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 recombinant adeno-associated virus (rAAV) or recombinant lentivirus containing nucleotides encoding thiamine pyrophosphokinase (TPK) and useful for treating Alzheimer's disease.

Background Art

[0002] Alzheimer's disease (AD) is a severe neurodegenerative disease that mainly affects the elderly. The onset of this disease is accompanied by the death of a large number of nerve cells and causes atrophy of the brain parenchyma. The main clinical symptoms of this disease are memory impairment, decline in language ability and spatial recognition 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 β-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, no effective treatment has been established.

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

[0004] Generally, the regulation of gene expression in cells can be carried out by methods such as calcium transformation, liposome transfection, electroporation, and infection with gene-introducing viruses.

[0005] Adeno-associated viruses (AAVs) are single-stranded DNA viruses widely present in humans, with humans and other primates being their natural hosts. Currently, there are no reports of AAVs being pathogenic. Recombinant adeno-associated viruses (rAAVs) are obtained through AAV-based manipulation and are widely used as gene therapy vectors due to their high safety, broad host range (infecting both dividing and non-dividing cells), and low immunogenicity.

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

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

Patent Document

[0009]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] Currently, it is desirable to develop new treatment methods for Alzheimer's disease, such as rAAV-based gene therapy, and to solve the problem of current drug shortages such as rAAV-based gene therapy.

Means for Solving the Problems

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

[0012] According to an embodiment, TPK comprises the amino acid sequence of SEQ ID NO: 1. According to an embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2.

[0013] According to an embodiment, the promoter is a neuron-specific promoter. According to an embodiment, the promoter comprises the nucleotide sequence of SEQ ID NO: 3.

[0014] According to an embodiment, the expression cassette further comprises a WPRE element. According to an embodiment, the WPRE element comprises the nucleotide sequence of SEQ ID NO: 4.

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

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

[0017] In a second embodiment, a modified thiamine pyrophosphokinase (TPK) polypeptide comprising, compared to the original TPK polypeptide, one or more amino acid substitutions selected from the group consisting of positions 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 exhibits improved catalytic activity in converting thiamine(TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide. A modified TPK polypeptide is provided, wherein the aforementioned positions are numbered with reference to sequence number 1 or 8.

[0018] According to an embodiment, the original TPK polypeptide is a wild-type TPK polypeptide. According to an embodiment, the original TPK polypeptide is a human TPK polypeptide, and the modified TPK polypeptide includes 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 amino acid substitutions at all of positions 13, 30, 31, 37, 85, 129, 158, and 181. According to an embodiment, the original TPK polypeptide is a mouse TPK polypeptide, and the modified TPK polypeptide includes an amino acid substitution at position 11 and / or 35. According to an embodiment, the modified TPK polypeptide includes 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 according to the present disclosure.

[0020] Also provided is an expression cassette comprising a polynucleotide according to the present disclosure, the expression cassette being operably linked to a promoter. According to an embodiment, the promoter is a neuron-specific promoter. According to an embodiment, the promoter includes the nucleotide sequence of SEQ ID NO: 3. According to an embodiment, the expression cassette further includes a WPRE element. According to an embodiment, the WPRE element includes the nucleotide sequence of SEQ ID NO: 4.

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

[0022] According to an embodiment, the rAAV is an rAAV of serotype AAV_PHP eB.

[0023] In a fifth aspect, there is provided a pharmaceutical composition comprising a modified TPK polypeptide, polynucleotide, rAAV or recombinant lentivirus according to the present disclosure, and a pharmaceutically acceptable carrier. By an embodiment, 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. By an embodiment, the modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition is administered intravenously, intracerebrally or intrathecally.

[0025] There is also provided the use of a modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure for the manufacture of a medicament for preventing or treating Alzheimer's disease. By an embodiment, the medicament is administered intravenously, intracerebrally or intraspinally.

