: 7, SEQ ID NO:14, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:5.
  • the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:4, SEQ ID NO:5.
  • the rAAV can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, avian AAV, bovine AAV, canine AAV, Equine AAV, primate AAV, non-primate AAV and ovine AAV.
  • the rAAV is rAAV of serotype AAV_PHP eB.
  • rAAV rAAV with a system comprising three plasmids including: 1) a transgenic plasmid comprising the genome of rAAV encoding a gene product of interest; ii) a packaging plasmid encoding REP and/or CAP protein; and iii) helper plasmid (see, e.g., Crosson SM et al., Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation. Mol Ther Methods Clin Dev. 2018; 10:1-7).
  • the packaging involves introducing the system into a host cell.
  • lentiviral proteins such as lentiviral GAG and POL proteins, especially mutated POL proteins in trans
  • transfer vectors transgenic plasmids
  • envelope vectors encoding selected envelope proteins
  • Lentiviral packaging system consisting of packaging vectors that prevent integration). Encapsulation of lentiviruses involves introducing the system into host cells.
  • the present invention also provides host cells comprising the polynucleotides, vectors, rAAV or recombinant lentiviruses of the present invention.
  • the host cells When used to package rAAV or recombinant lentiviral virions, the host cells are referred to as "packaging cells".
  • the host cell is stably genetically modified with a polynucleotide or vector of the invention. In other embodiments, the host cell is transiently genetically modified with a polynucleotide or vector of the invention.
  • a polynucleotide or vector of the invention is stably or transiently introduced into host cells using established techniques, including but not limited to electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like.
  • the polynucleotides or vectors of the invention will generally further comprise a selectable marker, such as any of several well known selectable markers, eg neomycin resistance and the like.
  • Host cells of the invention may be derived from mammalian cells.
  • Suitable mammalian cells include, but are not limited to, primary cells and cell lines, wherein suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts , CHO cells, etc.
  • suitable host cells include, for example, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos.
  • CRL9618, CCL61, CRL9096), 293 cells For example, ATCC No.CRL-1573), Vero cells, N1H3T3 cells (such as ATCC No.CRL-1658), Huh-7 cells, BHK cells (such as ATCC No.CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No.CRL1651), RAT1 cells, mouse L cells (ATCC No.CCL1.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, etc.
  • ATCC No.CRL-1573 Vero cells
  • N1H3T3 cells such as ATCC No.CRL-1658
  • Huh-7 cells BHK cells (such as ATCC No.CCL10)
  • PC12 cells ATCC No.CRL1721)
  • COS cells COS-7 cells
  • RAT1 cells mouse L cells (ATCC No.CCL1.3), human embryonic kidney (HEK) cells (ATCC No.CR
  • the present invention provides a pharmaceutical composition, comprising the polynucleotide, vector, rAAV or recombinant lentivirus of the present invention, and a pharmaceutically acceptable carrier, diluent, excipient or buffer.
  • a pharmaceutically acceptable carrier, diluent, excipient or buffer is suitable for use in humans.
  • excipients, carriers, diluents and buffers include any agent which can be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. These may include pharmaceutically acceptable salts such as mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, Benzoates, etc. Additionally, auxiliary substances may be present in such carriers, such as Such as wetting agents or emulsifiers, pH buffering substances, etc. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein.
  • the present invention provides a method for promoting glucose metabolism, or preventing or treating glucose metabolism disorders, preferably glucose metabolism disorders in the brain, comprising administering polynucleotides, vectors, rAAV, and recombinant lentiviruses of the present invention to subjects or patients in need or pharmaceutical compositions.
  • the present invention provides a method for treating or preventing Alzheimer's disease, comprising administering the polynucleotide, vector, rAAV or recombinant lentivirus of the present invention, or a pharmaceutical composition to a subject or patient in need.
  • the polynucleotide, vector, rAAV, recombinant lentivirus, or pharmaceutical composition is administered intravenously, intracerebrally, or intrathecally.
  • the subject or patient is a human.
  • the present invention also provides the use of polynucleotides, vectors, rAAV or recombinant lentiviruses of the present invention, or pharmaceutical compositions in the preparation of drugs for promoting glucose metabolism, or preventing or treating glucose metabolism disorders, preferably glucose metabolism disorders in the brain use.
  • the present invention also provides the use of the polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition of the present invention in the preparation of medicines for treating or preventing Alzheimer's disease.
  • the drug is administered intravenously, intracerebrally, or intrathecally.
  • the medicament is administered to a human.
  • the present invention also provides the use of the polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition of the present invention for promoting glucose metabolism, or preventing or treating glucose metabolism disorders, preferably glucose metabolism disorders in the brain.
  • the present invention also provides the polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition of the present invention, which is used for treating or preventing Alzheimer's disease.
  • the polynucleotide, vector, rAAV, recombinant lentivirus, or pharmaceutical composition is administered intravenously, intracerebrally, or intrathecally.
  • the polynucleotide, vector, rAAV, recombinant lentivirus, or pharmaceutical composition is administered to a human.
  • AD Alzheimer's disease
  • the prevention or treatment of Alzheimer's disease includes preventing, alleviating or eliminating AD symptoms, such as AD-like neurodegeneration, brain atrophy, pathological changes of A ⁇ and tau, neuroinflammation and neurovascular disorders, or slowing down the progress of AD.
  • AD involves damage to multiple brain regions, and the virus of the present invention can specifically and efficiently penetrate the blood-brain barrier and act on multiple brain regions extensively.
  • the rAAV of the present invention can be administered through direct injection (such as intracerebral injection or intrathecal injection) across the blood-brain barrier, or administered through peripheral intravenous injection, and can also achieve the same effect as direct injection into the brain.
  • direct injection such as intracerebral injection or intrathecal injection
  • peripheral intravenous injection can also achieve the same effect as direct injection into the brain.
  • the difference between the present invention and other gene therapies is that only exogenously mediated compensatory expression of TPK does not involve endogenous TPK, let alone editing of other genes, which can greatly reduce other risks.
  • the present invention introduces highly active exogenous TPK to achieve beneficial therapeutic effects.
  • GSK3 ⁇ is a very conserved serine/threonine kinase in evolution. In addition to regulating the activity of glycogen synthase, it can also act on other pathways to regulate cell differentiation, proliferation, survival and apoptosis. Studies have shown that the activation of GSK3 ⁇ can promote the phosphorylation of tau protein, increase A ⁇ deposition, activate microglia, damage neuron production, inhibit LTP, etc. to cause the pathogenesis of AD (Elisabetta Lauretti, Ozlem Dincer, Domenico Pratic ⁇ , Glycogen synthase kinase-3 signaling in Alzheimer's disease, Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, Volume 1867, Issue 5, 2020).
  • the rAAV of the present invention expresses TPK in vivo (such as in the brain), and its mediated "TPK-TDP-glucose metabolism" pathway interacts with the GSK3 ⁇ pathway to increase phosphorylated GSK3 ⁇ (p-GSK3 ⁇ ) levels and p- The GSK3 ⁇ /GSK3 ⁇ ratio, inhibiting the activity of GSK3 ⁇ , provides beneficial therapeutic effects in AD.
  • Example 1 preparation of rAAV and recombinant lentivirus
  • AAV-TPK laboratory-grade rAAV (serotype AAV_PHP eB) encoding TPK, hereinafter referred to as AAV-TPK, whose genome contains: i) neuron-specific promoter (SEQ ID NO: 3), ii) the nucleotide sequence (SEQ ID NO:2) and WPRE element (SEQ ID NO:4) of encoding mouse TPK.
  • AAV-TPK laboratory-grade rAAV (serotype AAV_PHP eB) encoding TPK, hereinafter referred to as AAV-TPK, whose genome contains: i) neuron-specific promoter (SEQ ID NO: 3), ii) the nucleotide sequence (SEQ ID NO:2) and WPRE element (SEQ ID NO:4) of encoding mouse TPK.
  • SEQ ID NO: 3 neuron-specific promoter
  • SEQ ID NO:2 the nucleotide sequence
  • WPRE element SEQ ID NO:4
  • TPK lentivirus whose genome contains: i) eukaryotic expression promoter (SEQ ID NO: 7), ii) Nucleotide sequence (SEQ ID NO:2) and WPRE element (SEQ ID NO:4) encoding mouse TPK.
  • SEQ ID NO: 7 eukaryotic expression promoter
  • SEQ ID NO:2 Nucleotide sequence
  • SEQ ID NO:4 Nucleotide sequence
  • SEQ ID NO:4 Nucleotide sequence
  • WPRE element SEQ ID NO:4
  • the purpose of this example is to conduct a preliminary evaluation of the effect of gene therapy through experiments at the cellular level.
  • Thermofisher/Gibco product number 25200072 Use trypsin (Thermofisher/Gibco product number 25200072) to digest the cerebral cortex of newborn mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) to obtain primary neurons, and culture them in 6-well plates (1.5x 10 6 /well ) for 4 days (10% FBS + 10% F12 + 1% Glutemax + 78% DMEM + 1% 100X penicillin-streptomycin double antibody (5,000U/mL) on the first day, then 96% Neurobasal medium + 2% B27 + 1% Glutemax + 1% 100X penicillin-streptomycin double antibody (5,000 U/mL), the above reagents are configured in volume ratio, and are all purchased from Thermofisher/Gibco).
  • the medium was removed, and the lentivirus suspension prepared in Example 1 (in PBS, 1.5 mL, MOI 5) was added to infect for 8 hr, and the lentivirus encoding GFP but not containing the TPK coding sequence was used as a control.
  • the liquid was removed and fresh medium was added and cultured for 7 days.
  • GFP expression was observed by fluorescence microscopy. As shown in Figure 2, a large number of infected cells expressed GFP, indicating a high infection rate of the virus.
  • the total protein concentration in the supernatant was determined by the BCA method; the extracted total protein was denatured at 95°C, and the TPK level in the sample was analyzed by Western Blot.
  • Membrane in the region of the size of the target protein TPK is 25-35KD, ⁇ -Actin is 40-55KD). Block the membrane with blocking solution (purchased from Takara, Cat. No.
  • the TDP content in cells infected with TPK lentivirus was significantly higher than that in uninfected cells and control virus-infected cells, while the TM content in AAV-TPK cells was significantly lower than that in uninfected cells and Control virus infected cells (Fig. 3C).
  • the results showed that the overexpression of TPK in cells infected with TPK lentivirus significantly promoted the conversion of TM to TDP, and the conversion rate was about twice that of the control group.
  • the purpose of this example is to verify whether gene therapy can significantly improve the expression of TPK protein in the brain of C57BL/6J mice by injecting AAV virus into the tail vein of mice, and increase the conversion rate of TM/TDP in the brain of mice, thereby enhancing glucose metabolism .
  • Tail vein injection gave each group the AAV-TPK (TPK group) prepared in Example 1 and the control rAAV (except that there was no TPK coding sequence) with a titer of 5.0E+12vg/mL (prepared in normal saline) and TPK-AAV respectively. same) suspension, 200 ⁇ L each.
  • the mouse fix the mouse on the tail vein injection fixator, wipe the tail of the mouse with an alcohol cotton ball for disinfection, use a 1ml syringe to inject 200 ⁇ L of virus suspension into the tail vein of the mouse, and press the dry cotton ball after injection. Spot for 30 seconds to prevent virus spillage.
  • the mice were fed in the experimental area for 4 weeks, and the body weight and food intake of the mice were recorded every week.
  • the heart was perfused with pre-cooled PBS, and then the mouse brain tissue was collected (the cerebellum, brainstem and olfactory bulb parts were removed, and the left and right brains were separated).
  • the right brain was fixed with 4% paraformaldehyde (PFA) for immunofluorescent staining. Specifically, after the right brain was fixed with 4% PFA for 12-24 hours, it was replaced with 30% sucrose for dehydration and sugar precipitation for 2 days. Store at -80°C.
  • the embedded right brain was sliced into 30 ⁇ m thick slices using a cryostat, and the slice angle was along the longitudinal section from the olfactory bulb to the cerebellum (coronal section).
  • the sliced slices were washed 3-5 times with 1X PBS, OCT was washed, and 0.5% Triton was added to rupture the membrane at room temperature for 30 min, and then replaced with 3% BSA (bovine serum albumin) for blocking at room temperature for 1 h, and primary antibody (anti-GFP) was added Antibody, purchased from Avessellabs, Cat. No.
  • GFP-1020 was incubated overnight at 4°C; the primary antibody was recovered, washed 3 times with 1X PBS, 5mins/time, and the secondary antibody Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary was added Antibody, Alexa Fluor 488, (purchased from Invitrogen, product number is A11008), incubate at room temperature in the dark for 2 hours, and finally stick the thin section on a glass slide, after a little drying, add a mounting agent dropwise to seal the section, and use a Nikon Ti2E inverted microscope Imaging to take pictures.
  • Alexa Fluor 488 purchased from Invitrogen, product number is A11008
  • the polypeptide encoded by rAAV virus can be widely expressed in brain tissue.
  • Example 2 Place the left brain or liver tissue (left upper lobe) in 1.5ml of cell lysate (see Example 2), and lyse it in a freezer grinder (70Hz, 60s, 2 times, with an interval of 20s); then centrifuge at 13,000rpm at 4°C 15 minutes, take the supernatant.
  • Western Blot and HPLC analysis were performed as described in Example 2 to detect TPK expression and TDP and TM content in the brain.
  • Example 4 TPK overexpression and TM/TDP conversion rate increase in the mouse brain of intracerebroventricular injection of AAV-TPK
  • the purpose of this example is to verify whether intracerebroventricular injection of AAV-TPK virus in mice can significantly increase the expression of TPK protein in the brain of C57BL/6J mice, and increase the conversion rate of TM/TDP in the brain of mice, thereby enhancing glucose metabolism.
  • the concentration of the virus stock solution used was 1.0E+13vg/1mL, and the volume injected into the lateral ventricle of the mouse was 4.0E+10vg/4 ⁇ L (2 ⁇ L on the left and right sides) per mouse.
  • Adult mice were anesthetized with Shutai 50, and the hair between the ears and eyes was shaved and fixed on the brain stereotaxic instrument.
  • the mouse brain skin was wiped with alcohol cotton balls for disinfection, and the skin was cut with scissors to expose the skull. Dry the surface of the skull until the anterior bregma can be clearly seen.
