Recombinant viruses expressing TPK and their use in the treatment of Alzheimer's disease

Abstract

Provided is a recombinant adeno-associated virus (rAAV) or a recombinant lentivirus, the rAAV or the recombinant lentivirus comprising an expression cassette in its genome comprising a polynucleotide encoding thiamine pyrophosphokinase (TPK) operably linked to a promoter. Also provided is a pharmaceutical composition comprising the rAAV or the recombinant lentivirus, and the use of the rAAV, the recombinant lentivirus, and the pharmaceutical composition in the preparation of a medicament for treating or preventing Alzheimer's disease.

Description

[Technical field]

[0001] The present disclosure relates to the field of gene therapy. In particular, the present disclosure relates to a recombinant adeno-associated virus (rAAV) or a recombinant lentivirus that contains a nucleotide encoding thiamine pyrophosphokinase (TPK) and is useful for treating Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease (AD) is a severe neurodegenerative disease that mainly affects elderly people. The onset of the disease is accompanied by the death of many neurons and causes brain parenchymal atrophy. The main clinical symptoms of the disease are memory impairment, decline in language and spatial ability, and mental abnormalities. Currently, the etiology of Alzheimer's disease is unknown, and there are various hypotheses regarding the mechanism of Alzheimer's disease, such as the beta-amyloid protein (Aβ) cascade hypothesis, the phosphorylated tau protein hypothesis, the neuroinflammatory response hypothesis, and the oxidative stress hypothesis. Although extensive research has been conducted based on these hypotheses over the past few decades, an effective treatment has not been established.

[0003] Although the brain accounts for only about 2% of body weight, the energy consumed by the brain accounts for about 20% of the total energy consumption of the human body, which indicates that glucose metabolism is extremely important for the normal function of the brain. Research has revealed that glucose metabolic abnormalities occur in the brains of Alzheimer's disease patients before clinical symptoms appear, and the glucose metabolic level of the brain of the patient gradually decreases as the disease progresses. Therefore, it is expected that the progression of Alzheimer's disease can be alleviated or stopped by regulating the glucose metabolic level of brain cells.

[0004] In general, regulation of gene expression in cells can be achieved by methods such as calcium transduction, liposome transfection, electroporation, and infection with gene-transducing viruses.

[0005] Adeno-associated virus (AAV) is a single-stranded DNA virus that is widespread in humans, and its natural hosts are humans and other primates. Currently, no reports have been found that AAV has pathogenicity. Recombinant adeno-associated virus (rAAV) has been obtained through engineering based on AAV, and is widely used as a vector for gene therapy due to its high safety, broad host range (capable of infecting both dividing and non-dividing cells), and low immunogenicity.

[0006] The FDA approved two rAAV-based gene therapy products, Luxturna and Zolgensma, in 2017 and 2019, respectively. Luxturna is a treatment for Burkholder's congenital black cataract, co-developed by Spark Therapeutics and Florida Children's Hospital. AAV2 is used as the vector in Luxturna, which carries human RPE65 cDNA and is administered by subretinal injection. Zolgensma is a treatment for pediatric spinal muscular atrophy, developed by Novartis. AAV9 is used as the vector in Zolgensma, which carries the SMN1 gene required for motor neuron survival and is administered by intravenous injection.

[0007] Vitamin B1, also known as thiamine (TM), is converted in the body by thiamine pyrophosphokinase (TPK) to the biologically active thiamine diphosphate (TDP). TDP is a necessary coenzyme for transketonase, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase in glucose metabolism. Studies have shown that transketonase activity is reduced by more than 45% and α-ketoglutarate dehydrogenase activity is reduced by more than 75% in brain tissue from patients with Alzheimer's disease; TDP levels in whole blood from patients with Alzheimer's disease are significantly lower than normal values, and whole blood TDP levels are highly correlated with reduced glucose metabolism in the brains of patients with Alzheimer's disease. Studies have reported that TPK mRNA and protein expression levels in brain tissue from patients with Alzheimer's disease are significantly lower than those in brain tissue from age- and sex-matched healthy individuals. More importantly, TPK mRNA levels are positively correlated in the brains of healthy subjects but negatively correlated in the brains of patients with Alzheimer's disease. This result suggests that TPK expression may be a protective factor that delays the onset and progression of Alzheimer's disease. Conditional knockout of the TPK gene in excitatory neurons of mouse brains results in significant glucose metabolic abnormalities and cognitive impairment, brain atrophy with synapse and neuronal loss, Aβ deposition and plaque formation, tau abnormal phosphorylation and neurofibrillary tangles, microglial and astrocyte activation and neuroinflammation, impaired microangiogenesis, and dysregulation of peripheral glucose metabolism in mice, all key pathophysiological features of human Alzheimer's disease (see, for example, Sang et al., Thiamine pyrophosphokinase deficiency induces Alzheimer's pathology, bioRxiv, 2020, and WO2021023069A1). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Sang et al., Thiamine pyrophosphokinase induces deficiency Alzheimer's pathology, bioRxiv, 2020 [Patent documents]

[0009] [Patent Document 1] WO2021023069A1 Summary of the Invention [Problem to be solved by the invention]

[0010] Currently, it is desirable to develop new treatments for Alzheimer's disease, such as rAAV-based gene therapy, to address the current shortage of drugs, such as rAAV-based gene therapy. [Means for solving the problem]

[0011] Summary of the Invention In a first aspect, a recombinant adeno-associated virus (rAAV) or recombinant lentivirus is provided, the rAAV or recombinant lentivirus comprising within its genome an expression cassette comprising a polynucleotide encoding thiamine pyrophosphokinase (TPK) operably linked to a promoter.

[0012] In some embodiments, the TPK comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:2.

[0013] In some embodiments, the promoter is a neuron-specific promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:3.

[0014] In some embodiments, the expression cassette further comprises a WPRE element. In some embodiments, the WPRE element comprises the nucleotide sequence of SEQ ID NO:4.

[0015] In some embodiments, the promoter is a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:7.

[0016] In some embodiments, the rAAV is of the serotype AAV_PHP eB.

[0017] In a second embodiment, there is provided a modified thiamine pyrophosphokinase (TPK) polypeptide, comprising, compared to a parent TPK polypeptide, substitutions of amino acids at one or more positions selected from the group consisting of 13, 30, 31, 37, 85, 129, 158, 181, 11 and 35, wherein the amino acid at position 13 is substituted with P, the amino acid at position 30 is substituted with A, the amino acid at position 31 is substituted with R, the amino acid at position 37 is substituted with K, the amino acid at position 85 is substituted with K, the amino acid at position 129 is substituted with G, the amino acid at position 158 is substituted with K, the amino acid at position 181 is substituted with S, the amino acid at position 11 is substituted with W, and the amino acid at position 35 is substituted with W, The modified TPK polypeptide has improved catalytic activity for converting thiamine (TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide; Modified TPK polypeptides are provided, wherein said positions are numbered with reference to SEQ ID NO:1 or 8.

[0018] In some embodiments, the original TPK polypeptide is a wild-type TPK polypeptide. In some embodiments, the original TPK polypeptide is a human TPK polypeptide and the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of 13, 30, 31, 37, 85, 129, 158 and 181, preferably at all of 13, 30, 31, 37, 85, 129, 158 and 181. In some embodiments, the original TPK polypeptide is a mouse TPK polypeptide and the modified TPK polypeptide comprises an amino acid substitution at 11 and / or 35. In some embodiments, the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11.

[0019] In a third aspect, there is provided a polynucleotide encoding a modified TPK polypeptide in accordance with the present disclosure.

[0020] Also provided is an expression cassette comprising a polynucleotide according to the present disclosure operably linked to a promoter. In some embodiments, 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 expression cassette further comprises a WPRE element. In some embodiments, the WPRE element comprises the nucleotide sequence of SEQ ID NO: 4.

[0021] In a fourth aspect, there is provided a rAAV or recombinant lentivirus, the rAAV or recombinant lentivirus comprising within its genome an expression cassette as provided in the third aspect of the present disclosure.

[0022] In some embodiments, the rAAV is of the serotype AAV_PHP eB.

[0023] In a fifth aspect, there is provided a pharmaceutical composition comprising a modified TPK polypeptide, a polynucleotide, a rAAV or a recombinant lentivirus according to the present disclosure, and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for intravenous, intracerebral, or intrathecal administration.

[0024] In a sixth aspect, there is provided a method of preventing or treating Alzheimer's disease, comprising administering to a subject in need thereof a modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure. In some embodiments, the modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition is administered intravenously, intracerebrally or intrathecally.

[0025] Also provided is the use of a modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure in the manufacture of a medicament for preventing or treating Alzheimer's disease. In some embodiments, the medicament is administered intravenously, intracerebrally, or intraspinally.

[0026] Further provided is a modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure for use in preventing or treating Alzheimer's disease. In some embodiments, the modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition is administered intravenously, intracerebrally or intraspinally. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 shows a map of the plasmid containing the genome of AAV-TPK (FIG. 1A) and a map of the plasmid containing the genome of the TPK lentivirus (FIG. 1B). [Diagram 2] FIG. 2 shows images of lentivirus-infected neurons taken by fluorescence microscopy. [Diagram 3] Figure 3 shows TPK expression (Figure 3A, representative parental cells without viral infection), TDP content (Figure 3B), and TM content (Figure 3C) in lentivirus-infected neuronal cells. [Figure 4] FIG. 4 shows the body weight (left and center panels) and food intake (right panel) of mice injected with rAAV via the tail vein. [Diagram 5] FIG. 5 shows the TDP content (left panel) and TM content (right panel) in the blood of mice injected with rAAV via the tail vein. [Figure 6] FIG. 6 shows immunofluorescence images of sections of different regions of the brain of mice injected with rAAV via the tail vein. [Figure 7] Figure 7 shows the expression of TPK (left) and TDP and TM content (right) in the brains of mice administered rAAV via the tail vein. [Figure 8] FIG. 8 shows TDP content (left) and TM content (right) in the liver of mice administered rAAV via the tail vein. [Figure 9] FIG. 9 shows the body weight (left and center panels) and food intake (right panel) of mice administered rAAV into the lateral ventricle. [Figure 10] FIG. 10 shows the TDP content (left panel) and TM content (right panel) in the blood of mice administered rAAV into the lateral ventricle. [Figure 11] FIG. 11 shows immunofluorescence images of sections from different regions of the brain of mice injected with rAAV into the lateral ventricle. [Figure 12] FIG. 12 shows the expression of TPK (left panel) and the content of TDP and TM (right panel) in the brains of mice injected with rAAV into the lateral ventricle. [Figure 13] FIG. 13 shows the TDP content (left panel) and TM content (right panel) in the liver of mice administered rAAV into the lateral ventricle. [Figure 14] FIG. 14 shows the changes in body weight of mice developed from embryos administered rAAV into the lateral ventricle over a period of two weeks after weaning, i.e., 21 days after birth. [Figure 15]FIG. 15 shows the TDP (left panel) and TM (right panel) content in the blood of mice that developed from embryos injected with rAAV into the lateral ventricle on postnatal day 21. [Figure 16] FIG. 16 shows immunofluorescence images of sections of different regions of mouse brains that developed from embryos injected with rAAV into the lateral ventricle at postnatal day 21. [Figure 17] FIG. 17 shows the expression of TPK (left panel) and the content of TDP and TM (right panel) in mouse brains that developed from embryos administered rAAV into the lateral ventricle on postnatal day 21. [Figure 18] FIG. 18 shows TDP (left panel) and TM (right panel) content in the livers of mice that developed from embryos administered rAAV into the lateral ventricle on postnatal day 21. [Figure 19] FIG. 19 shows the changes in body weight 21 days after rAAV injection into the lateral ventricle of neonatal mice, i.e., 2 weeks after weaning. [Figure 20] FIG. 20 shows TDP (left) and TM (right) content in blood 21 days after rAAV injection into the lateral ventricle of neonatal mice. [Figure 21] FIG. 21 shows immunofluorescence images of sections from different brain regions 21 days after injection of rAAV into the lateral ventricles of neonatal mice. [Figure 22] FIG. 22 shows TPK expression (left) and TDP and TM content (right) in the brain 21 days after rAAV injection into the lateral ventricle of neonatal mice. [Figure 23] FIG. 23 shows the changes in body weight 21 days after rAAV injection into the hippocampus of neonatal mice, i.e., 2 weeks after weaning. [Figure 24] FIG. 24 shows TDP (left) and TM (right) content in blood 21 days after rAAV injection into the hippocampus of neonatal mice. [Diagram 25] FIG. 25 shows immunofluorescence images of sections from different brain regions 21 days after rAAV injection into the hippocampus of neonatal mice. [Figure 26] FIG. 26 shows TPK expression in the brain 21 days after rAAV injection into the hippocampus of neonatal mice. [Figure 27] FIG. 27 shows TDP (left panel) and TM (right panel) content in liver 21 days after rAAV injection into the hippocampus of neonatal mice. [Figure 28] FIG. 28 shows a comparison of the activity of modified human TPK with wild-type human and mouse TPK. [Figure 29] FIG. 29 shows a comparison of the activity of modified mouse TPK with wild-type mouse TPK. [Diagram 30] FIG. 30 shows the results of a Western blot of TPK protein in brain tissue. [Diagram 31] FIG. 31 shows the results of HPLC of the TDP content in brain tissue. [Diagram 32] FIG. 32 shows the curves of body weight (A), food intake (B), and blood glucose level (C) of mice injected with AAV-hTPK. [Diagram 33] FIG. 33 shows the results of Western blot of TPK protein in brain tissue and liver of APP / PS1 mice administered AAV-TPK. [Diagram 34] FIG. 34 shows the contents of TDP and TM in brain tissue, liver, and blood of APP / PS1 mice administered AAV-TPK. [Diagram 35] FIG. 35 shows the results of Western blot of p-GSK3β and GSK3β in brain tissue of APP / PS1 mice injected with AAV-TPK into the lateral ventricle. [Diagram 36] FIG. 36 shows the ratio of p-GSK3β / GSK3β in brain tissue of APP / PS1 mice injected with AAV-TPK into the lateral ventricle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description of the Invention I. Definition All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs, unless otherwise defined.Any method and material similar or equivalent to those described herein can also be used in the implementation and teaching of this disclosure, but preferred method and material are described.All publications mentioned herein are incorporated by reference to disclose and describe the cited method and / or material.