  • the anterior bregma is the 0-point positioning coordinate (AP: -0.2mm, L/R: ⁇ 1.0mm, H: 2.5mm), mark the drilling point, and use 0.5 Drill the skull with a mm drill, inhale 2 ⁇ L of AAV virus with a 10 ⁇ L microinjection needle, and insert the needle to a depth of 2.5 mm at 0 point on the Z axis with the skull surface as the Z axis. Injection speed: 0.2 ⁇ L/min, stop the needle for 5 minutes, and slowly pull out the injection needle. After the skin was sutured, the mice were put into a cage with a heating pad, and after waking up, they were put into a breeding cage and raised in the experimental area for 4 weeks, and their body weight and food intake were recorded every week.
  • AP -0.2mm
  • TDP and TM content in blood After raising for 4 weeks, collect samples as described in Example 3 and detect TDP and TM content in blood, rAAV disease The expression of the polypeptide encoded by the virus in the brain, the expression level of TPK and the content of TDP and TM in the brain and liver tissue.
  • Example 5 TPK overexpression and TM/TDP conversion rate increase in the brain of mice developed from embryos injected with AAV-TPK into the lateral ventricle
  • the purpose of this example is to verify whether the gene therapy method of directly delivering AAV into the lateral ventricle of fetal mice can significantly increase the expression of TPK in neurons in the brain of C57BL6/J mice, and improve the conversion rate of TM/TDP in neurons, thereby Enhance sugar metabolism.
  • mice After the offspring mice were born, the head of the mice was irradiated with 480nm excitation light to detect the virus injection results, and the mice successfully injected with the virus were retained. After feeding for 21 days, the three mice were treated as described in Example 3 to collect samples and detect the TDP and TM content in the blood, the expression of the polypeptide encoded by the rAAV virus in the brain, and the expression levels of TPK in the brain and liver tissues As well as TDP and TM content; the other 12 mice were weaned and their body weight was recorded, they were housed in separate cages for two weeks, and their body weight was recorded again.
  • mice developed from embryos injected with rAAV gene products encoded by rAAV virus were widely expressed in the cortex and hippocampus, and also expressed in some other brain regions.
  • TPK overexpression and TM/TDP conversion rate increase in neonatal mouse brain stereotaxically injected with AAV-TPK
  • the purpose of this example is to verify whether the direct delivery of AAV-TPK to the neonatal mouse (Postnatal 0day, P0 mouse) lateral ventricle (Intracerebral ventricular, ICV) and hippocampus (Intraparenchymal, IP) can significantly increase the C57BL6/J mouse brain
  • the expression of TPK in inner neurons increases the conversion rate of TM/TDP in neurons, thereby enhancing glucose metabolism.
  • C57BL6/J mice (embryo 18.5days, E18.5) were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., a total of 11 mice. Experiments were performed after neonatal mice were born.
  • Injection site of lateral ventricle 1mm forward from Lambda, 1mm left and right, and 1mm deep.
  • Bilateral intracerebroventricular injection 800nL virus suspension (1.0E+13vg/mL) was injected on each side.
  • Hippocampal injection site 1 0.7mm forward from Lambda, 1.2mm left and right, and 0.9mm deep; hippocampal injection site 2: 0.1mm forward from Lambda, 2.2mm left and right, and 1.4mm deep. Both sides of the hippocampus were injected at 2 sites, a total of 4 injections, each injection of 200nL virus suspension (1.0E+13vg/mL).
  • the body of the newborn mouse was buried in ice, and the nose was not covered so that it could breathe for about 3 mins. After the body turned from pinkish purple to slightly white and no longer struggling, the P0 mice were taken out of the ice and placed on ice for subsequent experiments.
  • each of the 3 mice treated was processed as described in Example 3 to collect samples and detect the TDP and TM content in the blood, the expression of the polypeptide encoded by the rAAV virus in the brain, and the brain and liver tissue
  • the expression level of TPK and the content of TDP and TM; other mice were weaned and their body weight was recorded. They were kept in separate cages for two weeks, and their body weight was recorded again.
  • the inventors compared human TPK and mouse TPK protein structures, selected amino acids that may have a greater impact on TPK activity, and then carried out single point mutations and combined mutations on these amino acids, designed and prepared TPK and its components as shown in Table 2. mutant.
  • nucleotide sequence (SEQ ID NO: 1 and 14-19) encoding the mutated TPK was cloned into the pMCSG7-(+) backbone, and transformed into Escherichia coli BL21 (DE3) competent cells, coated Spread on LB agar medium (containing 50mg/L kanamycin), culture overnight at 37°C, then transfer single colonies to LB liquid medium (containing 50mg/L kanamycin) for cultivation, and sequence verification.
  • Validated clones were activated on LB agar medium. Then, a single colony was inoculated into LB liquid medium (containing 50 mg/L kanamycin), and incubated with shaking at 37° C. for 12 hours. Transfer 1 mL of the culture to 50 mL of fresh LB liquid medium (containing 50 mg/L kanamycin), incubate with shaking at 37 °C until the OD600 reaches about 0.6, add IPTG (final concentration: 0.5 mM) and incubate at 25 °C 16h to induce protein expression.
  • LB liquid medium containing 50 mg/L kanamycin
  • E. coli cells were collected.
  • the collected E. coli cells were resuspended in a pre-cooled protein balance solution (50mM HEPES, 2mM MgCl2, 250mM NaCl, 10% glycerol) at a ratio of 1:8, and the E. coli cells were sonicated at 4°C.
  • the cell lysate was centrifuged at 12,000 rpm at 4°C for 60 min, the supernatant was collected and passed through a nickel column (GE, 17-3712-02), and the nickel column was washed with a 30 mM imidazole solution prepared from protein balance solution to remove non-specifically bound impurities. Protein; use 500mM imidazole solution prepared by protein balance solution to elute the nickel column, collect the eluate containing the target protein; and use Merck Millipore 10KD pore concentration tube to dialyze to remove imidazole until the imidazole concentration is lower than 5mM.
  • the dialyzed product was further purified by FPLC molecular sieves (SEC column: superdex 200increase; PBS buffer, pH 7.4), the target protein solution was collected, and the protein was concentrated with a 10KD pore concentration tube of Merck Millipore.
  • TPK enzyme solution TDP (nM)/mg TPK/min.
  • the variant hTPK 8M showed a significantly increased activity of catalyzing the conversion of TM to TDP compared to human TPK (hTPK) (p ⁇ 0.05), but still lower than that of wild-type mouse TPK (mTPK). Activity (p ⁇ 0.05).
  • the introduced variants mTPK 11W and mTPK 35W showed higher catalytic activity of converting TM into TDP, among which the variant mTPK 11W showed the highest activity (p ⁇ 0.01vs. mTPK); while the introduction of 241W substitution reduced the activity.
  • Example 8 Effects of rAAV encoding TPK variants on the activity of TPK in vivo
  • This example investigates the therapeutic effect of rAAV encoding a TPK variant with increased activity in neonatal mice and APP/PS1 mice.
  • AAV-hTPK its genome comprises: i) neuron-specific promoter (SEQ ID NO: 3), ii) nucleotide sequence (SEQ ID NO: 3) encoding hTPK NO:14) and WPRE element (SEQ ID NO:4).
  • the purpose of this example is to verify whether the direct delivery of AAV-hTPK to the intracerebral ventricle (Intracerebral ventricle, ICV) of newborn mice (Postnatal 0day, P0 mice) can significantly increase the expression of TPK in neurons in the brain of C57BL6/J mice Expression, increase the conversion rate of TM/TDP in neurons, thereby enhancing glucose metabolism.
  • ICV Intracerebral ventricle
  • C57BL6/J mice (embryo 18.5days, E18.5) were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., a total of 11 mice.
  • the total amount of virus stock solution is 2E10vg (800nL), which is diluted to 1/5, 1/25, 1/100, and 1/200 of the stock solution concentration respectively.
  • rAAV not containing the TPK coding sequence was used as a control. Experiments were performed after neonatal mice were born.
  • Injection site of lateral ventricle 1mm forward from Lambda, 1mm left and right, and 1mm deep. Bilateral intracerebroventricular injection, 800nL virus suspension was injected on each side.
  • the body of the newborn mouse was buried in ice, and the nose was not covered so that it could breathe for about 3 mins. After the body turned from pinkish purple to slightly white and no longer struggling, the P0 mice were taken out of the ice and placed on ice for subsequent experiments.
  • mice of each treatment were treated as described in Example 3 to collect samples, and the expression level of TPK and the content of TDP in brain tissue were detected.
  • the expression level of TPK protein in brain tissue showed a dose-dependent change with the titer gradient of the injected virus.
  • the results of HPLC showed that after injecting AAV-hTPK (from stock solution to 100-fold dilution), the content of TDP in brain tissue decreased with the dilution of the virus, showing a dose-dependent effect (Figure 31).
  • AAV-TPK prepared in Example 1 Verify whether the gene therapy method of directly delivering AAV (AAV-TPK prepared in Example 1) to the lateral ventricle (Intracerebral ventricle, ICV) of adult APP/PS1 mice can significantly increase the number of neurons in the brains of APP/PS1 mice
  • the expression of TPK can improve the conversion rate of TM/TDP in neurons and enhance the process of glucose metabolism.
  • the concentration of the virus stock solution used was 1.0E+13vg/1mL, and the volume injected into the lateral ventricle of the mouse was 4.0E+10vg/4 ⁇ L (2 ⁇ L on the left and right sides) per mouse.
  • mice were anesthetized with isoflurane, and the hair between the ears and eyes was shaved and fixed on the brain stereotaxic apparatus.
  • the anterior bregma is the 0-point positioning coordinate (AP: -0.2mm, L/R: ⁇ 1.0mm, H: 2.5mm), mark the drilling point, and use 0.5 Drill the skull with a mm drill, inhale 2 ⁇ L of AAV virus with a 10 ⁇ L microinjection needle, and insert the needle to a depth of 2.5 mm at 0 point on the Z axis with the skull surface as the Z axis.
  • AP -0.2mm
  • L/R ⁇ 1.0mm
  • H 2.5mm
  • Injection speed 0.2 ⁇ L/min, stop the needle for 5 minutes, and slowly pull out the injection needle.
  • the mice were put into a cage with a heating pad, and after waking up, they were put into a breeding cage and raised in the experimental area for at least 8 weeks to wait for the expression of AAV.
  • the body weight, food intake, and blood sugar (fixed time point) were counted every week.
  • mice injected with AAV-TPK were not significantly different from those injected with control AAV, indicating the safety of AAV-TPK injection.
  • the expression level of TPK in the brain tissue of mice injected with AAV-TPK was high, while that of mice injected with control AAV was almost not expressed; while the mice injected with AAV-TPK and the mice injected with control AAV
  • the expression levels of TPK in the livers of mice were comparable, indicating that the AAV of the present invention did not show off-target phenomena.
  • the TDP content in the brain tissue of APP/PS1 mice injected with AAV-TPK was significantly higher than that of APP/PS1 mice injected with control AAV.
  • the TDP content in blood and liver tissue of APP/PS1 mice injected with AAV-TPK was equivalent to that of APP/PS1 mice injected with control AAV, indicating that the AAV of the present invention did not show off-target phenomena.
  • Example 3 After feeding for 6 months, collect samples as described in Example 3 and detect (the difference is only the primary antibody) the level of GSK3 ⁇ protein in the brain tissue (the primary antibody is GSK-3 ⁇ (27C10) (purchased from CST, catalog number 9315) and Phosphorylated GSK-3 ⁇ protein (p-GSK-3 ⁇ ) (Ser9) (the primary antibody was purchased from CST, Cat. No. 14630).
  • the primary antibody is GSK-3 ⁇ (27C10) (purchased from CST, catalog number 9315) and Phosphorylated GSK-3 ⁇ protein (p-GSK-3 ⁇ ) (Ser9) (the primary antibody was purchased from CST, Cat. No. 14630).
  • the level of p-GSK3 ⁇ and the ratio of p-GSK3 ⁇ /GSK3 ⁇ in brain tissue of APP/PS1 mice injected intracerebroventricularly with AAV-TPK were higher than those of APP/PS1 mice injected with control AAV. It is suggested that the "TPK-TDP-glucose metabolism" pathway interacts with the p-GSK3 ⁇ pathway.

Abstract

提供一种重组腺相关病毒(rAAV)或重组慢病毒，其在基因组中包含表达盒，所述表达盒包含与启动子可操作连接的编码硫胺素焦磷酸激酶(TPK)的多核苷酸。还提供包含所述rAAV或重组慢病毒的药物组合物以及用所述rAAV、重组慢病毒和所述药物组合物在制备用于治疗或预防阿尔茨海默病的药物中的用途。

Description

表达TPK的重组病毒及其治疗阿尔茨海默病的用途 技术领域
本发明涉及基因治疗领域。具体而言，本发明涉及病的重组腺相关病毒(rAAV)或重组慢病毒，所述rAAV包含编码硫胺素焦磷酸激酶(TPK)的多核苷酸，所述rAAV或重组慢病毒可以用于治疗阿尔茨海默。
背景技术
阿尔茨海默病(Alzheimer’s disease，AD)是一种严重的神经退行性疾病，多见于老年人，该病的发生伴随着大量神经元凋亡并导致脑实质萎缩。该病的主要临床表现为记忆力障碍，语言能力、空间定向能力降低，精神异常等。目前AD的发病机制仍不清楚，存在多种关于AD机制的假说，如β-淀粉样蛋白(Aβ)级联假说、磷酸化Tau蛋白假说、神经炎性反应假说和氧化应激假说等。在过去几十年，依据这些假说进行了大量研究，但仍未获得有效的治疗方案。
大脑占体重的约2％，但所消耗的能量却占人体消耗的总能量约20％，这说明葡萄糖代谢对于大脑正常行使其功能非常重要。研究发现，AD患者脑中葡萄糖代谢异常的发生早于在其临床症状，并且随着疾病进展，患者脑中葡萄糖代谢水平会逐渐降低。因此，调节脑内细胞的葡萄糖代谢水平有望缓解或终止AD的进展。
通常，调控细胞中的基因表达可通过钙转法、脂质体转染法、电转法和转基因病毒感染等方法来实现。
腺相关病毒(AAV)是在人类中广泛存在的单链DNA病毒，其天然宿主为人类及其它灵长类，目前尚未发现AAV可以致病的报道。重组的腺相关病毒(rAAV)是在AAV基础上进行工程化获得的，因其安全性高、宿主范围广(分裂细胞和非分裂细胞均可感染)、免疫原性低，被广泛用作基因治疗的载体。
FDA分别于2017年和2019年批准了两种基于rAAV的基因治疗产品：Luxturna和Zolgensma。Luxturna由Spark Therapeutics和弗罗里达儿童医院共同开发，用于治疗伯氏先天性黑内障。Luxturna使用AAV2作为载体携带了人源RPE65cDNA，给药方式为视网膜下注射。Zolgensma由诺华公司开发用于治疗小儿脊髓性肌肉萎缩症。Zolgensma使用AAV9作为载体携带运动神经元存活所必需的SMN1基因，给药方式为静脉注射。
维生素B1，又称为硫胺素(TM)，在体内由硫胺素焦磷酸激酶(TPK)转化为具有生物活性的二磷酸硫胺素(TDP)。TDP是葡萄糖代谢中转酮酶、丙酮酸脱氢酶、α-酮戊二酸脱氢酶的必要辅酶。研究表明，AD患者脑组织中转酮酶活性下降超过45％，α-酮戊二酸脱氢酶活性下降超过75％；AD患者全血中TDP水平显著低于正常水平，并且全血中的TDP水平与AD病人脑葡萄糖代谢下降高度相关。已有研究发现AD患者脑组织中TPK mRNA与蛋白表达水平显著低于年龄性别匹配的正常人脑组织，更为重要的是，在认知正常的被研究者脑内，TPK mRNA水平与AD病理评级的Braak评分成正相关，而在AD患者成负相关。结果表明TPK表达可能是一个延缓AD疾病发生和发展的保护性因素。条件性敲除小鼠脑兴奋性神经元中的TPK基因显著导致小鼠脑葡萄糖代谢异常和认知功能损害、突触和神经元丢失导致的脑萎缩、Aβ沉积和斑块形成、Tau异常磷酸化和神经纤维缠结形成、小胶质和星形胶质细胞激活和神经炎症、微血管形成障碍和外周糖代谢调控障碍等所有人类AD疾病的重要多病理生理特征(参见，例如，Sang et al.,Thiamine pyrophosphokinase deficiency induces Alzheimer’s pathology,Biorxiv, 2020；和WO2021023069A1)。
目前，需要开发新的阿尔茨海默病的疗法以解决当下药物缺乏的问题，例如，基于rAAV的基因疗法。
发明内容
在第一方面，本发明提供一种重组腺相关病毒(rAAV)或重组慢病毒，其在基因组中包含表达盒，所述表达盒包含与启动子可操作连接的编码硫胺素焦磷酸激酶(TPK)的多核苷酸。
在一些实施方案中，所述TPK包含SEQ ID NO:1的氨基酸序列。在一些实施方案中，所述多核苷酸包含SEQ ID NO:2的核苷酸序列。
在一些实施方案中，所述启动子是神经元特异性启动子。在一些实施方案中，所述启动子包含SEQ ID NO:3的核苷酸序列。
在一些实施方案中，所述表达盒还包含WPRE元件。在一些实施方案中，所述WPRE元件包含SEQ ID NO:4的核苷酸序列。
在一些实施方案中，所述启动子是真核强启动子，例如巨细胞病毒(CMV)启动子。在一些实施方案中，所述启动子包含SEQ ID NO:7的核苷酸序列。
在一些实施方案中，所述rAAV是血清型AAV_PHP eB的rAAV。
在第二方面，本发明提供一种经修饰的硫胺素焦磷酸激酶(TPK)多肽，与起始TPK多肽相比，包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代，其中位置13的氨基酸取代为P，位置30的氨基酸取代为A，位置31的氨基酸取代为R，位置37的氨基酸取代为K，位置85的氨基酸取代为K，位置129的氨基酸取代为G，位置158的氨基酸取代为K，位置181的氨基酸取代为S，位置11的氨基取代为W，位置35的氨基酸取代为W，其中与起始TPK多肽相比，所述经修饰的TPK多肽具有提高的催化硫胺素(TM)转化为二磷酸硫胺素(TDP)的活性，且其中所述位置参照SEQ ID NO:1或8编号。
在一些实施方案中，所述起始TPK多肽是野生型TPK多肽。在一些实施方案中，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158和181的一或多个位置，优选全部，的氨基酸取代。在一些实施方案中，所述起始TPK多肽是小鼠TPK多肽，且其中所述经修饰的TPK多肽包含位置11和/或35的氨基酸取代的氨基酸取代。在一些实施方案中，所述经修饰的TPK多肽包含SEQ ID NO:9、10或11的氨基酸序列。
在第三方面，本发明还提供一种多核苷酸，其编码本发明的经修饰的TPK多肽。
本发明还提供一种表达盒，其包含与启动子可操作连接的本发明的多核苷酸。在一些实施方案中，所述启动子是神经元特异性启动子。在一些实施方案中，所述启动子包含SEQ ID NO:3的核苷酸序列。在一些实施方案中，所述表达盒还包含WPRE元件。在一些实施方案中，所述WPRE元件包含SEQ ID NO:4的核苷酸序列。
在第四方面，本发明还提供一种rAAV或重组慢病毒，其在基因组中包含本发明第三方面所提供的表达盒。
在一些实施方案中，所述rAAV是血清型AAV_PHP eB的rAAV。
在第五方面，本发明提供一种药物组合物，其包含本发明的经修饰的TPK多肽、多核苷酸、rAAV或重组慢病毒和药学上可接受的载体。在一些实施方案中，所述药物组合物被配制用于静脉内施用、脑内施用或鞘内施用。
在第六方面，本发明提供一种治疗或预防阿尔茨海默病的方法，包括给有需要的对象施用本发明的经修饰的TPK多肽、多核苷酸、rAAV、重组慢病毒或药物组合物。在一些实施方案中，静脉内施用、脑内施用或鞘内施用所述经修饰的TPK多肽、多核苷 酸、rAAV、重组慢病毒或药物组合物。
本发明还提供本发明的经修饰的TPK多肽、多核苷酸、rAAV、重组慢病毒或药物组合物在制备用于治疗或预防阿尔茨海默病的药物中的用途。在一些实施方案中，所述药物是静脉内施用、脑内施用或鞘内施用的。
本发明还提供本发明的经修饰的TPK多肽、多核苷酸、rAAV、重组慢病毒或药物组合物，其用于治疗或预防阿尔茨海默病的用途。在一些实施方案中，所述rAAV、重组慢病毒或药物组合物是静脉内施用、脑内施用或鞘内施用的。
附图说明
图1显示包含AAV-TPK的基因组的质粒图谱(图1A)和包含TPK慢病毒的基因组的质粒图谱(图1B)。
图2显示用慢病毒感染的神经元细胞的荧光显微术的图像。
图3显示用慢病毒感染的神经元细胞中的TPK表达(图3A，parental代表未经病毒感染的细胞)、TDP含量(图3B)和TM含量(图3C)。
图4显示尾静脉注射rAAV的小鼠的体重(左幅和中幅)和摄食量(右幅)。
图5显示尾静脉注射rAAV的小鼠的血液中的TDP(左幅)和TM(右幅)含量。
图6显示尾静脉注射rAAV的小鼠脑中各区域的切片的免疫荧光的图像。
图7显示尾静脉注射rAAV的小鼠脑中TPK表达(左幅)和TDP及TM含量(右幅)。
图8显示尾静脉注射rAAV的小鼠肝脏中的TDP含量(左幅)和TM含量(右幅)。
图9显示侧脑室注射rAAV的小鼠的体重(左幅和中幅)和摄食量(右幅)。
图10显示侧脑室注射rAAV的小鼠血液中的TDP(左幅)和TM(右幅)含量。
图11显示侧脑室注射rAAV的小鼠脑中各区域的切片的免疫荧光的图像。
图12显示侧脑室注射rAAV的小鼠脑中TPK表达(左幅)和TDP及TM含量(右幅)。
图13显示侧脑室注射rAAV的小鼠肝脏中的TDP含量(左幅)和TM含量(右幅)。
图14显示侧脑室注射rAAV的胚胎发育成的小鼠出生21天断乳后的2周内体重变化。
图15显示侧脑室注射rAAV的胚胎发育成的小鼠出生21天后血液中的TDP(左幅)和TM(右幅)含量。
图16显示侧脑室注射rAAV的胚胎发育成的小鼠出生21天后脑中各区域的切片的免疫荧光的图像。
图17显示侧脑室注射rAAV的胚胎发育成的小鼠出生21天后脑中TPK表达(左幅)和TDP及TM含量(右幅)。
图18显示侧脑室注射rAAV的胚胎发育成的小鼠出生21天后肝脏中的TDP含量(左幅)和TM含量(右幅)。
图19显示新生小鼠侧脑室注射rAAV 21天断乳后的2周内体重变化。
图20显示新生小鼠侧脑室注射rAAV 21天后血液中的TDP(左幅)和TM(右幅)含量。
图21显示新生小鼠侧脑室注射rAAV 21天后脑中各区域的切片的免疫荧光的图像。
图22显示新生小鼠侧脑室注射rAAV 21天后脑中TPK表达(左幅)和TDP及TM含量(右幅)。
图23显示新生小鼠海马注射rAAV 21天断乳后的2周内体重变化。
图24显示新生小鼠海马注射rAAV 21天后血液中的TDP(左幅)和TM(右幅)含量。
图25显示新生小鼠海马注射rAAV 21天后脑中各区域的切片的免疫荧光的图像。
图26显示新生小鼠海马注射rAAV 21天后脑中TPK表达。
图27显示新生小鼠脑室注射rAAV 21天后肝脏中的TDP含量(左幅)和TM含量(右幅)。
图28显示经修饰的人TPK与野生型人TPK和小鼠TPK的活性比较。
图29显示经修饰的小鼠TPK与野生型小鼠TPK的活性比较。
图30显示脑组织中TPK蛋白的Western Blot结果。
图31显示脑组织中TDP的含量的HPLC结果。
图32显示注射AAV-hTPK的小鼠体重(A)、摄食量(B)以及血糖(C)的曲线。
图33显示注射AAV-TPK的APP/PS1小鼠脑组织和肝脏中TPK蛋白的Western Blot结果。
图34显示注射AAV-TPK的APP/PS1小鼠脑组织、肝脏和血液中TDP和TM的含量。
图35显示侧脑室注射AAV-TPK的APP/PS1小鼠脑组织中的p-GSK3β和GSK3β的Western Blot结果。
图36显示侧脑室注射AAV-TPK的APP/PS1小鼠脑组织中p-GSK3β/GSK3β的比例。发明详述
一、定义
除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域中普通技术人员通常所理解的相同含义。虽然在本发明的实践和教学中也可使用与本文所述类似或等效的任何方法和材料，但现在描述优选方法和材料。本文提及的所有出版物通过引用并入本文以公开和描述连同出版物一起引用的方法和/或材料。
“硫胺素焦磷酸激酶”或“TPK”是催化硫胺素(TM)转化成二磷酸硫胺素(TDP)的反应的酶。
“AAV”是腺相关病毒的缩写，并且可用于指病毒本身或其衍生物。除非另有需要，所述术语覆盖所有亚型及自然存在和重组形式。缩写“rAAV”指重组腺相关病毒，也称为重组AAV载体(或“rAAV载体”)。术语“AAV”包括1型AAV(AAV-1)、2型AAV(AAV-2)、3型AAV(AAV-3)、4型AAV(AAV-4)、5型AAV(AAV-5)、6型AAV(AAV-6)、7型AAV(AAV-7)、8型AAV(AAV-8)、禽AAV、牛AAV、犬AAV、马AAV、灵长类AAV、非灵长类AAV和羊AAV。“灵长类AAV”指感染灵长类动物的AAV，“非灵长类AAV”指感染非灵长类哺乳动物的AAV，“牛AAV”指感染牛哺乳动物的AAV等。
本领域已知不同亚型AAV的基因组序列，以及天然末端重复序列(TR)、Rep蛋白和衣壳亚基的序列。此类序列可在文献或公用数据库例如基因库中找到。参见，例如，Genbank登记号NC_002077(AAV-1)、AF063497(AAV-1)、NC_001401(AAV-2)、AF043303(AAV-2)、NC_001729(AAV-3)、NC_001829(AAV-4)、U89790(AAV-4)、NC_006152(AAV-5)、AF513851(AAV-7)、AF513852(AAV-8)和NC_006261(AAV-8)；其公开内容通过引用并入本文，用于教导AAV核酸和氨基酸序列。
如本文所使用，“rAAV载体”指包含非AAV来源的多核苷酸序列(即与AAV异源的多核苷酸)，通常是细胞遗传转化的目标序列的AAV载体。一般而言，异源多核苷酸两侧有至少一个，而通常有两个AAV反向末端重复序列(ITR)。术语rAAV载体涵盖rAAV载体颗粒和rAAV载体质粒。rAAV载体可为单链(ssAAV)或自身互补(scAAV)。
“包装”指导致AAV颗粒装配和衣壳化的一系列胞内事件。
AAV“rep”和“cap”基因指编码腺相关病毒的复制和衣壳化蛋白的多核苷酸序列。AAV rep和cap也称为AAV“包装基因”。
AAV的“辅助病毒”指允许哺乳动物细胞复制并包装AAV(例如野生型AAV)的病毒。在本领域中已知AAV的多种此类辅助病毒，包括腺病毒、疱疹病毒和痘病毒(例如牛痘)。虽然C亚类的5型腺病毒最常用，但是腺病毒涵盖许多不同亚类。已知人、非人类哺乳动物和禽类来源的许多腺病毒并且可从保藏机构例如ATCC获得。疱疹病毒科的病毒 包括(例如)单纯疱疹病毒(HSV)和埃-巴二氏病毒(Epstein-Barr viruses)(EBV)以及巨细胞病毒(CMV)和假狂犬病病毒(PRV)；也可从保藏机构例如ATCC获得。
“辅助病毒功能”指辅助病毒基因组中编码的允许AAV复制和包装(连同对本文所述复制和包装的其它要求)的功能。如本文所述，可以多种方式提供“辅助病毒功能”，包括通过提供辅助病毒或为生产细胞提供(例如)编码必需功能的反式多核苷酸序列。例如，将包含编码一种或多种腺病毒蛋白的核苷酸序列的质粒(也称为辅助质粒)或其它表达载体连同rAAV载体一起转染至生产细胞中。
慢病毒载体是衍生自HIV-1的病毒载体。“重组慢病毒载体”或“重组慢病毒载体颗粒”是指在质粒载体转染后宿主细胞或生产细胞中产生的重组病毒颗粒和重组病毒样颗粒，所述质粒载体由转移载体、编码选定包膜蛋白的包膜载体和根据本领域熟知方法反式提供慢病毒蛋白(如慢病毒GAG和POL蛋白，特别是突变的POL蛋白以防止整合)的包装载体组成。
病毒样颗粒由呈现用于重组慢病毒基因组衣壳化的蛋白不完整组装产生，其采用的方式不能形成真病毒颗粒。
术语“多核苷酸”指任何长度的核苷酸聚合形式，包括脱氧核糖核苷酸或核糖核苷酸或其类似物。多核苷酸可包含经修饰的核苷酸，例如甲基化核苷酸和核苷酸类似物，并且可被非核苷酸组分中断。若存在，可在聚合物装配之前或之后给予对核苷酸结构的修饰。如本文所使用，术语多核苷酸可交替地指双链和单链分子。除非另有说明或要求，本文所述为多核苷酸的本发明任何实施方案涵盖双链形式和已知或预测构成双链形式的两种互补单链形式的每一种。
核酸杂交反应可在不同“严格性”的条件下进行。增强杂交反应的严格性的条件众所周知并且在本领域中公开。见，例如，通过引用并入本文的Sambrook等Molecular Cloning,A Laboratory Manual，第2版，Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989。