[0029] "Thiamine pyrophosphokinase" or "TPK" refers to an enzyme that catalyzes the reaction of converting thiamine (TM) to thiamine diphosphate (TDP).

[0030] "AAV" is an abbreviation for adeno-associated virus and may refer to the virus itself or its derivatives. Unless otherwise specified, the term encompasses all subtypes and naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also called recombinant AAV vector (or "rAAV vector"). The term "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), poultry AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals or the like.

[0031] The genomic sequences of different subtypes of AAV, and the sequences of natural terminal repeats (TRs), Rep proteins, and capsid subunits are well known in the art.Such sequences can be found in the literature or in public databases such as gene libraries.See, for example, Genbank access numbers 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) and NC_006261 (AAV-8). These disclosures are incorporated herein by reference for their teaching of AAV nucleic acid and amino acid sequences.

[0032] "rAAV vector" as used herein refers to an AAV vector that contains a polynucleotide sequence from a non-AAV source (i.e., AAV is a heterologous polynucleotide), and is usually an AAV vector for targeting sequences for genetic transformation of cells. In general, the heterologous polynucleotide is flanked by at least one, usually two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be single-stranded (ssAAV) or self-complementary (scAAV).

[0033] "Packaging" induces a series of intracellular events that lead to the assembly and encapsidation of AAV particles.

[0034] The AAV "rep" and "cap" genes refer to the polynucleotide sequences that encode the replication and capsid proteins of the adeno-associated virus. AAV rep and cap are also called AAV packaging genes.

[0035] AAV "helper virus" refers to a virus that allows replication and packaging of AAV (such as wild-type AAV) in mammalian cells. Many helper viruses for AAV are known, for example, adenoviruses, herpesviruses, and poxviruses (such as vaccinia virus). The most commonly used is adenovirus type 5 of the C subclass, but there are many different subclasses of adenoviruses. Many adenoviruses of human, non-human mammalian, and avian origin are known to be available from depositories such as the ATCC. Viruses in the Herpesviridae family include herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV). These are available from depositories such as the ATCC.

[0036] "Helper virus functions" refers to functions encoded in the genome of a helper virus that allow replication and packaging of AAV (along with other requirements necessary for replication and packaging as described herein). As described herein, "helper virus functions" may be provided in a variety of ways, such as by providing a helper virus or by providing (for example) a trans-multiple nucleic acid sequence encoding the necessary functions to a producer cell. For example, a plasmid (also called an auxiliary plasmid) or other expression vector containing a nucleic acid sequence encoding one or more adenoviral proteins is transfected into a producer cell along with a rAAV vector.

[0037] Lentivirus is a viral vector derived from HIV-1. "Recombinant lentiviral vector" or "recombinant lentiviral vector particle" refers to recombinant viral and viral-like particles generated in host or producer cells after transfection of a plasmid vector, which is composed of a transfer vector, an envelope vector encoding a selected envelope protein, and a packaging vector that provides lentiviral proteins in trans (e.g., lentiviral GAG and POL proteins, particularly mutant POL proteins to prevent integration), according to methods well known in the art.

[0038] Virus-like particles are generated by incomplete assembly of proteins present for encapsidation of the recombinant lentiviral genome, and the methods used fail to form true virus particles.

[0039] The term "polynucleotide" refers to a polymer of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may contain modified nucleotides, such as methylated nucleotides or nucleotide analogs, and may be interposed by non-nucleotide components. Modifications to the nucleotide structure, if present, may be applied before or after assembly of the polymer. The term polynucleotide, as used herein, may refer to alternating double-stranded and single-stranded molecules. Unless otherwise specified or required, all embodiments of the polynucleotide of the present disclosure include both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to form a double-stranded form.

[0040] Nucleic acid hybridization reaction can be carried out under different "stringent" conditions.Conditions for increasing stringency of hybridization reaction are well known in the art and published in the art.See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; which is incorporated herein by reference.

[0041] A polynucleotide or polypeptide that has a percentage of "sequence identity" with another polynucleotide or polypeptide refers to the percentage of bases or amino acids that are identical when the two aligned sequences are compared. There are many methods for determining sequence identity. Methods and computer programs that can be used to align sequences to determine sequence identity include, for example, BLAST, available at NCBI.nlm.nih.gov / BLAST / , and FASTA, available in the Genetics Computing Group (GCG) package, a wholly owned subsidiary of Oxford Molecular Group, Inc., Madison, Wisconsin, USA.

[0042] A "gene" refers to a polynucleotide containing at least one open reading frame that has the capacity to encode a particular protein after transcription and translation.

[0043] "Recombinant," as applied to a polynucleotide, means that the polynucleotide is the product of a different combination of cloning, restriction, or ligation steps, and other procedures that produce a construct that is different from that found in nature. A recombinant virus is a viral particle that contains a recombinant polynucleotide. The term includes the progeny of a pro-polynucleotide or pro-viral construct.

[0044] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional control of a polynucleotide, such as the replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Control may affect the frequency, rate, or specificity of a process, and may in fact be either facilitating or inhibiting. Control elements known in the art include transcriptional control sequences, such as promoters and enhancers. A promoter is a DNA region that is capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region downstream (3' direction) of the promoter.

[0045] "Operably linked" refers to the juxtaposition of genetic elements in a relationship that allows them to function as expected. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region as long as such a functional relationship is maintained.

[0046] An "expression vector" is a vector that contains a region encoding a target polypeptide and is used to achieve expression of the protein in a predetermined target cell. The expression vector further contains control elements operably linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and genes that can be operably linked for expression is sometimes called an "expression cassette", and many expression cassettes are known and available in the art or can be readily constructed from components available in the art.

[0047] "Heterologous" refers to an entity that is derived from a different genotype compared to another entity. For example, a polynucleotide introduced into a plasmid or vector from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter that is removed from a natural coding sequence and operably linked to a coding sequence that is not naturally linked to the coding sequence is a heterologous promoter. Thus, for example, a rAAV that contains a heterologous nucleic acid that encodes a heterologous gene product is a rAAV that contains a nucleic acid that is not normally contained in the naturally occurring wild-type AAV, and the encoded heterologous gene product is not normally a gene product encoded by the wild-type AAV.

[0048] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. These terms also include amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipoylation, phosphorylation, or conjugation with a labeling component.

[0049] Modifications include, but are not limited to, the substitution, deletion, insertion, and / or addition of one or more amino acids to the polypeptide sequence.

[0050] The term "conservative substitution", also known as substitution with a "homologous" amino acid residue, refers to an amino acid residue that is replaced with another amino acid having a similar side chain, such as amino acids with alkaline side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), or aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0051] Conservative amino acid substitutions usually have little effect on the activity of the resulting protein. Such substitutions are described below. Conservative substitutions refer to substitutions of amino acids with similar size, hydrophobicity, charge, polarity, spatial properties, aromaticity, etc. When it is desired to fine-tune the properties of a protein, such substitutions are usually conservative.

[0052] "Homologous" amino acid residues, as used herein, refer to amino acid residues that have similar chemical properties, including hydrophobicity, charge, polarity, spatial properties, aromaticity, etc. Examples of amino acids that are homologous to each other include the positively charged lysine, arginine, and histidine; the negatively charged glutamic acid and aspartic acid; the hydrophobic glycine, alanine, valine, leucine, isoleucine, proline, and phenylalanine; the polar amino acids serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, and glutamine; the aromatic phenylalanine, tyrosine, and tryptophan; and serine and threonine, or glutamine and asparagine, or leucine and isoleucine, which have chemically similar side chain groups.

[0053] Examples of conservative amino acid 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, or Ile or Leu for Val.

[0054] An "isolated" plasmid, nucleic acid, vector, virus, viral entity, host cell, or other substance refers to a preparation that is free of at least that substance or other components of a similar substance as it occurs in nature or as originally prepared. Thus, for example, purification techniques may be used to enrich the isolated substance from an original mixture.

[0055] The term "treatment" as used herein means obtaining a desired pharmacological and / or physiological effect. The effect may be a prophylactic effect, which completely or partially prevents the disease or its symptoms, and / or a therapeutic effect, which partially or completely cures the disease and / or side effects caused by the disease. "Treatment" as used herein includes any treatment for a disease in a mammal, particularly a human, and includes (a) preventing the onset of the disease in a subject susceptible to or at risk for the disease, but not yet diagnosed with the disease, (b) inhibiting the disease, i.e., preventing its onset, and (c) palliating the disease, i.e., slowing its progression.

[0056] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to humans and non-human primates, including monkeys and humans, mammalian sports animals (e.g., horses), mammalian farm animals (e.g., sheep, goats, etc.), mammalian pets (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0057] II. Modified TPK Polypeptides The present inventors have found that the catalytic activity of mouse pyrophosphokinase (mTPK) converting TM to TDP thiamine is higher than that of human TPK (hTPK). The present inventors modify the sequences of hTPK and mTPK to enhance the catalytic activity of converting TM to TDP. For example, the surface charge characteristics of hTPK are modified based on being derived from mouse, i.e., if the charge characteristics of residue Xh in hTPK are different from the residue Xm at the corresponding position in mTPK, residue Xh in hTPK is replaced with Xm.

[0058] Accordingly, there is provided a modified thiamine pyrophosphokinase (TPK) polypeptide, comprising, as compared to a parent TPK polypeptide, an amino acid substitution at one or more positions selected from the group consisting of: 13, 30, 31, 37, 85, 129, 158, 181, 11 and 35; The modified TPK polypeptide has improved catalytic activity for converting thiamine (TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide; Modified TPK polypeptides are provided, wherein said positions are numbered with reference to SEQ ID NO:1 or 8.

[0059] Preferably, the amino acid at position 13 is substituted with P. Preferably, the amino acid at position 30 is substituted with A. Preferably, the amino acid at position 31 is substituted with R. Preferably, the amino acid at position 37 is substituted with K. Preferably, the amino acid at position 85 is substituted with K. Preferably, the amino acid at position 129 is substituted with G. Preferably, the amino acid at position 158 is substituted with K. Preferably, the amino acid at position 181 is substituted with S. Preferably, the amino acid at position 11 is substituted with W. Preferably, the amino acid at position 35 is substituted with W.

[0060] The TPK polypeptide from which the amino acids are modified is referred to as the original TPK polypeptide as used herein. The original TPK polypeptide may be a wild-type TPK polypeptide or a mutant thereof. For example, if the modification is performed on a polypeptide of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 1 becomes the "original TPK polypeptide" of the modified TPK polypeptide, and if the modification is performed on a mutant of the polypeptide of SEQ ID NO: 1, the mutant polypeptide becomes the "original TPK polypeptide" of the modified TPK polypeptide.

[0061] In some embodiments, the original TPK polypeptide is a wild-type TPK polypeptide, such as a human TPK polypeptide or a mouse TPK polypeptide.