多核苷酸或多肽与另一多核苷酸或多肽具有一定百分比的“序列相同性”，是指比对时，该百分比的碱基或氨基酸在比较两个序列时相同。可以许多不同方式测定序列相似性。为测定序列相同性，可用于比对序列的方法和计算机程序包括，例如，BLAST，可在ncbi.nlm.nih.gov/BLAST/获得，和FASTA，可在Genetics Computing Group(GCG)包中获得，其来自Oxford Molecular Group,Inc.的全资子公司Madison，Wisconsin，USA。
“基因”指含有至少一个能够在转录和翻译后编码特定蛋白的开放阅读框的多核苷酸。
如应用于多核苷酸，“重组”意指多核苷酸是克隆、限制或连接步骤的不同组合和产生不同于在自然界中发现的多核苷酸的构建体的其它程序的产物。重组病毒是包含重组多核苷酸的病毒颗粒。所述术语分别包括原多核苷酸构建体和原病毒构建体子代的复制品。
“控制元件”或“控制序列”是参与分子相互作用，有助于多核苷酸的功能调控，包括多核苷酸的复制、重复、转录、剪接、翻译或降解的核苷酸序列。调控可影响所述过程的频率、速度或特异性，并且实际上可为增强性或抑制性。本领域已知的控制元件包括(例如)转录调控序列例如启动子和增强子。启动子是在某些条件下能够结合RNA聚合酶并且引发通常位于启动子下游(3’方向)的编码区域转录的DNA区域。
“可操作地连接”指遗传元件的并列，其中所述元件呈允许其以预期方式操作的关系。例如，如果启动子帮助起始编码序列的转录，则启动子与编码区操作性连接。只要维持这种功能关系，在启动子和编码区域之间可能有插入残基。
“表达载体”是包含编码目标多肽的区域的载体，并且用于在预定的靶细胞中实现蛋白质的表达。表达载体还包含与编码区域操作性连接以促进蛋白质在靶标中的表达的控 制元件。控制元件和与之可操作地连接以便表达的基因的组合有时称为“表达盒”，许多表达盒在本领域中已知并且可获得或可易于由在本领域可获得的组分构建。
“异源”指源自和与之比较的其余实体基因型不同的实体。例如，通过基因工程技术引入源自不同物种的质粒或载体中的多核苷酸为异源多核苷酸。取自其天然编码序列并且与并非发现与之天然连接的编码序列操作性连接的启动子为异源启动子。因此，例如，包括编码异源基因产物的异源核酸的rAAV是包括在自然存在的野生型AAV中一般不包括的核酸的rAAV，并且编码的异源基因产物是一般并非自然存在的野生型AAV编码的基因产物。
术语“多肽”、“肽”和“蛋白质”在本文中可交换用于指任何长度的氨基酸聚合物。所述术语还涵盖经修饰的氨基酸聚合物；例如，二硫键形成、糖基化、脂化、磷酸化或与标记组分结合。
所述修饰也包括对多肽序列的修饰，包括但不限于，取代、删除、插入和/或添加一或多个氨基酸。
术语“保守取代”也称为由“同源”氨基酸残基取代，是指其中氨基酸残基由具有相似侧链的氨基酸残基置换的取代，例如，碱性侧链的氨基酸(例如赖氨酸、精氨酸和组氨酸)、酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、非荷电极性侧链氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及芳香侧链氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
保守氨基酸取代通常对所得蛋白质的活性的影响最小。这种取代在下文描述。保守取代是用大小、疏水性、电荷、极性、空间特征、芳香性等相似的氨基酸置换一个氨基酸。当希望精细调节蛋白质的特性时，这种取代通常是保守的。
如本文所用，“同源”氨基酸残基是指具有相似化学性质的氨基酸残基，所述化学性质涉及疏水性、电荷、极性、空间特征、芳香性特征等。彼此同源的氨基酸的例子包括正电荷的赖氨酸、精氨酸、组氨酸，负电荷的谷氨酸、天冬氨酸，疏水性的甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸，极性的丝氨酸、苏氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、天冬酰胺、谷氨酰胺，芳香性的苯丙氨酸、酪氨酸、色氨酸，化学相似侧链基团的丝氨酸与苏氨酸，或者谷氨酰胺和天冬酰胺，或者亮氨酸和异亮氨酸。
蛋白质中氨基酸保守取代的例子包括：Ser取代Ala，Lys取代Arg，Gln或His取代Asn，Glu取代Asp，Ser取代Cys，Asn取代Gln，Asp取代Glu，Pro取代Gly，Asn或Gln取代His，Leu或Val取代Ile，Ile或Val取代Leu，Arg或Gln取代Lys，Leu或Ile取代Met，Met、Leu或Tyr取代Phe，Thr取代Ser，Ser取代Thr，Tyr取代Trp，Trp或Phe取代Tyr，及Ile或Leu取代Val。
“分离的”质粒、核酸、载体、病毒、病毒体、宿主细胞或其它物质指没有在所述物质或类似物质自然存在或最初制备时也可能存在的至少一些其它组分的物质的制剂。因此，例如，可使用纯化计数制备分离物质以使其从源混合物中富集。
如本文所使用，术语“治疗”指获得需要的药理学和/或生理学作用。所述作用在完全或部分预防疾病或其症状方面可为预防性和/或在对疾病和/或可归因于所述疾病的副作用的部分或完全治愈方面可为治疗性。如本文所使用，“治疗”包括对哺乳动物(特别是人类)疾病的任何治疗，并且包括：(a)预防疾病在可能易患病或有患病风险但尚未诊断为患病的对象中出现；(b)抑制疾病，即阻止其发展；和(c)缓解疾病，即引起疾病消退。
术语“个体”、“宿主”、“对象”和“患者”在本文中可交换使用，并且指哺乳动物，包 括但不限于人和非人灵长类动物，包括猿和人；哺乳类运动动物(例如，马)；哺乳类家畜(例如，绵羊、山羊等)；哺乳类宠物(狗、猫等)；和啮齿动物(例如，小鼠、大鼠等)。
二、经修饰的TPK多肽
发明人发现，小鼠硫胺素焦磷酸激酶(mTPK)的催化TM转化为TDP的活性高于人TPK(hTPK)。发明人对hTPK和mTPK序列进行了修饰，以提高催化TM转化为TDP的活性。例如，将hTPK的表面电荷性质进行鼠源化修饰，即当hTPK中的残基Xh与mTPK中匹配的位置的残基Xm的电荷性质不同时，将hTPK中的残基Xh取代为Xm。
因此，本发明提供一种经修饰的硫胺素焦磷酸激酶(TPK)多肽，与起始TPK多肽相比，包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代，其中与起始TPK多肽相比，所述经修饰的TPK多肽具有提高的催化TM转化为TDP的活性，且其中所述位置参照SEQ ID NO:1或8编号。
优选地，位置13的氨基酸取代为P。优选地，位置30的氨基酸取代为A。优选地，位置31的氨基酸取代为R。优选地，位置37的氨基酸取代为K。优选地，位置85的氨基酸取代为K。优选地，位置129的氨基酸取代为G。优选地，位置158的氨基酸取代为K。优选地，位置181的氨基酸取代为S。优选地，位置11的氨基取代为W。优选地，位置35的氨基酸取代为W。
在本文中，在其基础上进行氨基酸修饰的TPK多肽称为起始TPK多肽。所述起始TPK多肽可以是野生型TPK多肽，也可以是野生型TPK多肽的变体。例如，从SEQ ID NO:1的多肽开始进行修饰，则相对于经修饰的TPK多肽，SEQ ID NO:1的多肽是“起始TPK多肽”；而如果从SEQ ID NO:1的变体多肽开始进行修饰，则相对于经修饰的TPK多肽，所述变体多肽是“起始TPK多肽”。
在一些实施方案中，所述起始TPK多肽是野生型TPK多肽，例如人TPK多肽和小鼠TPK多肽。
在一些实施方案中，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代的氨基酸取代。在一些实施方案中，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158和181的一或多个位置的氨基酸取代的氨基酸取代。优选地，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含位置13、30、31、37、85、129、158和181的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽是小鼠TPK多肽，且其中所述经修饰的TPK多肽包含位置11和/或35的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:8的氨基酸序列或与SEQ ID NO:8具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置，例如位置13、30、31、37、85、129、158和181，位置13、30、31、37、85、129、158、181和11，位置13、30、31、37、85、129、158、181和35，或位置13、30、31、37、85、129、158、181、11和35的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置11W和/或35W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置11W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置35W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述所述经修饰的TPK多肽包含以下氨基酸取代或氨基酸取代的组合：
11W；
35W；
13P-30A-31R-37K-85K-129G-158K-181S；
13P-30A-31R-37K-85K-129G-158K-181S-11W；
13P-30A-31R-37K-85K-129G-158K-181S-35W；或
13P-30A-31R-37K-85K-129G-158K-181S-11W-35W。
在一些实施方案中，所述经修饰的TPK多肽包含SEQ ID NO:9、10或11的氨基酸序列或与SEQ ID NO:9、10或11具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列。例如，与SEQ ID NO:9、10或11相比，所述经修饰的TPK多肽包含一或多个氨基酸的取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:9的氨基酸序列相比，在除位置13、30、31、37、85、129、158和181以及，优选地，241之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:10的氨基酸序列相比，在除位置11和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:11的氨基酸序列相比，在除位置35和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽催化TM转化为TDP的活性是所述起始TPK多肽的至少110％、120％、130％、140％、150％、200％、250％、300％或更高。
三、分离的多核苷酸和表达盒
本发明提供一种分离的多核苷酸，其编码硫胺素焦磷酸激酶(TPK)的多核苷酸。
所述TPK可以是来自人或非人动物，如非人哺乳动物的TPK。来自非人哺乳动物的TPK的实例包括但不限于来自非人灵长类动物如猴、马、牛、绵羊、山羊、猪、狗、猫、小鼠、大鼠的TPK。
在一些实施方案中，所述TPK是野生型TPK。在一些实施方案中，所述TPK是小鼠TPK或人TPK。
在一些实施方案中，所述TPK包含SEQ ID NO:1的氨基酸序列。在一些实施方案中，编码所述TPK的多核苷酸包含SEQ ID NO:2的核苷酸序列。在一些实施方案中，所述TPK由SEQ ID NO:1的氨基酸序列组成。在一些实施方案中，编码所述TPK的多核苷酸由SEQ ID NO:2的核苷酸序列组成。
在一些实施方案中，所述TPK是经修饰的TPK，其与起始TPK相比，包含一或多 个氨基酸的取代、插入、缺失和/或添加，其中所述经修饰的TPK的活性是所述起始TPK的至少10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、110％、120％、130％、140％、150％、200％、250％、300％或更高。在一些实施方案中，与起始TPK相比，所述经修饰的TPK包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸的取代、插入、缺失和/或添加。在一些实施方案中，所述经修饰的TPK与所述起始TPK具有至少80％、90％、95％、96％、97％、98％、99％或更高的相同性。
所述起始TPK可以是野生型TPK或经修饰的TPK。在一些实施方案中，所述起始TPK是野生型小鼠TPK或野生型人TPK。
在一些实施方案中，所述起始TPK包含SEQ ID NO:1的氨基酸序列。在一些实施方案中，所述起始TPK由SEQ ID NO:1的氨基酸序列组成。
在一些实施方案中，与起始TPK多肽相比，所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代，其中与起始TPK多肽相比，所述经修饰的TPK多肽具有提高的催化TM转化为TDP的活性，且其中所述位置参照SEQ ID NO:1或8编号。
优选地，位置13的氨基酸取代为P。优选地，位置30的氨基酸取代为A。优选地，位置31的氨基酸取代为R。优选地，位置37的氨基酸取代为K。优选地，位置85的氨基酸取代为K。优选地，位置129的氨基酸取代为G。优选地，位置158的氨基酸取代为K。优选地，位置181的氨基酸取代为S。优选地，位置11的氨基取代为W。优选地，位置35的氨基酸取代为W。
在一些实施方案中，所述起始TPK多肽是野生型TPK多肽，例如人TPK多肽和小鼠TPK多肽。
在一些实施方案中，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代的氨基酸取代。在一些实施方案中，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158和181的一或多个位置的氨基酸取代的氨基酸取代。优选地，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含位置13、30、31、37、85、129、158和181的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽是小鼠TPK多肽，且其中所述经修饰的TPK多肽包含位置11和/或35的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:8的氨基酸序列或与SEQ ID NO:8具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置，例如位置13、30、31、37、85、129、158和181，位置13、30、31、37、85、129、158、181和11，位置13、30、31、37、85、129、158、181和35，或位置13、30、31、37、85、129、158、181、11和35的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置11W和/或35W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始 TPK多肽相比，所述经修饰的TPK多肽包含位置11W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置35W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述所述经修饰的TPK多肽包含以下氨基酸取代或氨基酸取代的组合：
11W；
35W；
13P-30A-31R-37K-85K-129G-158K-181S；
13P-30A-31R-37K-85K-129G-158K-181S-11W；
13P-30A-31R-37K-85K-129G-158K-181S-35W；或
13P-30A-31R-37K-85K-129G-158K-181S-11W-35W。
在一些实施方案中，所述经修饰的TPK多肽包含SEQ ID NO:9、10或11的氨基酸序列或与SEQ ID NO:9、10或11具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列。例如，与SEQ ID NO:9、10或11相比，所述经修饰的TPK多肽包含一或多个氨基酸的取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:9的氨基酸序列相比，在除位置13、30、31、37、85、129、158和181以及，优选地，241之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:10的氨基酸序列相比，在除位置11和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:11的氨基酸序列相比，在除位置35和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽催化TM转化为TDP的活性是所述起始TPK多肽的至少110％、120％、130％、140％、150％、200％、250％、300％或更高。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:15、16或17的核苷酸序列，或与SEQ ID NO:15、16或17具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:15的核苷酸序列，或与SEQ ID NO:15具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。在一些实施方案中，所述多核苷酸包含SEQ ID NO:15，或与SEQ ID NO:15具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列，且所述核苷酸序列编码与SEQ ID NO:9相比，在除位置13、30、31、37、85、129、158和181以及，优选地，241之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代的氨基酸序列。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:16的核苷酸序列，或与SEQ ID NO:16具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。在一些实施方案中，所述多核苷酸包含SEQ ID NO:16，或与SEQ ID NO:16具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列，且所述核苷酸序列编码与SEQ ID NO:10相比，在除位置11和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、 3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代的氨基酸序列。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:17的核苷酸序列，或与SEQ ID NO:17具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。在一些实施方案中，所述多核苷酸包含SEQ ID NO:17，或与SEQ ID NO:17具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列，且所述核苷酸序列编码与SEQ ID NO:11相比，在除位置35和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代的氨基酸序列。
在一些实施方案中，所述多核苷酸是DNA或RNA，如mRNA。
本发明还提供一种表达盒，所述表达盒包含与启动子可操作连接的本发明的多核苷酸。
在一些实施方案中，所述启动子是神经元特异性启动子。在一些实施方案中，所述启动子包含SEQ ID NO:3的核苷酸序列。在一些实施方案中，所述启动子由SEQ ID NO:3的核苷酸序列组成。在一些实施方案中，所述启动子包含与SEQ ID NO:3具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述启动子由与SEQ ID NO:3具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述启动子是真核强启动子，例如巨细胞病毒(CMV)启动子。在一些实施方案中，所述启动子包含SEQ ID NO:7的核苷酸序列。在一些实施方案中，所述启动子由SEQ ID NO:7的核苷酸序列组成。在一些实施方案中，所述启动子包含与SEQ ID NO:7具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述启动子由与SEQ ID NO:7具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
表达盒还可以包含另外的控制序列，如增强子、内含子、mRNA稳定化序列和聚腺苷酸化信号序列。
在一些实施方案中，所述表达盒还包含mRNA稳定化序列，如WPRE元件。在一些实施方案中，所述WPRE元件包含SEQ ID NO:4的核苷酸序列。在一些实施方案中，所述WPRE元件由SEQ ID NO:4的核苷酸序列组成。在一些实施方案中，所述WPRE元件包含与SEQ ID NO:4具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述WPRE元件由与SEQ ID NO:4具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述WPRE元件在编码TPK的多核苷酸的下游。
在一些实施方案中，所述表达盒还包含聚腺苷酸化信号序列，例如，但不限于，人生长因子基因的聚腺苷酸化信号序列和兔球蛋白基因的聚腺苷酸化信号序列。在一些实施方案中，所述表达盒还包含人生长因子基因的聚腺苷酸化信号序列。在一些实施方案中，所述聚腺苷酸化信号序列包含SEQ ID NO:5的核苷酸序列。在一些实施方案中，所述聚腺苷酸化信号序列由SEQ ID NO:5的核苷酸序列组成。在一些实施方案中，所述聚腺苷酸化信号序列包含与SEQ ID NO:5具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述聚腺苷酸化信号序列由与SEQ ID NO:5具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述聚腺苷酸化信号序列在WPRE元件的下游。
所述表达盒可以是rAAV的基因组的部分。因此，在一些实施方案中，所述表达盒侧翼为AAV的ITR。所述ITR的非限制性实例包括衍生自AAV-1、AAV-2、AAV-3、 AAV-4、AAV-5、AAV-6、AAV-7、AAV-8、AAV-9、禽AAV、牛AAV、犬AAV、马AAV、灵长类AAV、非灵长类AAV和羊AAV的ITR。在一些实施方案中，所述ITR是AAV-2的ITR。在一些实施方案中，5’ITR和3’ITR相同。在一些实施方案中，5’ITR和3’ITR不同。
在一些实施方案中，5’ITR和3’ITR都是ITR130，例如，包含SEQ ID NO:6的核苷酸序列。在一些实施方案中，5’ITR和3’ITR分别由SEQ ID NO:6的核苷酸序列组成。在一些实施方案中，5’ITR和3’ITR分别包含与SEQ ID NO:6具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，5’ITR和3’ITR分别由与SEQ ID NO:6具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:14、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:14、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:15、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:15、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:16、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:16、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:17、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:17、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:14、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:16、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:17、SEQ ID NO:4、SEQ ID NO:5。
本发明还提供包含本发明的多核苷酸或表达盒的载体。在一些实施方案中，所述载体是表达载体。在一些实施方案中，所述载体是真核表达载体。
在一些实施方案中，所述载体是在rAAV包装中提供rAAV基因组的载体，即转基因质粒。
在一些实施方案中，所述载体是在慢病毒包装中提供慢病毒基因组的载体，即转基 因质粒。
四、重组病毒
本发明提供一种重组腺相关病毒(rAAV)或重组慢病毒，其在基因组中包含表达盒，所述表达盒包含与启动子可操作连接的编码TPK的多核苷酸。
所述TPK可以是来自人或非人动物，如非人哺乳动物的TPK。来自非人哺乳动物的TPK的实例包括但不限于来自非人灵长类动物如猴、马、牛、绵羊、山羊、猪、狗、猫、小鼠、大鼠的TPK。
在一些实施方案中，所述TPK是野生型TPK。在一些实施方案中，所述TPK是小鼠TPK或人TPK。
在一些实施方案中，所述TPK包含SEQ ID NO:1的氨基酸序列。在一些实施方案中，编码所述TPK的多核苷酸包含SEQ ID NO:2的核苷酸序列。在一些实施方案中，所述TPK由SEQ ID NO:1的氨基酸序列组成。在一些实施方案中，编码所述TPK的多核苷酸由SEQ ID NO:2的核苷酸序列组成。
在一些实施方案中，所述TPK是经修饰的TPK，其与起始TPK相比，包含一或多个氨基酸的取代、插入、缺失和/或添加，其中所述经修饰的TPK的活性是所述起始TPK的至少10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、110％、120％、130％、140％、150％、200％、250％、300％或更高。在一些实施方案中，与起始TPK相比，所述经修饰的TPK包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸的取代、插入、缺失和/或添加。在一些实施方案中，所述经修饰的TPK与所述起始TPK具有至少80％、90％、95％、96％、97％、98％、99％或更高的相同性。
所述起始TPK可以是野生型TPK或经修饰的TPK。在一些实施方案中，所述起始TPK是野生型小鼠TPK或野生型人TPK。
在一些实施方案中，所述起始TPK包含SEQ ID NO:1的氨基酸序列。在一些实施方案中，所述起始TPK由SEQ ID NO:1的氨基酸序列组成。
在一些实施方案中，与起始TPK多肽相比，所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代，其中与起始TPK多肽相比，所述经修饰的TPK多肽具有提高的催化TM转化为TDP的活性，且其中所述位置参照SEQ ID NO:1或8编号。
优选地，位置13的氨基酸取代为P。优选地，位置30的氨基酸取代为A。优选地，位置31的氨基酸取代为R。优选地，位置37的氨基酸取代为K。优选地，位置85的氨基酸取代为K。优选地，位置129的氨基酸取代为G。优选地，位置158的氨基酸取代为K。优选地，位置181的氨基酸取代为S。优选地，位置11的氨基取代为W。优选地，位置35的氨基酸取代为W。
在一些实施方案中，所述起始TPK多肽是野生型TPK多肽，例如人TPK多肽和小鼠TPK多肽。
在一些实施方案中，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代的氨基酸取代。在一些实施方案中，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158和181的一或多个位置的氨基酸取代的氨基酸取代。优选地，所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含位置13、30、31、37、85、129、158和181的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽是小鼠TPK多肽，且其中所述经修饰的TPK多肽包含位置11和/或35的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:8的氨基酸序列或与SEQ ID NO:8具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置，例如位置13、30、31、37、85、129、158和181，位置13、30、31、37、85、129、158、181和11，位置13、30、31、37、85、129、158、181和35，或位置13、30、31、37、85、129、158、181、11和35的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置11W和/或35W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置11W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述起始TPK多肽包含SEQ ID NO:1的氨基酸序列或与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列，其中与所述起始TPK多肽相比，所述经修饰的TPK多肽包含位置35W的氨基酸取代的氨基酸取代。
在一些实施方案中，所述所述经修饰的TPK多肽包含以下氨基酸取代或氨基酸取代的组合：
11W；
35W；
13P-30A-31R-37K-85K-129G-158K-181S；
13P-30A-31R-37K-85K-129G-158K-181S-11W；
13P-30A-31R-37K-85K-129G-158K-181S-35W；或
13P-30A-31R-37K-85K-129G-158K-181S-11W-35W。
在一些实施方案中，所述经修饰的TPK多肽包含SEQ ID NO:9、10或11的氨基酸序列或与SEQ ID NO:9、10或11具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性的氨基酸序列。例如，与SEQ ID NO:9、10或11相比，所述经修饰的TPK多肽包含一或多个氨基酸的取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:9的氨基酸序列相比，在除位置13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:10的氨基酸序列相比，在除位置11和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽与SEQ ID NO:11的氨基酸序列相比，在除位置35和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代。