[0062] In some embodiments, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35. In some embodiments, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of positions 13, 30, 31, 37, 85, 129, 158, and 181. Preferably, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at positions 13, 30, 31, 37, 85, 129, 158, and 181.

[0063] In some embodiments, the original TPK polypeptide is a mouse TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at position 11 and / or position 35.

[0064] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:8 or comprises an amino acid sequence having 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 to SEQ ID NO:8, wherein the modified TPK polypeptide has an amino acid sequence similar to that of SEQ ID NO:8, e.g., at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 37, 85, 129, 158, 181, 11, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 37, 85, 129, 158, 181, 1 ...9, 30, 30, 31, 37, 85, 129, 158, 181, 11, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 30, 30, 30, 30, 30, and 35, for example, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 35, or substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11 and 35.

[0065] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution at position 11 with W and / or at position 35 with W compared to the original TPK polypeptide.

[0066] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution of W at position 11 compared to the original TPK polypeptide.

[0067] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, and particularly preferably at least 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution of W at position 35 compared to the original TPK polypeptide.

[0068] In some embodiments, the modified TPK polypeptides include the following amino acid substitutions or combinations of amino acid substitutions: 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; or 13P-30A-31R-37K-85K-129G-158K-181S-11W-35W, Includes.

[0069] In some embodiments, the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11 or comprises an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 9, 10 or 11. For example, compared to SEQ ID NO: 9, 10 or 11, the modified TPK polypeptide comprises one or more amino acid substitutions.

[0070] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 13, 30, 31, 37, 85, 129, 158 and 181, preferably other than 241, compared to the amino acid sequence of SEQ ID NO:9.

[0071] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 11 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to the amino acid sequence of SEQ ID NO:10.

[0072] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 35 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to the amino acid sequence of SEQ ID NO:11.

[0073] In some embodiments, the modified TPK polypeptide has a catalytic activity for converting TM to TDP that is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or more of the activity of the original TPK polypeptide.

[0074] III. Isolated Polynucleotides and Expression Cassettes An isolated polynucleotide encoding thiamine pyrophosphokinase (TPK) is provided.

[0075] TPK can be from human or non-human animals, such as non-human mammalian TPK. Examples of non-human mammalian TPK include, but are not limited to, from non-human primates, such as monkeys, horses, cows, sheep, goats, pigs, dogs, cats, mice and rats.

[0076] In some embodiments, the TPK is wild-type TPK. In some embodiments, the TPK is mouse TPK or human TPK.

[0077] In some embodiments, 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.

[0078] In some embodiments, the TPK is a modified TPK that contains one or more amino acid substitutions, insertions, deletions and / or additions compared to the original TPK, and the activity of the modified TPK is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or more of that of the original TPK. In some embodiments, the modified TPK contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, insertions, deletions and / or additions compared to the original TPK. In some embodiments, the modified TPK has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the original TPK.

[0079] The original TPK may be a wild-type TPK or a modified TPK. In some embodiments, the original TPK is a wild-type mouse TPK or a wild-type human TPK.

[0080] In some embodiments, the original TPK comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the original TPK consists of the amino acid sequence of SEQ ID NO: 1.

[0081] In some embodiments, the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35 relative to the original TPK polypeptide, wherein the modified TPK polypeptide has improved catalytic activity for converting thiamine (TM) to thiamine diphosphate (TDP) relative to the original TPK polypeptide. The modified TPK polypeptide has improved catalytic activity for converting thiamine (TM) to thiamine diphosphate (TDP) relative to the original TPK polypeptide, wherein the positions are numbered with reference to SEQ ID NO: 1 or 8.

[0082] Preferably, the amino acid at position 13 is substituted with P. Preferably, the amino acid at position 30 is substituted with A. Preferably, the amino acid at position 31 is substituted with R. Preferably, the amino acid at position 37 is substituted with K. Preferably, the amino acid at position 85 is substituted with K. Preferably, the amino acid at position 129 is substituted with G. Preferably, the amino acid at position 158 is substituted with K. Preferably, the amino acid at position 181 is substituted with S. Preferably, the amino acid at position 11 is substituted with W. Preferably, the amino acid at position 35 is substituted with W.

[0083] In some embodiments, the original TPK polypeptide is a wild-type TPK polypeptide, such as a human TPK polypeptide or a mouse TPK polypeptide.

[0084] In some embodiments, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35. In some embodiments, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of positions 13, 30, 31, 37, 85, 129, 158, and 181. Preferably, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at positions 13, 30, 31, 37, 85, 129, 158, and 181.

[0085] In some embodiments, the original TPK polypeptide is a mouse TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at position 11 and / or position 35.

[0086] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:8 or comprises an amino acid sequence having 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 to SEQ ID NO:8, wherein the modified TPK polypeptide has an amino acid sequence similar to that of SEQ ID NO:8, e.g., at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 37, 85, 129, 158, 181, 11, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 37, 85, 129, 158, 181, 1 ...9, 30, 30, 31, 37, 85, 129, 158, 181, 11, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 30, 30, 30, 30, 30, and 35, for example, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 35, or substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11 and 35.

[0087] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution at position 11 with W and / or at position 35 with W compared to the original TPK polypeptide.

[0088] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution of W at position 11 compared to the original TPK polypeptide.

[0089] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution of W at position 35 compared to the original TPK polypeptide.

[0090] In some embodiments, the modified TPK polypeptides include the following amino acid substitutions or combinations of amino acid substitutions: 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; or 13P-30A-31R-37K-85K-129G-158K-181S-11W-35W, Includes.

[0091] In some embodiments, the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11 or comprises an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 9, 10 or 11. For example, compared to SEQ ID NO: 9, 10 or 11, the modified TPK polypeptide comprises one or more amino acid substitutions.

[0092] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 13, 30, 31, 37, 85, 129, 158 and 181, preferably other than 241, compared to the amino acid sequence of SEQ ID NO:9.

[0093] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 11 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to the amino acid sequence of SEQ ID NO:10.

[0094] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 35 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to the amino acid sequence of SEQ ID NO:11.

[0095] In some embodiments, the modified TPK polypeptide has a catalytic activity for converting TM to TDP that is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or more of the activity of the original TPK polypeptide.

[0096] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:15, 16 or 17, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:15, 16 or 17.

[0097] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, and such nucleotide sequence encodes an amino acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 13, 30, 31, 37, 85, 129, 158 and 181, preferably other than 241, compared to SEQ ID NO:9.

[0098] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16, and such nucleotide sequence encodes an amino acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 11 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to SEQ ID NO: 10.

[0099] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17, and such nucleotide sequence encodes an amino acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 35 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to SEQ ID NO: 11.

[0100] In some embodiments, the polynucleotide is DNA or RNA, such as mRNA.

[0101] Also provided is an expression cassette comprising a polynucleotide according to the present disclosure operably linked to a promoter.

[0102] In some embodiments, 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 a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3. In some embodiments, the promoter consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3.

[0103] In some embodiments, the promoter is a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the promoter consists of the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the promoter comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the promoter consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7.

[0104] Expression cassettes can include additional regulatory elements, such as enhancers, introns, mRNA stabilizing sequences, and polyadenylation signal sequences.

[0105] In some embodiments, the expression cassette further comprises an mRNA stabilizing sequence, e.g., a WPRE element. In some embodiments, the WPRE element comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the WPRE element consists of the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the WPRE element comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4. In some embodiments, the WPRE element consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4.

[0106] In some embodiments, the WPRE element is downstream of the polynucleotide encoding the TPK.

[0107] In some embodiments, the expression cassette further comprises a polyadenylation signal sequence, such as, but not limited to, a polyadenylation signal sequence of a human growth factor gene and a polyadenylation signal sequence of a 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. In some embodiments, the polyadenylation signal sequence comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:5. In some embodiments, the polyadenylation signal sequence consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:5.

[0108] In some embodiments, a polyadenylation signal sequence is downstream of the WPRE element.

[0109] The expression cassette can be part of the genome of rAAV.Therefore, the expression cassette is flanked by the ITRs of AAV.Non-limiting examples of ITRs include those from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, poultry AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV.

[0110] In some embodiments, the ITRs are AAV-2 ITRs. In some embodiments, the 5'ITR is identical to the 3'ITR. In some embodiments, the 5'ITR is different from the 3'ITR.

[0111] In some embodiments, the 5'ITR and 3'ITRR are both ITR130, e.g., comprising the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each consist of the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each comprise a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each consist of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6.

[0112] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5. In some embodiments, the rAAV genome 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, and SEQ ID NO:6.

[0113] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:4, and SEQ ID NO:5. In some embodiments, the rAAV genome 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, and SEQ ID NO:6.

[0114] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:4, and SEQ ID NO:5. In some embodiments, the rAAV genome 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, and SEQ ID NO:6.

[0115] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:4, and SEQ ID NO:5. In some embodiments, the rAAV genome 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, and SEQ ID NO:6.

[0116] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:4, and SEQ ID NO:5. In some embodiments, the rAAV genome 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, and SEQ ID NO:6.

[0117] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.

[0118] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:4, and SEQ ID NO:5.

[0119] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:4, and SEQ ID NO:5.

[0120] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:4, and SEQ ID NO:5.

[0121] In some embodiments, the expression cassette comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:4, and SEQ ID NO:5.

[0122] Also provided is a vector comprising a polynucleotide or expression cassette according to the disclosure.

[0123] In some embodiments, the vector is an expression vector. In some embodiments, the vector is a eukaryotic expression vector.

[0124] In some embodiments, the vector is a vector that provides the genome of the rAAV in rAAV packaging, i.e., a transgenic plasmid.

[0125] In some embodiments, the vector is a vector that provides the lentiviral genome for lentiviral packaging, i.e., a transgenic plasmid.

[0126] IV. Recombinant Viruses A recombinant adeno-associated virus (rAAV) or a recombinant lentivirus is provided, the rAAV or the recombinant lentivirus comprising within its genome an expression cassette comprising a polynucleotide encoding thiamine pyrophosphokinase (TPK) operably linked to a promoter.

[0127] TPK can be from human or non-human animals, such as non-human mammalian TPK. Examples of non-human mammalian TPK include, but are not limited to, from non-human primates, such as monkeys, horses, cows, sheep, goats, pigs, dogs, cats, mice and rats.

[0128] In some embodiments, the TPK is wild-type TPK. In some embodiments, the TPK is mouse TPK or human TPK.

[0129] In some embodiments, 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.

[0130] In some embodiments, the TPK is a modified TPK that contains one or more amino acid substitutions, insertions, deletions and / or additions compared to the original TPK, and the activity of the modified TPK is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or more of that of the original TPK. In some embodiments, the modified TPK contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, insertions, deletions and / or additions compared to the original TPK. In some embodiments, the modified TPK has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the original TPK.

[0131] The original TPK may be a wild-type TPK or a modified TPK. In some embodiments, the original TPK is a wild-type mouse TPK or a wild-type human TPK.

[0132] In some embodiments, the original TPK comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the original TPK consists of the amino acid sequence of SEQ ID NO: 1.

[0133] In some embodiments, the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35 relative to the original TPK polypeptide, wherein the modified TPK polypeptide has improved catalytic activity for converting thiamine (TM) to thiamine diphosphate (TDP) relative to the original TPK polypeptide. The modified TPK polypeptide has improved catalytic activity for converting thiamine (TM) to thiamine diphosphate (TDP) relative to the original TPK polypeptide, wherein the positions are numbered with reference to SEQ ID NO: 1 or 8.

[0134] Preferably, the amino acid at position 13 is substituted with P. Preferably, the amino acid at position 30 is substituted with A. Preferably, the amino acid at position 31 is substituted with R. Preferably, the amino acid at position 37 is substituted with K. Preferably, the amino acid at position 85 is substituted with K. Preferably, the amino acid at position 129 is substituted with G. Preferably, the amino acid at position 158 is substituted with K. Preferably, the amino acid at position 181 is substituted with S. Preferably, the amino acid at position 11 is substituted with W. Preferably, the amino acid at position 35 is substituted with W.

[0135] In some embodiments, the original TPK polypeptide is a wild-type TPK polypeptide, such as a human TPK polypeptide or a mouse TPK polypeptide.

[0136] In some embodiments, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35. In some embodiments, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of positions 13, 30, 31, 37, 85, 129, 158, and 181. Preferably, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at positions 13, 30, 31, 37, 85, 129, 158, and 181.

[0137] In some embodiments, the original TPK polypeptide is a mouse TPK polypeptide, and the modified TPK polypeptide comprises an amino acid substitution at position 11 and / or position 35.