在一些实施方案中，所述经修饰的TPK多肽催化TM转化为TDP的活性是所述起始TPK多肽的至少110％、120％、130％、140％、150％、200％、250％、300％或更高。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:15、16或17的核苷酸序列， 或与SEQ ID NO:15、16或17具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:15的核苷酸序列，或与SEQ ID NO:15具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。在一些实施方案中，所述多核苷酸包含SEQ ID NO:15，或与SEQ ID NO:15具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列，且所述核苷酸序列编码与SEQ ID NO:9相比，在除位置13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代的氨基酸序列。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:16的核苷酸序列，或与SEQ ID NO:16具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。在一些实施方案中，所述多核苷酸包含SEQ ID NO:16，或与SEQ ID NO:16具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列，且所述核苷酸序列编码与SEQ ID NO:10相比，在除位置11和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代的氨基酸序列。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:17的核苷酸序列，或与SEQ ID NO:17具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列。在一些实施方案中，所述多核苷酸包含SEQ ID NO:17，或与SEQ ID NO:17具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性的核苷酸序列，且所述核苷酸序列编码与SEQ ID NO:11相比，在除位置35和241以及，优选地，13、30、31、37、85、129、158和181之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代，优选保守取代的氨基酸序列。
在一些实施方案中，所述启动子是神经元特异性启动子。在一些实施方案中，所述启动子包含SEQ ID NO:3的核苷酸序列。在一些实施方案中，所述启动子由SEQ ID NO:3的核苷酸序列组成。在一些实施方案中，所述启动子包含与SEQ ID NO:3具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述启动子由与SEQ ID NO:3具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述启动子是真核强启动子，例如巨细胞病毒(CMV)启动子。在一些实施方案中，所述启动子包含SEQ ID NO:7的核苷酸序列。在一些实施方案中，所述启动子由SEQ ID NO:7的核苷酸序列组成。在一些实施方案中，所述启动子包含与SEQ ID NO:7具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述启动子由与SEQ ID NO:7具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
表达盒还可以包含另外的控制序列，如增强子、内含子、mRNA稳定化序列和聚腺苷酸化信号序列。
在一些实施方案中，所述表达盒还包含mRNA稳定化序列，如WPRE元件。在一些实施方案中，所述WPRE元件包含SEQ ID NO:4的核苷酸序列。在一些实施方案中，所述WPRE元件由SEQ ID NO:4的核苷酸序列组成。在一些实施方案中，所述WPRE元件包含与SEQ ID NO:4具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述WPRE元件由与SEQ ID NO:4具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述WPRE元件在编码TPK的多核苷酸的下游。
在一些实施方案中，所述表达盒还包含聚腺苷酸化信号序列，例如，但不限于，人生长因子基因的聚腺苷酸化信号序列和兔球蛋白基因的聚腺苷酸化信号序列。在一些实施方案中，所述表达盒还包含人生长因子基因的聚腺苷酸化信号序列。在一些实施方案中，所述人生长因子基因的聚腺苷酸化信号序列包含SEQ ID NO:5的核苷酸序列。在一些实施方案中，所述人生长因子基因的聚腺苷酸化信号序列由SEQ ID NO:5的核苷酸序列组成。在一些实施方案中，所述人生长因子基因的聚腺苷酸化信号序列包含与SEQ ID NO:5具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，所述人生长因子基因的聚腺苷酸化信号序列由与SEQ ID NO:5具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述聚腺苷酸化信号序列在WPRE元件的下游。
所述rAAV的基因组中的ITR的非限制性实例包括衍生自AAV-1、AAV-2、AAV-3、AAV-4、AAV-5、AAV-6、AAV-7、AAV-8、AAV-9、禽AAV、牛AAV、犬AAV、马AAV、灵长类AAV、非灵长类AAV和羊AAV的ITR。在一些实施方案中，所述ITR是AAV-2的ITR。在一些实施方案中，所述ITR是AAV-2的ITR。在一些实施方案中，5’ITR和3’ITR相同。在一些实施方案中，5’ITR和3’ITR不同。
在一些实施方案中，5’ITR和3’ITR都是ITR130，例如，包含SEQ ID NO:6的核苷酸序列。在一些实施方案中，5’ITR和3’ITR分别由SEQ ID NO:6的核苷酸序列组成。在一些实施方案中，5’ITR和3’ITR分别包含与SEQ ID NO:6具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列。在一些实施方案中，5’ITR和3’ITR分别由与SEQ ID NO:6具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高相同性的核苷酸序列组成。
在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:14、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:14、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:15、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:15、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:16、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:16、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述表达盒包含，从5’到3’，SEQ ID NO:3、SEQ ID NO:17、SEQ ID NO:4、SEQ ID NO:5。在一些实施方案中，所述rAAV的基因组包含，从5’到3’，SEQ ID NO:6、SEQ ID NO:3、SEQ ID NO:17、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6。
在一些实施方案中，所述重组慢病毒的基因组包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述重组慢病毒的基因组包含，从5’到3’，SEQ ID NO:7、 SEQ ID NO:14、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述重组慢病毒的基因组包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述重组慢病毒的基因组包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:16、SEQ ID NO:4、SEQ ID NO:5。
在一些实施方案中，所述重组慢病毒的基因组包含，从5’到3’，SEQ ID NO:7、SEQ ID NO:17、SEQ ID NO:4、SEQ ID NO:5。
所述rAAV可以是AAV-1、AAV-2、AAV-3、AAV-4、AAV-5、AAV-6、AAV-7、AAV-8、AAV-9、禽AAV、牛AAV、犬AAV、马AAV、灵长类AAV、非灵长类AAV和羊AAV。在一些实施方案中，所述rAAV是血清型AAV_PHP eB的rAAV。
本领域已知用包含三个质粒的系统包装rAAV，所述三个质粒包括：1)转基因质粒，其包含编码感兴趣的基因产物的rAAV的基因组；ii)包装质粒，其编码REP和/或CAP蛋白；和iii)辅助质粒(参见，例如，Crosson SM et al.,Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation.Mol Ther Methods Clin Dev.2018；10:1-7)。所述包装涉及将所述系统引入宿主细胞。
本领域已知由转移载体(转基因质粒)、编码选定包膜蛋白的包膜载体和根据本领域熟知方法反式提供慢病毒蛋白(如慢病毒GAG和POL蛋白，特别是突变的POL蛋白以防止整合)的包装载体组成的慢病毒包装系统。慢病毒的包转涉及将所述系统引入宿主细胞。
本发明还提供包含本发明的多核苷酸、载体、rAAV或重组慢病毒的宿主细胞。用于包装rAAV或重组慢病毒病毒体时，所述宿主细胞称为“包装细胞”。在一些实施方案中，所述宿主细胞经本发明的多核苷酸或载体稳定地遗传修饰。在其它实施方案中，所述宿主细胞经本发明的多核苷酸或载体暂时地遗传修饰。
使用确定技术，包括但不限于电穿孔、磷酸钙沉淀、脂质体介导的转染等，稳定或暂时地将本发明的多核苷酸或载体引入宿主细胞中。为了稳定转化，本发明的多核苷酸或载体通常将进一步包括可选择标记，例如几种众所周知的可选择标记的任一种，例如新霉素抗性等。
本发明的宿主细胞可以衍生自哺乳动物细胞。适合的哺乳动物细胞包括但不限于原代细胞和细胞系，其中适合的细胞系包括但不限于293细胞、COS细胞、HeLa细胞、Vero细胞、3T3小鼠成纤维细胞、C3H10T1/2成纤维细胞、CHO细胞等。适合宿主细胞的非限制性实例包括(例如)HeLa细胞(例如美国典型培养物保藏中心(ATCC)No.CCL-2)、CHO细胞(例如，ATCC No.CRL9618、CCL61、CRL9096)、293细胞(例如，ATCC No.CRL-1573)、Vero细胞、N1H3T3细胞(例如ATCC No.CRL-1658)、Huh-7细胞、BHK细胞(例如ATCC No.CCL10)、PC12细胞(ATCC No.CRL1721)、COS细胞、COS-7细胞(ATCC No.CRL1651)、RAT1细胞、小鼠L细胞(ATCC No.CCL1.3)、人胚肾(HEK)细胞(ATCC No.CRL1573)、HLHepG2细胞等。
五、药物组合物
本发明提供了一种药物组合物，包含本发明的多核苷酸、载体、rAAV或重组慢病毒，和药学上可接受的载体、稀释剂、赋形剂或缓冲液。在一些实施方案中，药学上可接受的载体、稀释剂、赋形剂或缓冲液适合用于人类。
此类赋形剂、载体、稀释剂和缓冲液包括可施用而无异常毒性的任何药剂。药学上可接受的赋形剂包括但不限于液体，例如水、盐水、甘油和乙醇。其中可包括药学上可接受的盐，例如矿物酸盐例如盐酸盐、氢溴酸盐、磷酸盐、硫酸盐等；和有机酸的盐例如醋酸盐、丙酸盐、丙二酸盐、苯甲酸盐等。另外，在此类载剂中可存在辅助物质，例 如润湿剂或乳化剂、pH缓冲物质等。在本领域中已知多种多样的药学上可接受的赋形剂而不需要在本文中详细讨论。在多种出版物中已经详尽地描述了药学上可接受的赋形剂，包括(例如)A.Gennaro(2000)“Remington:The Science and Practice of Pharmacy,”20th Edition,Lippincott,Williams,&Wilkins；Pharmaceutical Dosage Forms and Drug Delivery Systems(1999)Editing by HC Ansel et al,7th edition,Lippincott,Williams,&Wilkins；和Handbook of Pharmaceutical Excipients(2000)AH Kibbe et al.,3rd edition,Amer.Pharmaceutical Assoc。
六、预防和治疗疾病
本发明提供一种促进糖代谢，或预防或治疗葡萄糖代谢障碍，优选脑中的葡萄糖代谢障碍的方法，包括给有需要的对象或患者施用本发明的多核苷酸、载体、rAAV、重组慢病毒或药物组合物。本发明提供一种治疗或预防阿尔茨海默病的方法，包括给有需要的对象或患者施用本发明的多核苷酸、载体、rAAV或重组慢病毒，或药物组合物。在一些实施方案中，静脉内施用、脑内施用或鞘内施用所述多核苷酸、载体、rAAV、重组慢病毒或药物组合物。在一些实施方案中，所述对象或患者是人。
本发明还提供本发明的多核苷酸、载体、rAAV或重组慢病毒，或药物组合物在制备用于促进糖代谢，或预防或治疗葡萄糖代谢障碍，优选脑中的葡萄糖代谢障碍的药物中的用途。本发明还提供本发明的多核苷酸、载体、rAAV、重组慢病毒或药物组合物在制备用于治疗或预防阿尔茨海默病的药物中的用途。在一些实施方案中，所述药物是静脉内施用、脑内施用或鞘内施用的。在一些实施方案中，所述药物施用于人。
本发明还提供本发明的多核苷酸、载体、rAAV、重组慢病毒或药物组合物，其用于促进糖代谢，或预防或治疗葡萄糖代谢障碍，优选脑中的葡萄糖代谢障碍的用途。本发明还提供本发明的多核苷酸、载体、rAAV、重组慢病毒或药物组合物，其用于治疗或预防阿尔茨海默病的用途。在一些实施方案中，所述多核苷酸、载体、rAAV、重组慢病毒或药物组合物是静脉内施用、脑内施用或鞘内施用的。在一些实施方案中，所述多核苷酸、载体、rAAV、重组慢病毒或药物组合物施用于人。
所述预防或治疗阿尔茨海默病包括预防、减轻或消除AD症状，如AD样神经退行性病变、脑萎缩、Aβ和tau病理改变、神经炎症和神经血管紊乱，或减缓AD进展。
AD涉及多脑区损伤，而本发明的病毒可以特异性高效透过血脑屏障，广泛作用于多个脑区。临床应用时，本发明的rAAV可以通过跨血脑屏障直接注射(如脑内注射或鞘内注射)施用，或通过外周静脉注射施用，也可以达到与脑部直接注射相当的效果。另外，本发明和其他基因治疗的区别是：仅外源介导TPK的补偿表达，不牵涉内源TPK，更不会涉及到其他基因的编辑，可以大大降低其他风险。更重要的是，本发明引入高活性的外源TPK，实现了有益的治疗效果。
GSK3β是进化上非常保守的丝氨酸/苏氨酸激酶，除调节糖原合成酶活性外，还可作用于其它通路，调节细胞的分化、增殖、存活和凋亡。有研究表明，GSK3β的激活可以促进tau蛋白的磷酸化、增加Aβ沉积、激活小胶质细胞、损伤神经元生成、抑制LTP等来引起AD的发病(Elisabetta Lauretti,Ozlem Dincer,Domenico Praticò,Glycogen synthase kinase-3 signaling in Alzheimer's disease,Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,Volume 1867,Issue 5,2020)。有利地，本发明的rAAV在体内(如脑内)表达TPK，其介导的“TPK-TDP-糖代谢”通路和GSK3β通路相互作用，提高磷酸化的GSK3β(p-GSK3β)水平和p-GSK3β/GSK3β的比例，抑制GSK3β的活性，为AD提供有益的治疗效果。
实施例
通过以下实施例，本领域技术人员会更清楚地理解本发明。应理解，以下实施例旨在示例性说明本发明的实施方案，而非限制本发明的范围。如无特别说明，本发明所使用的方法是本领域常规方法，所使用的实验材料也是商业可获得的。
实施例1、制备rAAV和重组慢病毒
委托CRO公司武汉枢密脑科学技术有限公司制备实验室级的编码TPK的rAAV(血清型AAV_PHP eB)，以下称为AAV-TPK，其基因组包含：i)神经元特异性启动子(SEQ ID NO:3)，ii)编码小鼠TPK的核苷酸序列(SEQ ID NO:2)和WPRE元件(SEQ ID NO:4)。包含rAAV基因组的质粒如图1A所示。
委托CRO公司和元生物技术(上海)股份有限公司制备实验室级的编码TPK的慢病毒，以下称为TPK慢病毒，其基因组包含：i)真核表达启动子(SEQ ID NO:7)，ii)编码小鼠TPK的核苷酸序列(SEQ ID NO:2)和WPRE元件(SEQ ID NO:4)。包含慢病毒基因组的质粒如图1B所示。
实施例2、TPK慢病毒感染的神经元细胞中过表达TPK且TPD含量增加
此实施例的目的在于，通过细胞水平的实验对基因治疗效果进行初步评价。
用胰蛋白酶(Thermofisher/Gibco货号25200072)消化新生鼠(购自浙江维通利华实验动物技术有限公司)的大脑皮质获得原代神经元，并在6孔板中培养(1.5x 106/孔)4天(第一天用10％FBS+10％F12+1％Glutemax+78％DMEM+1％100X青霉素-链霉素双抗(5,000U/mL)，然后用96％Neurobasal medium+2％B27+1％Glutemax+1％100X青霉素-链霉素双抗(5,000U/mL)，以上试剂配置为体积比，且均购自Thermofisher/Gibco)。去除培养基，并加入实施例1制备的慢病毒悬液(在PBS中，1.5mL，MOI为5)感染8hr，以编码GFP但不包含TPK编码序列的慢病毒作为对照。移除液体并添加新鲜培养基，培养7天。通过荧光显微镜观察GFP表达。如图2所示，大量经感染的细胞表达GFP，说明了病毒的感染率高。
移除培养基，并用预冷的PBS洗细胞三次，5min/次。每孔加入100μL细胞裂解液，其包含比例(v:v:v)为100:1:1的RIPA(购自碧云天)、磷酸酶抑制剂(购自Bimake)和蛋白酶抑制剂(购自sigma)，将细胞全部刮下，并细胞悬液移至置于冰上的1.5mL离心管；每孔再加入50μL细胞裂解液冲洗孔的底部，并移至同一离心管。每管加入2粒研磨钢珠，进行研磨(60Hz，60s，-10℃)；13000rpm，4℃离心15min，取上清置于冰上。
BCA法测定上清中的总蛋白浓度；并将提取的总蛋白95℃变性后，通过Western Blot分析样品中的TPK水平。简而言，变性后将样品加载到聚丙烯酰胺凝胶的孔中(待测样品每孔15μg蛋白，蛋白大小指示剂(Marker)5μL)，电泳结束后进行转膜，然后根据Marker剪下对应于目的蛋白大小的区域的膜(TPK为25-35KD，β-Actin为40-55KD)。用封闭液(购自Takara，货号T7131A)将膜室温封闭1h，然后在4℃分别和以下一抗孵育：兔抗TPK1抗体(购自Proteintech公司，货号10942-1-AP)和小鼠抗β-Actin抗体(购自碧云天生物技术有限公司，货号AF0003)孵育过夜。回收一抗，用1X TBST清洗膜三次，每次5mins，再分别对应添加二抗辣根过氧化物酶标记山羊抗兔IgG(H+L)(购自碧云天生物技术有限公司，货号A0208)；辣根过氧化物酶标记山羊抗小鼠IgG(H+L)(购自碧云天生物技术有限公司，货号A0216)室温孵育2h，用1xTBST清洗三次，用辣根过氧化物酶试剂盒(购自赛默飞，货号34095)进行显影。如图3A所示，经TPK慢病毒感染的细胞显著过表达TPK。
取100μL上清并加入等体积的预冷的5.4％高氯酸(PCA)，震荡混匀，13000rpm，4℃离心15min；使用如表1所示的设备和条件对上清进行HPLC分析。
表1
如图3B所示，经TPK慢病毒感染的细胞中的TDP含量显著高于未经感染的细胞和对照病毒感染的细胞，而AAV-TPK的细胞中TM含量显著低于未经感染的细胞和对照病毒感染的细胞(图3C)。结果显示，经TPK慢病毒感染的细胞中过表达TPK明显促进了TM向TDP的转化，转化率是对照组的约两倍。
实施例3、尾静脉注射AAV-TPK的小鼠的脑中TPK过表达和TM/TDP转化率升高
此实施例目的在于通过小鼠尾静脉注射AAV病毒验证基因治疗手段是否能够显著改善C57BL/6J小鼠脑中TPK蛋白表达量，并且使得小鼠脑中TM/TDP转化率上升，从而增强糖代谢。
适应性饲养3天后，将成年C57BL/6J小鼠(2月龄)(购自浙江维通利华实验动物技术有限公司)随机分为两组(TPK组和对照组，n＝3)，通过尾静脉注射给各组分别施用滴度为5.0E+12vg/mL(生理盐水配制)的实施例1制备的AAV-TPK(TPK组)和对照rAAV(除了没有TPK编码序列之外与TPK-AAV相同)的悬液，每只200μL。具体而言，将小鼠固定在尾静脉注射固定器上，用酒精棉球擦拭小鼠尾巴进行消毒，使用1ml注射器将200μL病毒悬液注射到小鼠尾静脉中，注射后干棉球按压注射位点30秒防止病毒外溢。小鼠于实验区饲养4周，并每周记录小鼠的体重和摄食量。
如图4所示，成年小鼠尾静脉注射AAV-TPK后，体重增加量显著小于对照组，摄食量与对照组相比有降低的趋势。
饲养4周后，使用3mL EDTA抗凝管取小鼠全血后立即置于冰上，冰上完成如下 操作，10min之内将0.3mL全血样品移至2mL离心管，加入30μL纯水，然后加入0.3mL预冷的PCA(5.7％)以沉淀蛋白。冰浴约30min后，样品在4℃12,000rpm离心10min，取上清，并如实施例2所述进行HPLC分析TDP和TM含量。
如图5所示，成年小鼠尾静脉注射AAV-TPK 4周后，外周血液TDP/TM含量无显著变化。
使小鼠安乐死后，使用预冷的PBS进行心脏灌流，然后收集小鼠脑组织(去除小脑、脑干和嗅球部分，左右脑分开)。
右脑用4％多聚甲醛(PFA)固定后进行免疫荧光染色。具体而言，右脑经过4％PFA固定12-24h后，更为换30％蔗糖进行脱水沉糖2天，沉糖完成后的右脑用冷冻切片包埋剂(OCT)包埋好后冻存于-80℃。使用冷冻切片机将包埋好的右脑切成30μm厚的薄片，切片角度为沿从嗅球向小脑的纵向切(冠状切)。切片后的薄片用1X PBS清洗3-5次，将OCT洗净，加入0.5％Triton进行室温破膜30min，再更换为3％BSA(牛血清白蛋白)室温封闭1h，加入一抗(抗GFP抗体，购自Avessellabs公司，货号为GFP-1020)4℃孵育过夜；回收一抗，用1X PBS清洗3次，5mins/次，加入二抗Goat anti-Rabbit IgG(H+L)Cross-Adsorbed Secondary Antibody,Alexa Fluor 488，(购自Invitrogen公司，货号为A11008)，避光室温孵育2h，最后将薄片贴于载玻片上，稍稍干燥后，滴加封片剂进行封片，用Nikon Ti2E倒置显微镜进行成像拍照。
如图6所示，成年小鼠尾静脉注射AAV-TPK 4周后，rAAV病毒所编码的多肽可在脑组织中广泛表达。
将左脑或肝组织(左上叶)置于1.5ml细胞裂解液(参见实施例2)中，用冷冻研磨仪中裂解(70Hz、60s，2次，中间间隔20s)；然后13,000rpm 4℃离心15分钟，取上清。如实施例2所述进行Western Blot和HPLC分析以检测脑中的TPK表达以及TDP和TM含量。
如图7所示，成年小鼠尾静脉注射AAV-TPK 4周后，脑组织TPK表达量显著增高，TDP含量显著增高，而TM含量于对照相当。
如图8所示，肝脏中TM和TDP均无显著变化，说明该病毒具有良好的脑特异性，即特异性靶向脑神经元，而不影响外周组织，该病毒的副作用完全可控。
实施例4、侧脑室注射AAV-TPK的小鼠脑中TPK过表达和TM/TDP转化率升高
此实施例目的在于验证小鼠侧脑室注射AAV-TPK病毒是否能够显著提高C57BL/6J小鼠脑中TPK蛋白表达量，并且使得小鼠脑中TM/TDP转化率上升，从而增强糖代谢。
适应性饲养3天后，将成年C57BL/6J小鼠(2月龄)(购自浙江维通利华实验动物技术有限公司)随机分为两组(TPK组和对照组，n＝5)，进行脑立体定位侧脑室注射AAV。使用病毒原液浓度为1.0E+13vg/1mL，小鼠侧脑室注射量为4.0E+10vg/4μL(左右各2μL)/只。将成年小鼠使用舒泰50麻醉后，剃去双耳双眼间毛发后固定在脑立体定位仪上，用酒精棉球擦拭小鼠脑部皮肤消毒，剪刀剪开皮肤暴露颅骨，使用干棉棒擦干颅骨表面至可以清晰看到前囟点，以前囟点为0点定位坐标(AP：-0.2mm，L/R：±1.0mm，H：2.5mm)，标注钻孔点，并使用0.5mm钻头颅骨钻钻孔，用10μL微量注射针吸入2μL AAV病毒，以颅骨面为Z轴0点进针深度2.5mm。注射速度：0.2μL/min，停针5min，缓慢拔出注射针。缝合皮肤后，将小鼠放进含加热垫的笼中待苏醒后放入饲养笼于实验区饲养4周，每周记录体重和摄食量。
如图9所示，成年小鼠侧脑室注射AAV-TPK后，体重和摄食量与对照相比没有显著差异。
饲养4周后，如实施例3所述收集样品并检测血液中的TDP和TM含量，rAAV病 毒所编码的多肽在脑中的表达，和脑和肝组织中的TPK表达水平以及TDP和TM含量。
如图10所示，成年小鼠侧脑室注射AAV-TPK 4周后，外周血TDP/TM含量无显著变化。
如图11所示，成年小鼠侧脑室注射rAAV 4周后，rAAV病毒编码的基因产物在侧脑室周围强表达，在部分其他脑区也有表达。
如图12所示，成年小鼠侧脑室注射AAV-TPK 4周后，与对照相比，脑组织TPK表达量显著增高，TDP含量显著增高，而TM含量降低，说明TM/TDP转化率提高。
如图13所示，肝脏中TM和TDP均无显著变化，说明该病毒具有良好的脑特异性，即特异性靶向脑神经元，而不影响外周组织，该病毒的副作用完全可控。
实施例5、侧脑室注射AAV-TPK的胚胎发育成的小鼠脑中TPK过表达和TM/TDP转化率升高
此实施例目的在于验证直接将AAV递送到胎鼠侧脑室中的基因治疗手段能否显著增加C57BL6/J小鼠大脑内神经元中TPK的表达，提高神经元中TM/TDP的转化率，从而增强糖代谢。
将采购自浙江维通利华实验动物技术有限公司的C57BL6/J小鼠(怀孕13.5天(embryo 13.5days，E13.5)适应性饲养1天(E14.5)，随机分为两组(n＝3)：TPK组(注射AAV-TPK)和对照组(注射对照病毒)。
将小鼠深度麻醉后，腹部向上平放用蘸有75％酒精的纸巾擦拭孕鼠腹部，并剪去腹部中心部分毛发，再用蘸有PBS的纸巾擦拭孕鼠腹部，去除残余酒精与碎毛发。用75％酒精擦拭手术器械进行消毒。用剪刀沿着孕鼠腹部中间剪开皮肤，大约1.5cm，沿着腹白线剪开孕鼠腹膜，暴露腹腔。用镊子小心地将胚胎从孕鼠腹腔中取出，铺在孕鼠腹部(以下手术过程中，用PBS保持小鼠腹腔和胚胎表面湿润)。将吸取了rAAV病毒悬液的玻璃电极轻轻刺入胚胎脑部，通过吹气将病毒悬液转移至胚胎的侧脑室中，每只胚胎每侧脑室注射0.5～1μL(5.0E+9～1.0E+10vg)病毒悬液。将胚胎放回孕鼠腹腔，用缝合线依次缝合小鼠腹膜与皮肤。缝合完成后，涂抹林可霉素利多卡因凝胶进行镇痛，并防止伤口发炎感染，然后将小鼠置于加热垫上直至醒转，转移回笼中继续饲养。
子代小鼠出生后用480nm的激发光照射小鼠头部检测病毒注射结果，保留成功注射病毒的小鼠。饲养21天后，3只小鼠如实施例3所述处理以收集样品并检测血液中的TDP和TM含量，rAAV病毒所编码的多肽在脑中的表达，和脑以及肝组织中的TPK表达水平以及TDP和TM含量；另外12只小鼠断乳并记录体重，分笼饲养两周，再次记录体重。
如图14所示，注射AAV-TPK的胚胎发育成的小鼠的断乳后2周内体重变化与对照相比没有显著差异。
如图15所示，注射AAV-TPK的胚胎发育成的小鼠外周血TDP/TM含量无显著变化。
如图16所示，注射rAAV的胚胎发育成的小鼠中，rAAV病毒编码的基因产物在皮层和海马中广泛表达，在部分其他脑区也有表达。
如图17所示，与对照相比，注射AAV-TPK的胚胎发育成的小鼠脑组织TPK表达量显著提高，TDP含量显著提高，而TM含量降低，说明TM/TDP转化率提高。
如图18所示，肝脏中TM和TDP均无显著变化，说明该病毒具有良好的脑特异性，即特异性靶向脑神经元，而不影响外周组织，该病毒的副作用完全可控。
实施例6、脑立体定位注射AAV-TPK的新生小鼠脑中TPK过表达和TM/TDP转化率升高
此实施例目的在于验证直接将AAV-TPK递送到新生小鼠(Postnatal 0day，P0小鼠)侧脑室(Intracerebral ventricular，ICV)和海马(Intraparenchymal，IP)中能否显著增加C57BL6/J小鼠大脑内神经元中TPK的表达，提高神经元中TM/TDP的转化率，从而增强糖代谢。
C57BL6/J小鼠(怀孕18.5天(embryo 18.5days，E18.5))采购自上海杰思捷实验动物有限公司，共计11只。在新生小鼠出生后进行实验。
侧脑室注射位点：从Lambda向前1mm，左右1mm，深1mm。双侧侧脑室注射，每侧注射800nL病毒悬液(1.0E+13vg/mL)。
海马注射位点①：从Lambda向前0.7mm，左右1.2mm，深0.9mm；海马注射位点②：从Lambda向前0.1mm，左右2.2mm，深1.4mm。两侧海马均注射2个位点，共注射4次，每次注射200nL病毒悬液(1.0E+13vg/mL)。
在玻璃电极中灌满石蜡油后，将玻璃电极用热熔枪固定在微量注射器上，微量注射器的针头插入玻璃电极中约1/3的位置，将微量注射器固定到自动注射泵上。
将新生小鼠的身体埋在冰中，不遮盖鼻子使其能够呼吸，持续约3mins。待其身体由粉紫色转为稍微变白并且不再挣扎运动后，将P0小鼠从冰中取出置于冰面上用于后续实验。
通过注射泵将病毒悬液吸到玻璃电极中。将通过冰处理彻底麻醉的新生小鼠用医用胶带固定在一个水平器皿上，使其头部保持水平即可。在体式显微镜下，确定小鼠头部的后囟(Lambda)为原点，通过操作臂移动注射器到达指定位置后，通过垂直操作臂(Z轴)调节注射器的上下位置，以针头碰到头部后微微下陷的位置为0点，向下送针到指定位置。最后通过注射泵控制器将病毒自动递送到新生小鼠脑内。注射完成后，使针在新生小鼠脑中停留约3mins，再缓慢将针拔出。
注射完成后，将新生小鼠迅速转移到加热垫上。在新生小鼠体温恢复开始活动后，将其放回母鼠笼中，并用笼中垫料敷在新生小鼠上一段时间使其沾染气味，待母鼠主动将新生小鼠叼回窝中时立体定位实验结束。
饲养21天后，每种处理的3只小鼠如实施例3所述处理以收集样品并检测血液中的TDP和TM含量，rAAV病毒所编码的多肽在脑中的表达，和脑以及肝组织中的TPK表达水平以及TDP和TM含量；另外的小鼠(侧脑室注射n＝11，海马注射n＝8)断乳并记录体重，分笼饲养两周，再次记录体重。