[0138] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:8 or comprises an amino acid sequence having 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 to SEQ ID NO:8, wherein the modified TPK polypeptide has an amino acid sequence similar to that of SEQ ID NO:8, e.g., at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 37, 85, 129, 158, 181, 11, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 37, 85, 129, 158, 181, 1 ...9, 30, 30, 31, 37, 85, 129, 158, 181, 11, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 30, 30, 30, 30, 30, and 35, for example, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 35, or substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11 and 35.

[0139] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution at position 11 with W and / or at position 35 with W compared to the original TPK polypeptide.

[0140] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution of W at position 11 compared to the original TPK polypeptide.

[0141] In some embodiments, the original TPK polypeptide comprises the amino acid sequence of SEQ ID NO:1 or comprises an amino acid sequence having 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 to SEQ ID NO:1, and the modified TPK polypeptide herein comprises an amino acid substitution of W at position 35 compared to the original TPK polypeptide.

[0142] In some embodiments, the modified TPK polypeptides include the following amino acid substitutions or combinations of amino acid substitutions: 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; or 13P-30A-31R-37K-85K-129G-158K-181S-11W-35W, Includes.

[0143] In some embodiments, the modified TPK polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11 or comprises an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 9, 10 or 11. For example, compared to SEQ ID NO: 9, 10 or 11, the modified TPK polypeptide comprises one or more amino acid substitutions.

[0144] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 13, 30, 31, 37, 85, 129, 158 and 181, preferably other than 241, compared to the amino acid sequence of SEQ ID NO:9.

[0145] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 11 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to the amino acid sequence of SEQ ID NO:10.

[0146] In some embodiments, the modified TPK polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 35 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to the amino acid sequence of SEQ ID NO:11.

[0147] In some embodiments, the modified TPK polypeptide has a catalytic activity for converting TM to TDP that is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300% or more of the activity of the original TPK polypeptide.

[0148] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:15, 16 or 17, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:15, 16 or 17.

[0149] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, and such nucleotide sequence encodes an amino acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 13, 30, 31, 37, 85, 129, 158 and 181, preferably other than 241, compared to SEQ ID NO:9.

[0150] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16, and such nucleotide sequence encodes an amino acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 11 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to SEQ ID NO: 10.

[0151] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17, and such nucleotide sequence encodes an amino acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than 35 and 241, preferably other than 13, 30, 31, 37, 85, 129, 158 and 181, compared to SEQ ID NO: 11.

[0152] In some embodiments, 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 a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3. In some embodiments, the promoter consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3.

[0153] In some embodiments, the promoter is a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the promoter consists of the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the promoter comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the promoter consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7.

[0154] Expression cassettes can include additional regulatory elements, such as enhancers, introns, mRNA stabilizing sequences, and polyadenylation signal sequences.

[0155] In some embodiments, the expression cassette further comprises an mRNA stabilizing sequence, e.g., a WPRE element. In some embodiments, the WPRE element comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the WPRE element consists of the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the WPRE element comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4. In some embodiments, the WPRE element consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4.

[0156] In some embodiments, the WPRE element is downstream of the polynucleotide encoding the TPK.

[0157] In some embodiments, the expression cassette further comprises a polyadenylation signal sequence, such as, but not limited to, a polyadenylation signal sequence of a human growth factor gene and a polyadenylation signal sequence of a 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. In some embodiments, the polyadenylation signal sequence comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:5. In some embodiments, the polyadenylation signal sequence consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:5.

[0158] In some embodiments, a polyadenylation signal sequence is downstream of the WPRE element.

[0159] Non-limiting examples of ITRs in the genome of rAAV include ITRs from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, poultry AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV. In some embodiments, the ITR is the ITR of AAV-2. In some embodiments, the 5'ITR is the same as the 3'ITR. In some embodiments, the 5'ITR is different from the 3'ITR.

[0160] In some embodiments, the 5'ITR and 3'ITRR are both ITR130, e.g., comprising the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each consist of the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each comprise a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each consist of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6.

[0161] In some embodiments, the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, and 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, and SEQ ID NO:6.

[0162] In some embodiments, the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:4, and 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:14, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0163] In some embodiments, the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:4, and 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:15, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0164] In some embodiments, the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:4, and 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:16, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0165] In some embodiments, the genome of the rAAV comprises, from 5' to 3', SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:4, and 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:17, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0166] In some embodiments, the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.

[0167] In some embodiments, the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:4, and SEQ ID NO:5.

[0168] In some embodiments, the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:4, and SEQ ID NO:5.

[0169] In some embodiments, the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:4, and SEQ ID NO:5.

[0170] In some embodiments, the genome of the recombinant lentivirus comprises, from 5' to 3', SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:4, and SEQ ID NO:5.

[0171] The rAAV can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, poultry AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV. In some embodiments, the rAAV is of the serotype AAV_PHP eB.

[0172] It is known in the art that rAAV is packaged using a system that includes three plasmids: 1) a gene transfer plasmid that contains the genome of the rAAV encoding a gene product of interest, ii) a packaging plasmid that encodes the REP and / or CAP proteins, and iii) a 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). Packaging involves the introduction of the system into a host cell.

[0173] Retroviral packaging systems are known in the art and consist of a transfer vector (transgenic plasmid), an envelope vector encoding a selected envelope protein, and a packaging vector that provides the retroviral proteins (such as retroviral GAG and POL proteins, particularly mutant POL proteins to prevent integration) according to methods well known in the art. Packaging of a retrovirus involves the introduction of the system into a host cell.

[0174] Also provided is a host cell comprising a polynucleotide, vector, rAAV or recombinant lentivirus according to the present disclosure. When the host cell is used to package rAAV or recombinant lentivirus virions, it is called a "packaging cell". In some embodiments, the host cell is stably engineered with a polynucleotide or vector according to the present disclosure. In other embodiments, the host cell is transiently engineered with a polynucleotide or vector according to the present disclosure.

[0175] Certain techniques, including but not limited to electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like, are used to stably or transiently introduce a polynucleotide or vector according to the present disclosure into a host cell.

[0176] For stable transformation, a polynucleotide or vector according to the present disclosure will usually further comprise a selectable marker, such as any of a number of well-known selectable markers, such as neomycin resistance.

[0177] The host cell according to the present disclosure may be derived from mammalian cells.Suitable mammalian cells include, but are not limited to, primary cells and cell lines.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. Non-limiting examples of host cells include (for example) HeLa cells (e.g., ATCC No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, N1H3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., 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, and the like.

[0178] V. Pharmaceutical Compositions Provided is a pharmaceutical composition comprising a polynucleotide, vector, rAAV or recombinant lentivirus according to the present disclosure and a pharma- ceutically acceptable carrier, diluent, excipient or buffer. In some embodiments, the pharma- ceutically acceptable carrier, diluent, excipient or buffer is suitable for use in humans.

[0179] Such excipients, carriers, diluents and buffers include any drug that can be used without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol, which may include pharma- ceutical acceptable salts, such as mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, and the like; and organic acid salts, e.g., acetate, propionate, malonate, benzoate, and the like. In addition, auxiliary substances, such as wetting agents, emulsifying agents, pH buffering substances, and the like, may be present in such carriers. A variety of pharma- ceutical acceptable excipients are known in the art that do not require detailed description. Pharmaceutically acceptable excipients are described in detail in various publications, e.g., 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 H. C. Ansel et al, 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., 3rd edition, Amer. Pharmaceutical Assoc.

[0180] VI. Disease Prevention and Treatment A method for promoting sugar metabolism or for preventing or treating impaired glucose metabolism, preferably impaired glucose metabolism in the brain, is provided, comprising administering to a subject in need thereof a polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure. A method for treating or preventing Alzheimer's disease is provided, comprising administering to a subject or patient in need thereof a polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure. In some embodiments, the polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition is administered intravenously, intracerebrally or intraspinally. In some embodiments, the subject or patient is a human.

[0181] Also provided is the use of a polynucleotide, vector, rAAV or recombinant lentivirus, or pharmaceutical composition according to the present disclosure in the manufacture of a medicament for promoting sugar metabolism or preventing or treating glucose metabolic disorders, preferably glucose metabolic disorders in the brain. Also provided is the use of a polynucleotide, vector, rAAV, recombinant lentivirus, or pharmaceutical composition according to the present disclosure in the manufacture of a medicament for treating or preventing Alzheimer's disease. In some embodiments, such medicaments are administered intravenously, intracerebrally, or intrathecally. In some embodiments, such medicaments are administered to humans.

[0182] Further provided is a polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure for use in promoting sugar metabolism or preventing or treating glucose metabolism disorders, preferably glucose metabolism disorders in the brain. Also provided is a polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition for use in treating or preventing Alzheimer's disease. In some embodiments, the polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition is administered intravenously, intracerebrally or intraspinally. In some embodiments, the polynucleotide, vector, rAAV, recombinant lentivirus or pharmaceutical composition is administered to a human.

[0183] Prevention or treatment of Alzheimer's disease includes preventing, alleviating, or eliminating symptoms of AD such as neurodegenerative pathologies such as Alzheimer's disease, brain atrophy, pathological changes of Aβ and tau, neuroinflammation, and neurovascular disorders, or slowing the progression of AD.

[0184] Alzheimer's disease involves damage to multiple brain regions, and the virus according to the present disclosure can selectively and efficiently penetrate the blood-brain barrier and affect multiple brain regions over a wide area. In clinical applications, the rAAV according to the present disclosure can be administered by direct injection (e.g., intracerebral injection or intrathecal injection) across the blood-brain barrier or by peripheral vein injection, and peripheral vein injection can also achieve the same effect as direct injection into the brain. Furthermore, the difference between the present disclosure and other gene therapies is that it only mediates the compensatory expression of TPK exogenously, without involving endogenous TPK, much less editing other genes, and therefore other risks can be significantly reduced. More importantly, the present disclosure introduces highly active exogenous TPK to achieve beneficial therapeutic effects.

[0185] GSK3β is a serine / threonine kinase that is highly conserved during evolution, and in addition to regulating glycogen synthase activity, it also acts on other pathways and can regulate cell differentiation, proliferation, survival, and apoptosis. It has been reported that activation of GSK3β can promote phosphorylation of tau protein, increase Aβ deposition, activate microglia, damage neuronal generation, inhibit LTP, etc., which may lead to the development of Alzheimer's disease (Elisabetta Lauretti, Ozlem Dincer, Domenico Pratico, Glycogen synthase kinase-3 signaling in Alzheimer's disease, Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, Volume 1867, Issue 5, 2020). Advantageously, rAAV according to the present disclosure expresses TPK in vivo (e.g., in the brain), thereby mediating the interaction between the "TPK-TDP glucose metabolism" pathway and the GSK3β pathway, increasing phosphorylated GSK3β (p-GSK3β) levels and the p-GSK3β / GSK3β ratio, and inhibiting GSK3β activity, resulting in beneficial therapeutic effects on AD. EXAMPLES

[0186] Through the following examples, those skilled in the art will understand the present disclosure more clearly. It should be understood that the following examples are intended to illustrate exemplary embodiments of the present disclosure, and are not intended to limit its scope. Unless otherwise specified, the methods used herein are routine in the art, and the experimental materials used are commercially available.

[0187] Example 1 Preparation of rAAV and Recombinant Lentiviruses We commissioned BrainVTA, a CRO company in Wuhan, to prepare a laboratory-scale rAAV encoding TPK (serotype AAV_PHP eB), designated AAV-TPK, whose genome includes i) a neuron-specific promoter (SEQ ID NO: 3), ii) a nucleotide sequence encoding mouse TPK (SEQ ID NO: 2) and a WPRE element (SEQ ID NO: 4). The plasmid containing the genome of such a rAAV is shown in FIG. 1A.

[0188] We commissioned a CRO company, Shanghai OBiO, to prepare a laboratory-scale lentivirus encoding TPK, designated as TPK lentivirus, whose genome includes i) a eukaryotic expression promoter (SEQ ID NO: 7), ii) a nucleotide sequence encoding mouse TPK (SEQ ID NO: 2) and a WPRE element (SEQ ID NO: 4). The plasmid containing the genome of such a lentivirus is shown in FIG. 1B.

[0189] Example 2 Overexpression of TPK and increased TPD content in neural cells infected with TPK lentivirus The purpose of this example was to preliminarily evaluate the effectiveness of gene therapy through cell-level experiments.