如图19所示，P0小鼠侧脑室注射AAV-TPK 3周后，断乳后的2周内体重改变与对照组相比无显著变化，而P0小鼠海马注射AAV-TPK 3周后，断乳后的2周内体重变化与对照组相比亦无显著变化(图23)。
如图24所示，P0小鼠海马注射AAV-TPK 3周后，外周血TDP和TM含量无显著变化。
如图21所示，P0小鼠侧脑室注射rAAV 3周后，rAAV编码的基因产物在皮层和海马中广泛表达，在部分其他脑区也有表达，而P0小鼠海马注射rAAV 3周后，rAAV编码的基因产物在海马中强表达，在部分其他脑区也有表达(图25)。
如图22所示，P0小鼠侧脑室注射rAAV 3周后，与对照相比，脑组织TPK表达量显著提高，TDP含量显著提高，而TM含量降低，说明TM/TDP转化率提高。如图26所示，P0小鼠海马注射AAV-TPK 3周后，与对照相比，脑组织TPK表达量显著增高。
如图27所示，肝脏中TM和TDP均无显著变化，说明该病毒具有良好的脑特异性，即特异性靶向脑神经元，而不影响外周组织，该病毒的副作用完全可控。
实施例7、筛选高活性的TPK变体
本实施例通过在TPK多肽中引入氨基酸取代，筛选比野生型TPK多肽活性更高的 突变体。
发明人通过比较人TPK和鼠源TPK蛋白结构，选定可能对TPK活性影响比较大的氨基酸，然后对这些氨基酸进行单点突变和组合突变，设计并制备了如表2所示的TPK及其突变体。
表2
具体而言，将编码突变上述TPK的核苷酸序列(SEQ ID NO:1和14-19)克隆到pMCSG7-(+)骨架中，并转化至大肠杆菌BL21(DE3)感受态细胞中，涂布于LB琼脂培养基(含有50mg/L的卡那霉素)，37℃培养过夜，然后将单菌落转移至LB液体培养基(含有50mg/L的卡那霉素)中培养，测序验证。
将经验证的克隆在LB琼脂培养基上活化。然后，将单菌落接种至LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡孵育12h。将1mL培养物转接至50mL新鲜的LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡孵育至OD600达到0.6左右，加入IPTG(终浓度为0.5mM)在25℃孵育16h以诱导蛋白质表达。
孵育后，将培养物以5,000g在4℃离心5min，弃上清，收集大肠杆菌细胞。将收集的大肠杆菌细胞1:8重悬于预冷的蛋白平衡溶液(50mM HEPES，2mM MgCl2，250mM NaCl，10％甘油)中，在4℃超声破碎大肠杆菌细胞。细胞破碎液以12,000rpm在4℃离心60min，收集上清液过镍柱(GE,17-3712-02)，用蛋白平衡溶液配制的30mM的咪唑溶液对镍柱进行清洗除去非特异结合的杂蛋白；用蛋白平衡溶液配制的500mM的咪唑溶液对镍柱进行洗脱，收集包含目的蛋白的洗脱液；并用Merck Millipore的10KD孔径浓缩管，透析去咪唑，至咪唑浓度低于5mM。透析产物经FPLC分子筛(SEC柱：superdex 200increase；PBS缓冲液，PH 7.4)进一步纯化，收集目的蛋白溶液，并用Merck Millipore的10KD孔径浓缩管浓缩蛋白。
用于检测酶活性的实验材料如表3所示。
表3

将50mM TPK酶溶液与150mM Tris-HCl缓冲液和10mM ATP溶液等比混合，并将混合物取210μL分装至离心管中。向每个离心管中加入DMSO 30μL，最后向其中加入20μM硫胺素溶液30μL，于37℃孵育0.5小时。然后，加入270μL 5％PCA终止液混匀终止反应。将150μL上述混合物转移至1.5mL离心管中，加入30μL 10mM铁氰化钾衍生化试剂进行对样品衍生化，最后加入15μL 1M磷酸终止液停止反应。如实施例2所述，通过高效液相色谱法检测TDP/TM的含量。TPK酶活性＝TDP(nM)/mg TPK/min。
如图28所示，与人TPK(hTPK)相比，变体hTPK 8M显示出显著提高的催化TM转化为TDP的活性(p<0.05)，但仍低于野生型小鼠TPK(mTPK)的活性(p<0.05)。如图29所示，与野生型mTPK相比，引入变体mTPK 11W和mTPK 35W显示出更高的催化TM转化为TDP的活性，其中变体mTPK 11W显示出最高的活性(p<0.01vs.mTPK)；而引入241W取代使活性降低。
实施例8、编码TPK变体的rAAV对TPK体内活性的影响
本实施例在新生小鼠和APP/PS1小鼠中研究编码活性提高的TPK变体的rAAV的治疗效果。
如实施例1所述制备编码hTPK的rAAV，命名为AAV-hTPK，其基因组包含：i)神经元特异性启动子(SEQ ID NO:3)，ii)编码hTPK的核苷酸序列(SEQ ID NO:14)和WPRE元件(SEQ ID NO:4)。
8.1.侧脑室注射AAV-hTPK的新生小鼠脑中TPK过表达和TM/TDP转化率升高
此实施例的目的在于验证直接将AAV-hTPK递送到新生小鼠(Postnatal 0day，P0小鼠)侧脑室(Intracerebral ventricular，ICV)中能否显著增加C57BL6/J小鼠大脑内神经元中TPK的表达，提高神经元中TM/TDP的转化率，从而增强糖代谢。
C57BL6/J小鼠(怀孕18.5天(embryo 18.5days，E18.5))采购自上海杰思捷实验动物有限公司，共计11只。病毒原液总量为2E10vg(800nL)，分别稀释成原液浓度的1/5、1/25、1/100、1/200。使用不包含TPK编码序列的rAAV作为对照。在新生小鼠出生后进行实验。
侧脑室注射位点：从Lambda向前1mm，左右1mm，深1mm。双侧侧脑室注射，每侧注射800nL病毒悬液。
在玻璃电极中灌满石蜡油后，将玻璃电极用热熔枪固定在微量注射器上，微量注射器的针头插入玻璃电极中约1/3的位置，将微量注射器固定到自动注射泵上。
将新生小鼠的身体埋在冰中，不遮盖鼻子使其能够呼吸，持续约3mins。待其身体由粉紫色转为稍微变白并且不再挣扎运动后，将P0小鼠从冰中取出置于冰面上用于后续实验。
通过注射泵将病毒悬液吸到玻璃电极中。将通过冰处理彻底麻醉的新生小鼠用医用胶带固定在一个水平器皿上，使其头部保持水平即可。在体式显微镜下，确定小鼠头部的后囟(Lambda)为原点，通过操作臂移动注射器到达指定位置后，通过垂直操作臂(Z 轴)调节注射器的上下位置，以针头碰到头部后微微下陷的位置为0点，向下送针到指定位置。最后通过注射泵控制器将病毒自动递送到新生小鼠脑内。注射完成后，使针在新生小鼠脑中停留约3mins，再缓慢将针拔出。
注射完成后，将新生小鼠迅速转移到加热垫上。在新生小鼠体温恢复开始活动后，将其放回母鼠笼中，并用笼中垫料敷在新生小鼠上一段时间使其沾染气味，待母鼠主动将新生小鼠叼回窝中时立体定位实验结束。
饲养21天后，每种处理的3只小鼠如实施例3所述处理以收集样品，并检测脑组织中的TPK表达水平以及TDP含量。
如图30所示，脑组织中TPK蛋白的表达量随所注射的病毒的滴度梯度表现出剂量依赖性变化。HPLC结果显示，注射AAV-hTPK(原液至100倍稀释液范围内)，脑组织中TDP的含量随着病毒的稀释而下降，呈现剂量依赖效应(图31)。
8.2.侧脑室注射AAV-TPK对APP/PS1小鼠的作用
验证通过直接将AAV(实施例1中制备的AAV-TPK)递送到成年APP/PS1小鼠侧脑室(Intracerebral ventricular，ICV)的基因治疗手段能否显著增加APP/PS1小鼠大脑内神经元中TPK的表达，从而改善神经元中TM/TDP的转化率，增强其糖代谢过程。
适应性饲养3天后，将成年APP/PS1雄性小鼠(13月龄)(购自是卡文斯百格(苏州)模式动物研究有限公司)随机分为两组(TPK组和对照组，n＝12)，进行脑立体定位侧脑室注射AAV。使用病毒原液浓度为1.0E+13vg/1mL，小鼠侧脑室注射量为4.0E+10vg/4μL(左右各2μL)/只。将成年小鼠使用异氟烷麻醉后，剃去双耳双眼间毛发后固定在脑立体定位仪上，用酒精棉球擦拭小鼠脑部皮肤消毒，剪刀剪开皮肤暴露颅骨，使用干棉棒擦干颅骨表面至可以清晰看到前囟点，以前囟点为0点定位坐标(AP：-0.2mm，L/R：±1.0mm，H：2.5mm)，标注钻孔点，并使用0.5mm钻头颅骨钻钻孔，用10μL微量注射针吸入2μL AAV病毒，以颅骨面为Z轴0点进针深度2.5mm。注射速度：0.2μL/min，停针5min，缓慢拔出注射针。缝合皮肤后，将小鼠放进含加热垫的笼中待苏醒后放入饲养笼于实验区饲养至少8周等待AAV表达，每周统计体重和摄食量以及血糖(固定时间点)，如实施例3所述收集血液、脑组织及肝组织样品，检测血液中的TDP和TM含量，脑和肝组织中的TPK表达水平以及TDP和TM含量(n＝4)。
如图32所示，注射AAV-TPK的小鼠体重和摄食量以及血糖与注射对照AAV的小鼠没有显著差异，说明注射AAV-TPK的安全性。
如图33所示，注射AAV-TPK的小鼠脑组织中的TPK表达水平高，而注射对照AAV的小鼠脑组织中的TPK几乎没有表达；而注射AAV-TPK的小鼠和注射对照AAV的小鼠肝脏中的TPK表达水平相当，表明本发明的AAV没有显示脱靶现象。
如图34所示，注射AAV-TPK的APP/PS1小鼠脑组织中TDP含量显著高于注射对照AAV的APP/PS1小鼠。而注射AAV-TPK的APP/PS1小鼠的血液和肝脏组织TDP含量与注射对照AAV的APP/PS1小鼠相当，表明本发明的AAV没有显示脱靶现象。
饲养6个月后，如实施例3所述收集样品并检测(区别仅在于一抗)脑组织中的GSK3β蛋白水平(一抗为GSK-3β(27C10)(购自CST，货号为9315)和磷酸化的GSK-3β蛋白(p-GSK-3β)(Ser9)(一抗购自CST，货号为14630)。
如图35和36所示，侧脑室注射AAV-TPK的APP/PS1小鼠脑组织中的p-GSK3β水平和p-GSK3β/GSK3β的比例高于注射对照AAV的APP/PS1小鼠。提示“TPK-TDP-糖代谢”通路和p-GSK3β通路有相互作用。
序列
SEQ ID NO:1小鼠TPK氨基酸序列
SEQ ID NO:2小鼠TPK核苷酸序列
SEQ ID NO:3启动子核苷酸序列
SEQ ID NO:4WPRE核苷酸序列

SEQ ID NO:5人生长因子基因的聚腺苷酸化信号序列
SEQ ID NO:6ITR
SEQ ID NO:7真核启动子
SEQ ID NO:8人TPK氨基酸序列
SEQ ID NO:9人TPK 8M氨基酸序列
SEQ ID NO:10小鼠TPK 11W氨基酸序列
SEQ ID NO:11小鼠TPK 35W氨基酸序列

SEQ ID NO:12小鼠TPK 241W氨基酸序列
SEQ ID NO:13小鼠TPK 11W+241W氨基酸序列
SEQ ID NO:14人TPK核苷酸序列
SEQ ID NO:15人TPK 8M核苷酸序列

SEQ ID NO:16小鼠TPK 11W核苷酸序列
SEQ ID NO:17小鼠TPK 35W核苷酸序列
SEQ ID NO:18小鼠TPK 241W核苷酸序列

SEQ ID NO:19小鼠TPK 11W+241W核苷酸序列

Claims (33)

  1. 一种重组腺相关病毒(rAAV)或重组慢病毒，其在基因组中包含表达盒，所述表达盒包含与启动子可操作连接的编码硫胺素焦磷酸激酶(TPK)的多核苷酸。
  2. 权利要求1的rAAV或重组慢病毒，其中所述TPK包含SEQ ID NO:1的氨基酸序列。
  3. 权利要求1或2的rAAV或重组慢病毒，其中所述多核苷酸包含SEQ ID NO:2的核苷酸序列。
  4. 权利要求1-3任一项的rAAV或重组慢病毒，其中所述启动子是神经元特异性启动子。
  5. 权利要求1-4任一项的rAAV或重组慢病毒，其中所述启动子包含SEQ ID NO:3的核苷酸序列。
  6. 权利要求1-5任一项的rAAV或重组慢病毒，其中所述表达盒还包含WPRE元件。
  7. 权利要求6的rAAV或重组慢病毒，其中所述WPRE元件包含SEQ ID NO:4的核苷酸序列。
  8. 权利要求1-7任一项的rAAV，其是血清型AAV_PHP eB的rAAV。
  9. 一种药物组合物，其包含权利要求1-8任一项的rAAV或重组慢病毒和药学上可接受的载体。
  10. 权利要求9的药物组合物，其配制用于静脉内施用、脑内施用或鞘内施用。
  11. 一种治疗或预防阿尔茨海默病的方法，包括给有需要的对象施用权利要求1-8任一项的rAAV或重组慢病毒或权利要求9或10的药物组合物。
  12. 权利要求11的方法，其中静脉内施用、脑内施用或鞘内施用所述rAAV或重组慢病毒或药物组合物。
  13. 权利要求1-8任一项的rAAV或重组慢病毒或权利要求9或10的药物组合物在制备用于治疗或预防阿尔茨海默病的药物中的用途。
  14. 权利要求13的用途，其中所述药物是静脉内施用、脑内施用或鞘内施用的。
  15. 一种经修饰的硫胺素焦磷酸激酶(TPK)多肽，与起始TPK多肽相比，包含选自位置13、30、31、37、85、129、158、181、11和35的一或多个位置的氨基酸取代，其中位置13的氨基酸取代为P，位置30的氨基酸取代为A，位置31的氨基酸取代为R，位置37的氨基酸取代为K，位置85的氨基酸取代为K，位置129的氨基酸取代为G，位置158的氨基酸取代为K，位置181的氨基酸取代为S，位置11的氨基取代为W，位置35的氨基酸取代为W，其中与起始TPK多肽相比，所述经修饰的TPK多肽具有提高的催化硫胺素(TM)转化为二磷酸硫胺素(TDP)的活性，且其中所述位置参照SEQ ID NO:1或8编号。
  16. 权利要求15的经修饰的TPK多肽，其中所述起始TPK多肽是野生型TPK多肽。
  17. 权利要求15或16的经修饰的TPK多肽，其中所述起始TPK多肽是人TPK多肽，且其中所述经修饰的TPK多肽包含选自位置13、30、31、37、85、129、158和181的一或多个位置，优选全部的氨基酸取代。
  18. 权利要求15或16的经修饰的TPK多肽，其中所述起始TPK多肽是小鼠TPK多肽，且其中所述经修饰的TPK多肽包含位置11和/或35的氨基酸取代的氨基酸取代。
  19. 权利要求15-18任一项的经修饰的TPK多肽，其包含SEQ ID NO:9、10或11的氨基酸序列。
  20. 一种多核苷酸，其编码权利要求15-19任一项的经修饰的TPK多肽。
  21. 一种表达盒，其包含与启动子可操作连接的权利要求20的多核苷酸。
  22. 权利要求21的表达盒，其中所述启动子是神经元特异性启动子。
  23. 权利要求21或22的表达盒，其中所述启动子包含SEQ ID NO:3的核苷酸序列。
  24. 权利要求21-23任一项的表达盒，其还包含WPRE元件。
  25. 权利要求24的表达盒，其中所述WPRE元件包含SEQ ID NO:4的核苷酸序列。
  26. 一种rAAV或重组慢病毒，其在基因组中包含权利要求21-25任一项的表达盒。
  27. 权利要求26的rAAV，其是血清型AAV_PHP eB的rAAV。
  28. 一种药物组合物，其包含权利要求26或27的rAAV或重组慢病毒和药学上可接受的载体。
  29. 权利要求28的药物组合物，其配制用于静脉内施用、脑内施用或鞘内施用。
  30. 一种治疗或预防阿尔茨海默病的方法，包括给有需要的对象施用权利要求15-19任一项TPK多肽、权利要求20的多核苷酸、权利要求26或27的rAAV或重组慢病毒或权利要求28或29的药物组合物。
  31. 权利要求30的方法，其中静脉内施用、脑内施用或鞘内施用所述多肽、多核苷酸、rAAV或重组慢病毒或药物组合物。
  32. 权利要求15-19任一项TPK多肽、权利要求20的多核苷酸、权利要求26或27的rAAV或重组慢病毒或权利要求28或29的药物组合物在制备用于治疗或预防阿尔茨海默病的药物中的用途。
  33. 权利要求32的用途，其中所述药物是静脉内施用、脑内施用或鞘内施用的。
PCT/CN2023/073317 2022-01-29 2023-01-20 表达tpk的重组病毒及其治疗阿尔茨海默病的用途 WO2023143435A1 (zh)

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