[0190] The cerebral cortex of newborn mice (purchased from Zhejiang Charlesriver) was treated with trypsin (Thermofisher / Gibco, Catalog No.: 25200072) to collect primary neurons, which were then plated in a 6-well plate (1.5x10 6The cells were cultured in Neurobasal medium + 2% B27 + 1% Glutemax + 78% DMEM + 1% 100X penicillin-streptomycin double antibody (5000U / mL) for 4 days (on day 1, 10% FBS + 10% F12 + 1% Glutemax + 78% DMEM + 1% 100X penicillin-streptomycin double antibody (5000U / mL) was added, where the above reagents were mixed in volume ratios and all purchased from Therofisher / Gibco). The medium was removed, and the lentivirus suspension prepared in Example 1 (MOI 5, 1.5mL / well) was added and infected for 8 hours, where the lentivirus encoding GFP without the coding sequence of TPK was used as a control. The liquid was removed, and fresh culture liquid was added for 7 days of culture. The expression of GFP was observed under a fluorescent microscope. As shown in Figure 2, a large number of infected cells expressed GFP, indicating a high infection rate of the virus.

[0191] The medium was removed and the cells were washed three times with pre-chilled PBS for 5 min each time. 100 μL of cell lysis solution was added to each well and RIPA (purchased from Beyotime), phosphatase inhibitor (purchased from Bimake), and protease inhibitor (purchased from Sigma) were added in a ratio of 100:1:1 (v:v:v). All cells were scraped off and the cell suspension was transferred to a 1.5 mL centrifuge tube on ice. 50 μL of cell lysis solution was added to each well, washed the bottom of the well and transferred to the same centrifuge tube. Two grinding steel balls were added to each tube for grinding (60 Hz, 60 s, -10°C), centrifuged at 13000 rpm, 4°C for 15 min, and the supernatant was stored on ice.

[0192] The total protein concentration in the supernatant was determined by the BCA method. After the extracted total protein was denatured at 95°C, the TPK level in the samples was analyzed by Western blotting. Briefly, after denaturation, the samples were loaded into the wells of a polyacrylamide gel (the sample to be tested was 15 μg protein per well, and the protein size indicator (marker) was 5 μL), and after electrophoresis, the membrane was transferred and the membrane corresponding to the molecular weight region of the target protein was cut out according to the marker (TPK is 25-35 KD, β-actin is 40-55 KD). The membrane was blocked with blocking solution (purchased from Takara, catalog number: T7131A) for 1 hour at room temperature, and then incubated overnight at 4°C with the following primary antibodies: anti-TPK1 antibody (purchased from Proteintech, catalog number: 10942-1-AP) and mouse anti-β-actin antibody (purchased from Beyotime, catalog number: AF0003) overnight at 4°C. The primary antibody was removed, and the membrane was washed three times with 1XTBST for 5 min each. The corresponding secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (purchased from Beyotime, Catalog No.: A0208) and horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (purchased from Beyotime, Catalog No.: A0216), were added and incubated at room temperature for 2 h, then washed three times with 1xTBST and developed with a horseradish peroxidase kit (Cat. No.: 34095, purchased from Thermo Fisher). As shown in Figure 3A, TPK was significantly overexpressed in cells infected with the TPK retrovirus.

[0193] 100 μL of the supernatant was collected, and an equal volume of pre-cooled 5.4% perchloric acid (PCA) was added, followed by shaking well and centrifuging at 13,000 rpm at 4° C. for 15 minutes. HPLC analysis of the supernatant was performed using the apparatus and conditions shown in Table 1.

[0194] [Table 1]

[0195] As shown in Fig. 3B, the TDP content in cells infected with the TPK retrovirus was significantly higher than that in uninfected cells and cells infected with the control virus. Meanwhile, the TM content in AAV-TPK cells was significantly lower than that in uninfected cells and cells infected with the control virus (Fig. 3C). These results indicate that overexpression of TPK significantly promoted the conversion of TM to TDP in cells infected with the TPK retrovirus, and the conversion rate was approximately twice that of the control group.

[0196] Example 3: Overexpression of TPK and increased TM / TDP conversion rate in the brains of mice injected with AAV-TPK via the tail vein 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 and increase the TM / TDP conversion rate in the mouse brain, thereby improving glucose metabolism by tail vein injection of AAV into mice.

[0197] After 3 days of adaptive feeding, adult C57BL / 6J mice (2 months old) (purchased from Zhejiang Charlesriver) were randomly divided into two groups (TPK group and control group, n=3), and AAV-TPK (TPK group) and control rAAV (same as TPK-AAV except for the coding sequence of TPK) were administered to mice in each group via tail vein injection at a titer of 5.0E+12vg / mL (prepared with saline), 200μL each. Specifically, mice were fixed in a tail vein injection fixture and their tails were disinfected with alcohol cotton. Using a 1ml syringe, 200μL of virus suspension was injected into the tail vein of the mice, and the injection site was pressed with a dry cotton ball for 30 seconds after injection to prevent virus leakage. The mice were kept in the experimental area for 4 weeks, and their body weight and food intake were recorded every week.

[0198] As shown in FIG. 4, after AAV-TPK was injected into the tail vein of adult mice, body weight gain was significantly smaller than in the control group, and food intake also tended to be reduced compared to the control group.

[0199] After 4 weeks of feeding, whole blood of the mice was collected using 3 mL EDTA anticoagulant tubes, which were immediately placed on ice. The following procedures were performed on ice. In 10 minutes, 0.3 mL of whole blood sample was transferred to a 2 mL centrifuge tube, to which 30 μL of pure water was added, followed by the addition of 0.3 mL of pre-cooled PCA (5.7%) to precipitate proteins. After about 30 minutes of ice bath, the sample was centrifuged at 4°C, 12000 rpm for 10 minutes. The supernatant was collected and analyzed for TDP and TM content by HPLC as described in Example 2.

[0200] As shown in Figure 5, after AAV-TPK was administered via the tail vein to adult mice for 4 weeks, there was no significant change in the TDP / TM content in the peripheral blood.

[0201] Mice were euthanized and then perfused cardiacly with pre-chilled PBS. Mouse brain tissue was harvested (cerebellum, brain stem, and olfactory bulbs were removed, and the left and right brains were separated).

[0202] The right brain was fixed with 4% paraformaldehyde (PFA) and immunofluorescence stained. Specifically, after fixation with 4% PFA for 12-24 hours, the right brain was dehydrated and sugar precipitated with 30% sucrose for 2 days. After sugar precipitation, the right brain was embedded in OCT (Oxygen-Cortaneous Tissue) and frozen at -80°C. The embedded right brain was sliced ​​into thin sections (30 μm) in the longitudinal (coronal) direction from the olfactory bulb to the cerebellum using a Cryoslicer. The slices were washed 3-5 times with 1XPBS to remove the OCT. 0.5% Triton was added for 30 minutes at room temperature to destroy the membrane, and then replaced with 3% BSA (bovine serum albumin) and blocked for 1 hour at room temperature. The primary antibody (anti-GFP antibody, purchased from Avessellabs, catalog number GFP-1020) was added and incubated overnight at 4°C. The primary antibody was removed and washed with 1x PBS three times for 5 min each time. Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (purchased from Invitrogen, Cat. No. A11008) was added as the secondary antibody and incubated for 2 h in the dark. Finally, the thin sections were applied to glass slides, dried a little, and then mounted with a drop of mounting medium. Images were taken using a Nikon Ti2E inverted microscope.

[0203] As shown in Figure 6, 4 weeks after tail vein injection of AAV-TPK into adult mice, the polypeptides encoded by the rAAV virus could be widely expressed in brain tissues.

[0204] The left brain or liver tissue (upper left lobe) was placed in 1.5 ml of cell lysis solution (see Example 2), lysed by freeze grinder (70 Hz, 60 seconds, twice, 20 seconds interval), centrifuged at 13000 rpm, 4°C for 15 minutes, and the supernatant was collected. Western blot and HPLC analysis were performed to detect the TPK expression and TDP and TM content in the brain as described in Example 2.

[0205] As shown in Figure 7, 4 weeks after tail vein injection of AAV-TPK in adult mice, TPK expression in brain tissue was significantly increased, TDP content was significantly increased, and TM content was comparable to that of the control.

[0206] As shown in Figure 8, TM and TDP in the liver showed no significant changes, indicating that the virus had good brain specificity and specifically targeted brain neurons without affecting peripheral tissues. The side effects of the virus were completely controllable.

[0207] Example 4: Overexpression of TPK and increased TM / TDP conversion rate in the brains of mice injected with AAV-TPK into the lateral ventricle The purpose of this example is to verify whether injection of AAV-TPK virus into the lateral ventricle of mice can significantly increase the expression of TPK protein in the brains of C57BL / 6J mice and increase the TM / TDP conversion rate in the mouse brain, thereby improving glucose metabolism.

[0208] After 3 days of adaptation feeding, adult C57BL / 6J mice (2 months old) (purchased from Zhejiang Charlesriver) were randomly divided into two groups (TPK group and control group, n = 5) for stereotaxic injection of AAV into the lateral ventricles of the brain.

[0209] The concentration of the virus stock solution used was 1.0E+13vg / 1mL, and the amount injected into the lateral ventricles of the mice was 4.0E+10vg / 4μL per mouse (2μL each into the left and right lateral ventricles).

[0210] Adult mice were anesthetized with Zoletil 50, the hair between the ears and eyes was shaved, and the mouse was fixed in a stereotaxic apparatus. The skin of the mouse brain area was wiped with an alcohol cotton ball and disinfected. The skin was opened with scissors to expose the skull. The surface of the skull was wiped dry with a dry cotton swab until the frontal fontanelle was clearly visible. The coordinates of the frontal fontanelle were designated as 0 (AP: -0.2 mm, L / R: ±1.0 mm, H: 2.5 mm) and the drill point was marked. A hole was drilled in the skull with a 0.5 mm drill bit, and 2 μL of AAV virus was aspirated with a 10 μL microinjection needle. The needle depth was 2.5 mm, and the skull surface was designated as the 0 point of the Z axis. Injection speed: 0.2 μL / min, injection was stopped for 5 min, and the injection needle was slowly removed. After the skin was sutured, the mice were placed in a cage equipped with a heating pad and fed in the experimental area of ​​the feeding cage for 4 weeks after awakening. Body weight and food intake were recorded weekly.

[0211] As shown in FIG. 9, there was no significant difference in body weight and food intake between adult mice injected with AAV-TPK into the lateral ventricle and the control group.

[0212] After 4 weeks of feeding, samples were taken and the levels of TDP and TM in the blood, the expression of polypeptides encoded by the rAAV virus in the brain, and the expression levels of TPK and the levels of TDP and TM in brain and liver tissues were detected using the methods described in Example 3.

[0213] As shown in Figure 10, 4 weeks after AAV-TPK injection into the lateral ventricle of adult mice, there was no significant change in the TDP / TM content in the peripheral blood.

[0214] As shown in Figure 11, 4 weeks after AAV injection into the lateral ventricles of adult mice, the gene products encoded by the rAAV viruses were strongly expressed around the lateral ventricles and were also expressed in several other brain regions.

[0215] As shown in Figure 12, the expression of TPK in brain tissue was significantly increased, the TDP content was significantly increased, and the TM content was decreased, indicating that 4 weeks after AAV-TPK injection into the lateral ventricle of adult mice, the TM / TDP conversion rate was increased compared with the control group.

[0216] As shown in Figure 13, the TM and TDP in the liver were not significantly changed, indicating that the virus has brain specificity and specifically targets brain nerve cells without affecting peripheral tissues. The side effects of the virus were completely controllable.

[0217] Example 5: Overexpression of TPK and increased TM / TDP conversion rate in mouse brains developed from embryos injected into the lateral ventricle with AAV-TPK The purpose of this example is to verify whether gene therapy by direct delivery of AAV to the lateral ventricles of fetal mice can significantly increase the expression of TPK in brain neurons of C57BL6 / J mice and improve the TM / TDP conversion rate in neurons, thereby enhancing glucose metabolism.

[0218] C57BL6 / J mice (embryonic day 13.5, E13.5) purchased from Zhejiang Charlesriver were adapted and housed for 1 day (E14.5) and randomly divided into two groups (n = 3), the TPK group (injected with AAV-TPK) and the control group (injected with control virus).

[0219] Mice were deeply anesthetized and placed on their ventral side, and the abdomen of the pregnant mouse was wiped with tissue soaked in 75% alcohol. The hair in the center of the abdomen was clipped off, and the abdomen of the pregnant mouse was wiped with tissue soaked in PBS to remove residual alcohol and loose hair.

[0220] The surgical instruments were wiped with 75% alcohol for disinfection. The skin of the pregnant mouse was incised approximately 1.5 cm along the center of the abdomen with scissors, and the peritoneum of the pregnant mouse was incised along the white line of the abdomen to expose the abdominal cavity. Using tweezers, the embryos were carefully removed from the abdominal cavity of the pregnant mouse and placed in the abdominal cavity of the pregnant mouse (the abdominal cavity and the surface of the embryo were kept moist with PBS during the following surgical procedures). A glass electrode absorbed with rAAV virus suspension was lightly pierced into the brain of the embryo, air was blown in to move the virus suspension into the lateral ventricle of the embryo, and 0.5 to 1 μL (5.0E+9 to 1.0E+10 vg) of the virus suspension was injected into the lateral ventricle of each embryo. The embryos were returned to the abdominal cavity of the pregnant mouse, and the peritoneum and skin of the mouse were sutured in order. After suturing, lidocaine-lincomycin ointment was applied to the wound to prevent inflammation and infection, and the mouse was placed on a heating pad until it woke up, after which it was returned to its cage for continued care.

[0221] After birth, the pups were irradiated with 480 nm excitation light on the head to test the results of the virus injection, and the mice with successful virus injection were left. After 21 days of breeding, three mice were treated as described in Example 3, and samples were taken to detect the levels of TDP and TM in blood, the expression of polypeptides encoded by the rAAV virus in the brain, and the expression levels of TPK, TDP and TM in brain and liver tissues. The other 12 mice were weaned and their weights were recorded. They were kept in separate cages for two weeks, and their weights were recorded again.

[0222] As shown in FIG. 14, there was no significant difference in body weight change during the 2 weeks after weaning between mice developed from embryos injected with AAV-TPK and the control group.

[0223] As shown in FIG. 15, there was no significant change in peripheral blood TDP / TM content in mice developed from embryos injected with AAV-TPK.

[0224] As shown in Figure 16, in mice that developed from rAAV-injected embryos, the gene products encoded by the rAAV virus were widely expressed in several brain regions, including the cerebral cortex and hippocampus.

[0225] As shown in Figure 17, compared with the control, the expression of TPK and TDP content in brain tissues of mice developed from AAV-TPK-injected embryos were significantly increased, while the TM content was decreased, indicating an increased TM / TDP conversion rate.

[0226] As shown in Figure 18, TM and TDP in the liver showed no significant changes, indicating that the virus has high brain specificity and specifically targets brain neurons without affecting peripheral tissues. The side effects of the virus were completely controllable.

[0227] Example 6: Overexpression of TPK and increased TM / TDP conversion rates in the brains of neonatal mice stereotaxically injected with AAV-TPK The purpose of this example is to verify whether direct delivery of AAV-TPK to the lateral ventricles (intracerebroventricular, ICV) and (intracerebral parenchyma, IP) of neonatal mice (P0 mice on postnatal day 0) can significantly increase TPK expression in brain neurons of C57BL6 / J mice and improve the TM / TDP conversion rate in neurons, thereby enhancing glucose metabolism.

[0228] A total of 11 C57BL6 / J mice (embryonic day 18.5, E18.5) were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. Experiments were performed after the newborn mice were born.

[0229] Lateral ventricle injection site: 1 mm anterior to lambda, 1 mm left and right, 1 mm deep. 800 nL of virus suspension (1.0E+13 vg / mL) was injected into both lateral ventricles.

[0230] Hippocampal injection site (1): 0.7 mm anterior, 1.2 mm left and right from lambda, 0.9 mm deep. Hippocampal injection site (2): 0.1 mm anterior, 2.2 mm left and right from lambda, 1.4 mm deep. 200 nL of the virus suspension (1.0E+13 vg / mL) was injected into 4 sites in total, 2 sites on each side of the hippocampus.

[0231] After filling the glass electrode with paraffin oil, it was attached to the microinjector using a hot melt gun. The needle of the microinjector was inserted approximately one-third of the way into the glass electrode, and the microinjector was attached to an automatic injection pump.

[0232] The body of the newborn mouse was buried in ice for about 3 minutes with its nose uncovered to allow breathing. After the P0 mouse turned from purplish pink to slightly whitish without struggling, it was removed from the ice and placed on ice for the next experiment.

[0233] The virus suspension was sucked into the glass electrode using a syringe pump. A neonatal mouse was deeply anesthetized with ice treatment and fixed to a horizontal container with medical tape so that the head was kept horizontal.

[0234] Under a stereomicroscope, the syringe was moved to the specified position by moving the operating arm, starting from the occipital fontanelle (lambda) of the newborn mouse, and the vertical operating arm (Z-axis) adjusted the up and down position of the syringe. The position where the needle sank slightly after touching the head was set as the zero point, and the needle was lowered to the specified position. Finally, the injection pump controller automatically injected the virus into the brain of the newborn mouse. After the injection was completed, the needle remained in the brain of the newborn mouse for about 3 minutes and then was slowly withdrawn.

[0235] After injection, the newborn mice were quickly transferred to a heating pad. After the newborn mice had regained body temperature and started to move, the animals were returned to their mother's cage, covered with a cage pad, and left for a period of time to allow the odor to soak in. The stereotaxic experiment ended when the mother spontaneously returned the newborn mice to the nest.

[0236] After 21 days of feeding, three mice from each treatment group were treated as described in Example 3, and samples were taken to detect TDP and TM levels in blood, expression of polypeptides encoded by rAAV virus in brain, TPK expression levels, and TDP and TM levels in brain and liver tissue. The other mice (n=11 injected into the lateral ventricle, n=8 injected into the hippocampus) were weaned and their weights were recorded. The animals were then separated into cages, fed for 2 weeks, and their weights were recorded again.

[0237] As shown in Figure 19, 3 weeks after AAV-TPK was injected into the lateral ventricle, there was no significant change in body weight within 2 weeks after weaning compared to the control group of P0 mice. Also, 3 weeks after AAV-TPK was injected into the hippocampus, there was no significant change in body weight within 2 weeks after weaning compared to the control group of P0 mice (Figure 23).

[0238] As shown in Figure 24, 3 weeks after AAV-TPK injection into the hippocampus of P0 mice, there was no significant change in the contents of TDP and TM in the peripheral blood.

[0239] As shown in Figure 21, 3 weeks after injection of rAAV into the lateral ventricle of P0 mice, the gene products encoded by rAAV were widely expressed in the cerebral cortex, hippocampus, and several other brain regions, and 3 weeks after injection of rAAV into the hippocampus of P0 mice, the gene products encoded by rAAV were strongly expressed in the hippocampus and several other brain regions (Figure 25).

[0240] As shown in Figure 22, 3 weeks after rAAV administration into the lateral ventricle in P0 mice, the expression of TPK and the content of TDP were significantly increased in brain tissue compared with the control group, while the content of TM was decreased, indicating that the TM / TDP conversion rate was increased.

[0241] As shown in FIG. 26, 3 weeks after AAV-TPK injection into the hippocampus of P0 mice, the expression level of TPK in brain tissues was significantly increased compared with the control group.

[0242] As shown in Figure 27, TM and TDP in the liver did not change significantly, indicating that the virus was highly brain specific and specifically targeted brain neurons without affecting peripheral tissues. The side effects of the virus were completely controllable.

[0243] Example 7: Screening for TPK mutants with high activity In this example, mutants having higher activity than the wild-type TPK polypeptide were screened for by introducing amino acid substitutions into the TPK polypeptide.

[0244] By comparing the structures of human and mouse TPK proteins, the inventors selected amino acids that may have a significant effect on TPK activity, and designed and prepared the TPKs and their mutants shown in Table 2 by single and multiple mutations in these amino acids.

[0245] [Table 2]

[0246] Specifically, the nucleotide sequences encoding the above TPKs (SEQ ID NOs: 1 and 14-19) were cloned into the pMCSG7-(+) backbone and transformed into E. coli BL21(DE3) competent cells. The cells were then spread onto LB agar medium (containing 50 mg / L kanamycin) and cultured overnight at 37°C. A single colony was then transferred to LB liquid medium (containing 50 mg / L kanamycin) for sequence confirmation.

[0247] Confirmed clones were grown on LB agar medium. A single colony was then inoculated into LB broth (containing 50 mg / L kanamycin) and grown at 37°C for 12 hours. 1 mL of the culture was transferred to 50 mL of fresh LB broth (containing 50 mg / L kanamycin) and grown at 37°C until the OD600 reached approximately 0.6. IPTG (final concentration 0.5 mM) was added and grown at 25°C for 16 hours to induce protein expression.

[0248] After the incubation, the cells were collected by centrifugation at 5000g for 5 minutes at 4°C and the supernatant was discarded. The collected cells were resuspended in pre-cooled protein equilibration solution (50mM HEPES, 2mM MgCl2, 250mM NaCl, 10% glycerol) at a ratio of 1:8, and the cells were sonicated at 4°C. The cell lysate was centrifuged at 12000rpm for 60 minutes at 4°C. The supernatant was collected and passed through a nickel column (GE, 17-3712-02). The nickel column was washed with 30mM imidazole solution prepared in the protein equilibration solution to remove non-specifically bound impurity proteins. The nickel column was eluted with 500mM imidazole solution prepared in the protein equilibration solution, and the eluate containing the target protein was collected and dialyzed in a 10KD aperture concentration tube from Merck Millipore, and the imidazole concentration was removed until it was less than 5mM. The dialysate was further purified by FPLC molecular sieving (SEC column: superdex 200 increase; PBS buffer, pH 7.4), the target protein solution was recovered, and the protein was concentrated using a 10KD open-cell concentration tube from Merck Millipore.

[0249] The experimental materials for detecting the enzyme activity are shown in Table 3.

[0250] [Table 3]

[0251] 50 mM TPK enzyme solution was mixed with equal amounts of 150 mM Tris HCl buffer and 10 mM ATP solution, and 210 μL of the mixture was transferred to a centrifuge tube. 30 μL of DMSO was added to each centrifuge tube, and finally 30 μL of thiamine solution (20 μM) was added, and the mixture was incubated at 37 ° C for 0.5 hours. Then, 270 μL of 5% PCA quenching solution was added and mixed well to stop the reaction. 150 μL of the above mixture was transferred to a 1.5 mL centrifuge tube, and 30 μL of 10 mM potassium ferricyanide derivatization reagent was added for sample derivatization, and finally 15 μL of 1 M phosphoric acid neutralization solution was added to stop the reaction. As described in Example 2, the TDP / TM content was detected by high performance liquid chromatography. TPK enzyme activity = TDP (nM) / mg TPK / min

[0252] As shown in Figure 28, compared with human TPK (hTPK), the mutant hTPK 8M had significantly increased catalytic activity in the conversion of TM to TDP (p<0.05), but the catalytic activity was still lower than that of wild-type mouse TPK (mTPK) (p<0.05). As shown in Figure 29, compared with wild-type mTPK, the mutant mTPK 11W and mTPK 35W had higher catalytic activity in the conversion of TM to TDP, of which the mutant mTPK 11W showed the highest activity (p<0.01 vs. mTPK), but the introduction of the 241W substitution reduced the activity.

[0253] Example 8: Effect of rAAV encoding TPK mutants on TPK activity in vivo In this example, the therapeutic efficacy of rAAV encoding a TPK mutant with increased activity in neonatal and APP / PS1 mice was investigated.

[0254] An rAAV encoding hTPK, designated AAV hTPK, whose genome comprises i) a neuron-specific promoter (sequence number 3), ii) a nucleotide sequence encoding hTPK (sequence number 14) and a WPRE element (sequence number 4), was prepared as described in Example 1.

[0255] 8.1.Overexpression of TPK and increased TM / TDP conversion rate in the brains of neonatal mice injected with AAV-hTPK into the lateral ventricle The purpose of this example is to verify whether direct delivery of AAV-hTPK into the lateral ventricle (intracerebroventricular, ICV) of neonatal mice (P0 mice on postnatal day 0) can significantly increase TPK expression in brain neurons of C57BL6 / J mice and improve the TM / TDP conversion rate in neurons, thereby enhancing glucose metabolism.

[0256] A total of 11 C57BL6 / J mice (embryonic day 18.5, E18.5) were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. The total amount of virus stock solution was 2E10vg (800nL), which was diluted to 1 / 5, 1 / 25, 1 / 100, and 1 / 200 of the stock solution concentration, respectively. rAAV without the TPK coding sequence was used as a control. The experiment was performed after the birth of newborn mice.

[0257] Lateral ventricle injection site: 1 mm anterior to lambda, 1 mm left and right, 1 mm deep. 800 nL of virus suspension was injected into the left and right lateral ventricles.

[0258] After filling the glass electrode with paraffin oil, it was attached to the microinjector using a hot melt gun. The needle of the microinjector was inserted approximately one-third of the way into the glass electrode, and the microinjector was attached to an automatic injection pump.

[0259] The body of the newborn mouse was buried in ice for about 3 minutes with its nose uncovered to allow breathing. After the P0 mouse turned from purplish pink to slightly whitish without struggling, it was removed from the ice and placed on ice for the next experiment.

[0260] The virus suspension was sucked into the glass electrode using a syringe pump. A neonatal mouse was deeply anesthetized with ice treatment and fixed to a horizontal container with medical tape so that the head was kept horizontal.

[0261] Under a stereomicroscope, the syringe was moved to the specified position by moving the operating arm, starting from the occipital fontanelle (lambda) of the newborn mouse, and the vertical operating arm (Z-axis) adjusted the up and down position of the syringe. The position where the needle sank slightly after touching the head was set as the zero point, and the needle was lowered to the specified position. Finally, the injection pump controller automatically injected the virus into the brain of the newborn mouse. After the injection was completed, the needle remained in the brain of the newborn mouse for about 3 minutes and then was slowly withdrawn.

[0262] After injection, the newborn mice were quickly transferred to a heating pad. After the newborn mice had regained body temperature and started to move, the animals were returned to their mother's cage, covered with a cage pad, and left for a period of time to allow the odor to soak in. The stereotaxic experiment ended when the mother spontaneously returned the newborn mice to the nest.

[0263] After 21 days of feeding, three mice from each treatment group were treated as described in Example 3, samples were taken, and the TPK expression level and TDP content in brain tissue were detected.

[0264] As shown in Figure 30, the expression of TPK protein in brain tissues changed in a dose-dependent manner according to the titer gradient of the injected virus. HPLC results showed that injection of AAV-hTPK (ranging from stock solution to 100-fold dilution) dose-dependently decreased the TDP content in brain tissues, similar to the virus dilution (Figure 31).

[0265] 8.2.Effects of lateral ventricle injection of AAV-TPK on APP / PS1 mice In this example, we verify whether direct delivery of AAV (AAV-TPK prepared in Example 1) into the lateral ventricle (intracerebroventricular, ICV) of adult APP / PS1 mice significantly increases the expression of TPK in brain neurons of APP / PS1 mice, improves the conversion rate of TM / TDP in neurons, and thereby enhances glucose metabolism.

[0266] After 3 days of adaptive feeding, adult APP / PS1 male mice (13 months) (purchased from Cavens Biogle (Suzhou) Model Animal Research Co. Ltd.) were randomly divided into two groups (TPK group and control group, n = 12) and stereotaxic injection of AAV into the lateral ventricle was performed. The concentration of the virus stock solution used was 1.0E+13vg / 1mL, and the injection volume into the lateral ventricle was 4.0E+10vg / 4μL per mouse (2μL each for the left and right). Adult mice were anesthetized with isoflurane, the hair between the ears and eyes was shaved, and the mice were fixed in a stereotaxic apparatus. The skin of the mouse brain was wiped with an alcohol cotton ball and disinfected. The skin was cut open with scissors to expose the skull. The surface of the skull was wiped dry with a dry cotton swab until the frontal fontanelle was clearly visible. The coordinates of the frontal fontanelle were designated as 0 (AP: -0.2 mm, L / R: ±1.0 mm, H: 2.5 mm) and the drill point was marked. A hole was drilled into the skull with a 0.5 mm drill bit, and 2 μL of AAV virus was aspirated with a 10 μL microinjection needle. The needle depth was 2.5 mm, and the skull surface was designated as the 0 point of the Z axis. Injection rate: 0.2 μL / min, injection was stopped for 5 min, and the needle was slowly removed. After suturing the skin, the mice were placed in a cage equipped with a heating pad and fed in the experimental area of ​​the feeding cage for at least 8 weeks after awakening, waiting for the expression of AAV. Body weight, food intake, and blood glucose level were recorded every week (at specific time points).

[0267] As described in Example 3, blood, brain and liver tissue samples were collected to detect TDP and TM levels in blood, TPK expression levels, and TDP and TM contents in brain and liver tissues (n=4).

[0268] As shown in Figure 32, there was no significant difference in body weight, food intake, or blood glucose level between mice administered AAV-TPK and mice administered control AAV, indicating that administration of AAV-TPK is safe.

[0269] As shown in FIG. 33, the expression level of TPK was high in the brain tissue of mice injected with AAV-TPK, while there was little TPK expression in the brain tissue of mice injected with control AAV; the expression level of TPK in the liver of mice injected with AAV-TPK was almost equivalent to that of mice injected with control AAV, indicating that the AAV according to the present disclosure does not exhibit off-target effects.

[0270] As shown in Figure 34, the TDP content in brain tissue of APP / PS1 mice injected with AAV-TPK was significantly higher than that of APP / PS1 mice injected with control AAV, indicating that the TDP content in blood and liver tissue of APP / PS1 mice injected with AAV-TPK was comparable to that of APP / PS1 mice injected with control AAV, and that the AAV according to the present disclosure does not exhibit off-target effects.

[0271] After 6 months of feeding, samples were taken and GSK3β protein levels were detected in brain tissue (the only difference was the primary antibody) as described in Example 3. The primary antibodies were GSK-3β (27C10) (purchased from CST, catalog number 9315) and phosphorylated GSK-3β protein (p-GSK-3β) (serine 9) (primary antibody purchased from CST, catalog number 14630).

[0272] As shown in Figures 35 and 36, the p-GSK3β level and the p-GSK3β / GSK3β ratio in the brain tissue of APP / PS1 mice administered AAV-TPK into the lateral ventricle were higher than those in APP / PS1 mice administered control AAV, indicating an interaction between the "TPK-TDP-glucose metabolism pathway" and the p-GSK3β pathway. [Sequence List Free Text]

[0273] SEQ ID NO: 1: Amino acid sequence of mouse TPK MEHAFTPLEPLLPTGNLKYCLVVLNQPLDARFRHLWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAIKS

[0274] SEQ ID NO:2 Nucleotide sequence of mouse TPK ATGGAGCATGCCTTTACCCCGTTGGAACCCCTGCTACCTACGGGGAACTTGAAATACTGCCTTGTGGTTCTTAATCAGCCTTTGGATGCACGATTTCGCCATCTTTGGAAAAAGCTCTCTTAAGAGCCTGTGCTGATGGGGGTGCCAACCACTTATGATCTCACTGAAGGAGAGAGAGAA AGCTTCTTGCCTGAATTCGTCAGTGGGGACTTTGATTCTATTAGGCCTGAAGTCAAAGAGTACTACACCAAGAAGGGCTGTGATCTTATTTCAACTCCTGACCAAGACCACACTGACTTTACCAAGTGTCTTCAAGTGCTCCAAAGGAAGATAGAGGAAAGGAACTGCAGGTTGACGTG ATTGTGACACTGGGAGGTCTCGGTGGGCGTTTTGACCAAATCATGGCCTCTGTGAATACCCTTTTCCAAGCCACTCACATCACTCCTGTGCCGATTATAATAATCCAAAAGGACTCTCTCATCTACCTCCTCCAACCCGGGAAGCACAGGCTCCATGTAGACACTGGAATGGAAGGGAGCTGGTGTGG CCTGATTCCTGTTGGACAGCCTTGCAACCAGGTGACGACAACAGGCCTGAATGGAACCTCACAAATGATGTCTTGGCTTTGGAACACTGGTCAGTACTTCTAACACCTACGATGGGTCCGGCCTTGTCACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCATCAAGAGCTAA

[0275] sequence number 3 プロマーマーのnucleotide sequence AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGAAACAGGATGCGGCGAGGC GCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTGCCCGCCTGGCGGCGCGCGCCCGCCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCGCAAACTCCCCTCCGGCCACCTTGGTCGCGTCCGGCCC GCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCGGCGACTCAGCGCTGCCTCAGTCTGCGTGGGCAGCGGAGGAGTCTCGTGCCTGAGAGCGCAG

[0276] sequence number 4 WPRE's nucleotide sequence AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTAT AAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCC CTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC

[0277] SEQ ID NO: 5 Polyadenylation signal sequence of human growth factor gene CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGC ATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0278] SEQ ID NO: 6 ITR AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0279] SEQ ID NO:7 Eukaryotic promoter GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCCCCCATTGACGTCAATGGGAGTTTGTTTT GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTTACGTGGGAGGTCTATATAAGCAGAGCT

[0280] SEQ ID NO:8 Amino acid sequence of human TPK MEHAFTPLEPLLSTGNLKYCLVILNQPLDNYFRHLWNKALLRACADGGANRLYDITEGERESFLPEFINGDFDSIRPEVREYYATKGCELISTPDQDHTDFTKCLKMLQKKIEEKDLKVDVIVT LGGLAGRFDQIMASVNTLFQATHITPFPIIIIQEESLIYLLQPGKHRLHVDTGMEGDWCGLIPVGQPCMQVTTTGLKWNLTNDVLAFGTLVSTSNTYDGSGVVTVETDHPLLWTMAIKS

[0281] SEQ ID NO: 9 Human TPK 8M amino acid sequence MEHAFTPLEPLLPTGNLKYCLVILNQPLDARFRHLWKKALLRACADGGANRLYDITEGERESFLPEFINGDFDSIRPEVREYYAKKGCELISTPDQDHTDFTKCLKMLQKKIEEKDLKVDVIVTLG GLGGRFDQIMASVNTLFQATHITPFPIIIIQKESLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCMQVTTTGLKWNLTNDVLAFGTLVSTSNTYDGSGVVTVETDHPLLWTMAIKS

[0282] SEQ ID NO: 10 Mouse TPK 11W amino acid sequence MEHAFTPLEPWLPTGNLKYCLVVLNQPLDARFRHLWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAIKS

[0283] SEQ ID NO: 11 Mouse TPK 35W amino acid sequence MEHAFTPLEPLLPTGNLKYCLVVLNQPLDARFRHWWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAIKS

[0284] SEQ ID NO: 12 Mouse TPK 241W amino acid sequence MEHAFTPLEPLLPTGNLKYCLVVLNQPLDARFRHLWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAWKS

[0285] SEQ ID NO: 13 Mouse TPK 11W+241W amino acid sequence MEHAFTPLEPWLPTGNLKYCLVVLNQPLDARFRHLWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAWKS

[0286] SEQ ID NO: 14 Human TPK nucleotide sequence ATGGAGCATGCCTTTACCCCGTTGGAGCCCCTGCTTTCCACTGGGAATTTGAAGTACTGCCTTGTAATTCTTAATCAGCCTTTGGACAACTATTTTCGTCATCTTTGGAACAAAGCTCTTTTAAGAGCCTGTGCCGATGGAGGTGCCAACCGCTTATATGATATCACCGAAGGAGAGAGAGAAAGCTTTTTGCCTGAATTCATCAATGGAGACTTTGATTCTATTAGGCCTGAAGTCAGAGAATACTATGCTACTAAGGGATGTGAGCTCATTTCAACTCCTGATCAAGACCACACTGACTTTACTAAGTGCCTTAAAATGCTCCAAAAGAAGATAGAAGAAAAAGACTTAAAGGTTGATGTGATCGTGACACTGGGAGGCCTTGCTGGGCGTTTTGACCAGATTATGGCATCTGTGAATACCTTGTTCCAAGCGACTCACATCACTCCTTTTCCAATTATAATAATCCAAGAGGAATCGCTGATCTACCTGCTCCAACCAGGAAAGCACAGGTTGCATGTAGACACTGGAATGGAGGGTGATTGGTGTGGCC TTATTCCTGTTGGACAGCCTTGTATGCAGGTTACAACCACAGGCCTCAAGTGGAACCTCACAAATGATGTGCTTGCTTTTGGAACATTGGTCAGTACTTCCAATACCTACGACGGGTCTGGTGTTGTGACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCATCAAAAGCTAA

[0287] SEQ ID NO:15 Nucleotide sequence of human TPK 8M ATGGAGCATGCCTTTACCCCGTTGGAGCCCCTGCTTCCTACTGGGAATTTGAAGTACTGCCTTGTAATTCTTAATCAGCCTTTGGACGCGCGCTTTCGTCATCTTTGGAAGAAAGCTCTTTTAAGAGCCTGTGCCGATGGAGGTGCCAACCGCTTATATGATATCACCGAAGGAGAGAGAGAAAGCTTTTTGCCTGAATTCATCAATGGAGACTTTGATTCTATTAGGCCTGAAGTCAGAGAATACTATGCTAAGAAGGGATGTGAGCTCATTTCAACTCCTGATCAAGACCACACTGACTTTACTAAGTGCCTTAAAATGCTCCAAAAGAAGATAGAAGAAAAAGACTTAAAGGTTGATGTGATGGTGACACTGGGAGGCCTTGGAGGGCGTTTTGACCAGATTATGGCATCTGTGAATACCTTGTTCCAAGCGACTCACATCACTCCTTTTCCAATTATAATAATCCAAAAAGAATCGCTGATCTACCTGCTCCAACCAGGAAAGCACAGGTTGCATGTAGACACTGGAATGGAGGGTTCTTGGTGTGGCC TTATTCCTGTTGGACAGCCTTGTATGCAGGTTACAACCACAGGCCTCAAGTGGAACCTCACAAATGATGTGCTTGCTTTTGGAACATTGGTCAGTACTTCCAATACCTACGACGGGTCTGGTGTTGTGACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCATCAAAAGCTAA

[0288] SEQ ID NO: 16 Nucleotide sequence of mouse TPK 11W ATGGAGCATGCCTTTACCCCGTTGGAACCCTGGCTACCTACGGGGAACTTGAAATACTGCCTTGTGGTTCTTAATCAGCCTTTGGATGCACGATTTCGCCATCTTTGGAAAAAAGCTCTCTTAAGAGCCTGTGCTGATGGGGGTGCCAACCACTTATATGATCTCACTGAAGGAGAGAGAGAA AGCTTCTTGCCTGAATTCGTCAGTGGGGACTTTGATTCTATTAGGCCTGAAGTCAAAGAGTACTACACCAAGAAGGGCTGTGATCTTATTTCAACTCCTGACCAAGACCACACTGACTTTACCAAGTGTCTTCAAGTGCTCCAAAGGAAGATAGAGGAAAAGGAACTGCAGGTTGACGTG ATTGTGACACTGGGAGGTCTCGGTGGGCGTTTTGACCAAATCATGGCCTCTGTGAATACCCTTTTCCAAGCCACTCACATCACTCCTGTGCCGATTATAATAATCCAAAAGGACTCTCTCATCTACCTCCTCCAACCCGGGAAGCACAGGCTCCATGTAGACACTGGAATGGAAGGGAGCTGGTGTGG CCTGATTCCTGTTGGACAGCCTTGCAACCAGGTGACGACAACAGGCCTGAAATGGAACCTCACAAATGATGTTCTTGGCTTTGGAACACTGGTCAGTACTTCTAACACCTACGATGGGTCCGGCCTTGTCACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCATCAAGAGCTAA

[0289] SEQ ID NO: 17 Nucleotide sequence of mouse TPK 35W ATGGAGCATGCCTTTACCCCGTTGGAACCCCTGCTACCTACGGGGAACTTGAAATACTGCCTTGTGGTTCTTAATCAGCCTTTGGATGCACGATTTCGCCATTGGTGGAAAAAAGCTCTCTTAAGAGCCTGTGCTGATGGGGGTGCCAACCACTTATATGATCTCACTGAAGGAGAGAGAGAA AGCTTCTTGCCTGAATTCGTCAGTGGGGACTTTGATTCTATTAGGCCTGAAGTCAAAGAGTACTACACCAAGAAGGGCTGTGATCTTATTTCAACTCCTGACCAAGACCACACTGACTTTACCAAGTGTCTTCAAGTGCTCCAAAGGAAGATAGAGGAAAAGGAACTGCAGGTTGACGTG ATTGTGACACTGGGAGGTCTCGGTGGGCGTTTTGACCAAATCATGGCCTCTGTGAATACCCTTTTCCAAGCCACTCACATCACTCCTGTGCCGATTATAATAATCCAAAAGGACTCTCTCATCTACCTCCTCCAACCCGGGAAGCACAGGCTCCATGTAGACACTGGAATGGAAGGGAGCTGGTGTGG CCTGATTCCTGTTGGACAGCCTTGCAACCAGGTGACGACAACAGGCCTGAAATGGAACCTCACAAATGATGTTCTTGGCTTTGGAACACTGGTCAGTACTTCTAACACCTACGATGGGTCCGGCCTTGTCACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCATCAAGAGCTAA

[0290] SEQ ID NO: 18 Nucleotide sequence of mouse TPK 241W ATGGAGCATGCCTTTACCCCGTTGGAACCCCTGCTACCTACGGGGAACTTGAAATACTGCCTTGTGGTTCTTAATCAGCCTTTGGATGCACGATTTCGCCATCTTTGGAAAAAAGCTCTCTTAAGAGCCTGTGCTGATGGGGGTGCCAACCACTTATATGATCTCACTGAAGGAGAGAGAGAA AGCTTCTTGCCTGAATTCGTCAGTGGGGACTTTGATTCTATTAGGCCTGAAGTCAAAGAGTACTACACCAAGAAGGGCTGTGATCTTATTTCAACTCCTGACCAAGACCACACTGACTTTACCAAGTGTCTTCAAGTGCTCCAAAGGAAGATAGAGGAAAAGGAACTGCAGGTTGACGTG ATTGTGACACTGGGAGGTCTCGGTGGGCGTTTTGACCAAATCATGGCCTCTGTGAATACCCTTTTCCAAGCCACTCACATCACTCCTGTGCCGATTATAATAATCCAAAAGGACTCTCTCATCTACCTCCTCCAACCCGGGAAGCACAGGCTCCATGTAGACACTGGAATGGAAGGGAGCTGGTGTGG CCTGATTCCTGTTGGACAGCCTTGCAACCAGGTGACGACAACAGGCCTGAAATGGAACCTCACAAATGATGTTCTTGGCTTTGGAACACTGGTCAGTACTTCTAACACCTACGATGGGTCCGGCCTTGTCACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCTGGAAGAGCTAA

[0291] SEQ ID NO: 19 Nucleotide sequence of mouse TPK 11W+241W ATGGAGCATGCCTTTACCCCGTTGGAACCCTGGCTACCTACGGGGAACTTGAAATACTGCCTTGTGGTTCTTAATCAGCCTTTGGATGCACGATTTCGCCATCTTTGGAAAAAAGCTCTCTTAAGAGCCTGTGCTGATGGGGGTGCCA ACCACTTATATGATCTCACTGAAGGAGAGAGAGAAAGCTTCTTGCCTGAATTCGTCAGTGGGGACTTTGATTCTATTAGGCCTGAAGTCAAAGAGTACTACACCAAGAAGGGCTGTGATCTTATTTCAACTCCTGACCAAGACCACACTGACTTTACCAAGTGTCTTCAAGTGCTCCAAAGGAAGATAGAGGAAA AGGAACTGCAGGTTGACGTGATTGTGACACTGGGAGGTCTCGGTGGGCGTTTTGACCAAATCATGGCCTCTGTGAATACCCTTTTCCAAGCCACTCACATCACTCCTGTGCCGATTATAATAATCCAAAAGGACTCTCTCATCTACCTCCTCCAACCCGGGAAGCACAGGCTCCATGTAGACACTGGAATGGAAGGGAG CTGGTGTGGCCTGATTCCTGTTGGACAGCCTTGCAACCAGGTGACGACAACAGGCCTGAAATGGAACCTCACAAATGATGTTCTTGGCTTTGGAACACTGGTCAGTACTTCTAACACCTACGATGGGTCCGGCCTTGTCACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCTGGAAGAGCTAA

Claims

1. A recombinant adeno-associated virus (rAAV) or a recombinant lentivirus, comprising within its genome an expression cassette comprising a polynucleotide encoding thiamine pyrophosphokinase (TPK) operably linked to a promoter.

2. 2. The rAAV or recombinant lentivirus of claim 1 , wherein the TPK comprises the amino acid sequence of SEQ ID NO:

1.

3. 3. The rAAV or recombinant lentivirus of claim 1 or 2, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:

2.

4. The rAAV or recombinant lentivirus of any one of claims 1 to 3, wherein the promoter is a neuron-specific promoter.

5. 5. The rAAV or recombinant lentivirus of any one of claims 1 to 4, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:

3.

6. The rAAV or recombinant lentivirus of any one of claims 1 to 5, wherein the expression cassette further comprises a WPRE element.

7. The rAAV or recombinant lentivirus of claim 6, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO:

4.

8. The rAAV according to any one of claims 1 to 5, which is an rAAV of serotype AAV_PHP eB.

9. A pharmaceutical composition comprising the rAAV or recombinant lentivirus of any one of claims 1 to 8 and a pharma- ceutical acceptable carrier.

10. 10. The pharmaceutical composition of claim 9, formulated for intravenous, intracerebral, or intrathecal administration.

11. A method for preventing or treating Alzheimer's disease, comprising administering to a subject in need thereof an rAAV or recombinant lentivirus according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9 or 10.

12. The method of claim 11 , wherein the rAAV or recombinant lentivirus, or pharmaceutical composition is administered intravenously, intracerebrally, or intrathecally.

13. Use of an rAAV or recombinant lentivirus according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9 or 10, in the manufacture of a medicament for preventing or treating Alzheimer's disease.

14. 14. The use according to claim 13, wherein the medicament is administered intravenously, intracerebrally or intrathecally.

15. A modified thiamine pyrophosphokinase (TPK) polypeptide, comprising an amino acid substitution at one or more positions selected from the group consisting of: 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35, compared to a parent TPK polypeptide, wherein the amino acid at position 13 is substituted with P, the amino acid at position 30 is substituted with A, the amino acid at position 31 is substituted with R, the amino acid at position 37 is substituted with K, the amino acid at position 85 is substituted with K, the amino acid at position 129 is substituted with G, the amino acid at position 158 is substituted with K, the amino acid at position 181 is substituted with S, the amino acid at position 11 is substituted with W, and the amino acid at position 35 is substituted with W; The modified TPK polypeptide has improved catalytic activity for converting thiamine (TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide; A modified TPK polypeptide, wherein said positions are numbered with reference to SEQ ID NO:1 or 8.

16. 16. The modified TPK polypeptide of claim 15, wherein the original TPK polypeptide is a wild-type TPK polypeptide.

17. A modified TPK polypeptide, wherein the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide comprises amino acid substitutions at one or more positions selected from the group consisting of 13, 30, 31, 37, 85, 129, 158 and 181, preferably all of the amino acid substitutions at positions 13, 30, 31, 37, 85, 129, 158 and 181.

18. The modified TPK polypeptide according to claim 15 or 16, wherein the original TPK polypeptide is a mouse TPK polypeptide and the modified TPK polypeptide comprises a substitution of amino acids at positions 11 and / or 35.

19. A modified TPK polypeptide according to any one of claims 15 to 18, comprising the amino acid sequence of SEQ ID NO: 9, 10 or 11.

20. A polynucleotide encoding the modified TPK polypeptide of any one of claims 15 to 19.

21. 21. An expression cassette comprising the polynucleotide of claim 20 operably linked to a promoter.

22. 22. The expression cassette of claim 21, wherein the promoter is a neuron-specific promoter.

23. 23. The expression cassette of claim 21 or 22, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:

3.

24. 24. An expression cassette according to any one of claims 21 to 23, further comprising a WPRE element.

25. 25. The expression cassette of claim 24, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO:

4.

26. 26. A rAAV or recombinant lentivirus comprising within its genome an expression cassette according to any one of claims 21 to 25.

27. The rAAV of claim 26, which is an rAAV of serotype AAV_PHP eB.

28. 28. A pharmaceutical composition comprising the rAAV or recombinant lentivirus of claim 26 or 27 and a pharma- ceutical acceptable carrier.

29. 29. The pharmaceutical composition of claim 28, formulated for intravenous, intracerebral, or intrathecal administration.

30. A method for treating or preventing Alzheimer's disease, comprising administering to a subject in need thereof a TPK polypeptide according to any one of claims 15 to 19, a polynucleotide according to claim 20, an AAV or recombinant lentivirus according to claim 26 or 27, or a pharmaceutical composition according to claim 28 or 29.

31. 31. The method of claim 30, wherein the polypeptide, polynucleotide, rAAV or recombinant lentivirus, or pharmaceutical composition is administered intravenously, intracerebrally, or intraspinally.

32. Use of a TPK polypeptide according to any one of claims 15 to 19, a polynucleotide according to claim 20, an AAV or recombinant lentivirus according to claim 26 or 27, or a pharmaceutical composition according to claim 28 or 29 in the manufacture of a medicament for treating or preventing Alzheimer's disease.

33. 33. The use of claim 32, wherein the medicament is administered intravenously, intracerebrally, or intrathecally.

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