[0026] Furthermore, there is provided a modified TPK polypeptide, polynucleotide, rAAV, recombinant lentivirus or pharmaceutical composition according to the present disclosure for use in the prevention or treatment of Alzheimer's disease. By an embodiment, 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 maps of plasmids containing the genome of AAV-TPK (FIG. 1A) and plasmids containing the genome of TPK lentivirus (FIG. 1B). [Figure 2] FIG. 2 shows an image of nerve cells infected with lentivirus by a fluorescence microscope. [Figure 3] Figure 3 shows TPK expression (Figure 3A, parental representative cells without virus infection), TDP content (Figure 3B), and TM content (Figure 3C) in neurons infected with lentivirus. [Figure 4] Figure 4 shows the body weight (left panel and middle panel) and food intake (right panel) of mice injected with rAAV via the tail vein. [Figure 5] Figure 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] Figure 6 shows immunofluorescence images of sections of various regions of the brains of mice injected with rAAV via the tail vein. [Figure 7] Figure 7 shows the expression of TPK (left) and the content of TDP and TM (right) in the brains of mice administered rAAV via the tail vein. [Figure 8] Figure 8 shows the TDP content (left) and TM content (right) in the livers of mice administered rAAV via the tail vein. [Figure 9] Figure 9 shows the body weight (left panel and middle panel) and food intake (right panel) of mice administered rAAV into the lateral ventricle. [Figure 10] Figure 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] Figure 11 shows immunofluorescence images of sections of various regions of the brains of mice injected with rAAV into the lateral ventricle. [Figure 12] Figure 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] Figure 13 shows the TDP content (left panel) and TM content (right panel) in the livers of mice administered rAAV into the lateral ventricle. [Figure 14] Figure 14 shows the change in body weight of mice generated from embryos administered rAAV into the lateral ventricle, 2 weeks after weaning, that is, 21 days after birth. [Figure 15]Figure 15 shows the TDP content (left panel) and TM content (right panel) in the blood of mice developed from embryos injected with rAAV into the lateral ventricle at 21 days postnatal. [Figure 16] Figure 16 shows immunofluorescence images of sections of various regions of the brain of mice developed from embryos that were injected with rAAV into the lateral ventricle at 21 days postnatal. [Figure 17] Figure 17 shows TPK expression (left panel) and TDP and TM content (right panel) in the brains of mice developed from embryos that received rAAV into the lateral ventricle at 21 days postnatality. [Figure 18] Figure 18 shows the TDP content (left panel) and TM content (right panel) in the liver of mice developed from embryos that received rAAV into the lateral ventricle at 21 days postnatality. [Figure 19] Figure 19 shows the weight changes of neonatal mice 21 days after injection of rAAV into the lateral ventricle, i.e., 2 weeks after weaning. [Figure 20] Figure 20 shows the TDP content (left) and TM content (right) in the blood of neonatal mice 21 days after injection of rAAV into the lateral ventricle. [Figure 21] Figure 21 shows immunofluorescence images of sections of various parts of the brain 21 days after injection of rAAV into the lateral ventricle of neonatal mice. [Figure 22] Figure 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] Figure 23 shows the weight changes of neonatal mice 21 days after injection of rAAV into the hippocampus, i.e., 2 weeks after weaning. [Figure 24] Figure 24 shows the TDP content (left) and TM content (right) in the blood of neonatal mice 21 days after injection of rAAV into the hippocampus. [Figure 25] Figure 25 shows immunofluorescence images of sections from various regions of the brain of neonatal mice 21 days after injection of rAAV into the hippocampus. [Figure 26] Figure 26 shows TPK expression in the brain of neonatal mice 21 days after injection of rAAV into the hippocampus. [Figure 27] Figure 27 shows the TDP content (left panel) and TM content (right panel) in the liver of neonatal mice 21 days after injection of rAAV into the hippocampus. [Figure 28] Figure 28 shows a comparison of the activity of modified human TPK, wild-type human TPK, and mouse TPK. [Figure 29] Figure 29 shows a comparison of the activity of modified mouse TPK and wild-type mouse TPK. [Figure 30] Figure 30 shows the results of a Western blot of TPK protein in brain tissue. [Figure 31] Figure 31 shows the HPLC results for TDP content in brain tissue. [Figure 32] Figure 32 shows curves of body weight (A), food intake (B), and blood glucose level (C) of mice injected with AAV-hTPK. [Figure 33] Figure 33 shows the results of Western blotting of TPK protein in brain tissue and liver of APP / PS1 mice administered AAV-TPK. [Figure 34] Figure 34 shows the TDP and TM content in brain tissue, liver, and blood of APP / PS1 mice administered AAV-TPK. [Figure 35] Figure 35 shows the results of Western blots for p-GSK3β and GSK3β in brain tissue from APP / PS1 mice injected with AAV-TPK into the lateral ventricle. [Figure 36] Figure 36 shows the ratio of p-GSK3β / GSK3β in the brain tissue of APP / PS1 mice injected with AAV-TPK into the lateral ventricle. [Modes for carrying out the invention]

[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 methods and materials similar or equivalent to those described herein may also be used in the practice and teaching of this disclosure, but preferred methods and materials are described. All publications referenced herein are invoked by reference to disclose and describe the cited methods and / or materials.

[0029] "Thiamine pyrophosphokinase" or "TPK" refers to an enzyme that catalyzes the reaction that converts 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, this term encompasses all subtypes and naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as 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, cattle AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep 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 mammals of the Bovidae family or similar species.

[0031] The genome sequences of different AAV subtypes, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are well known in the industry. Such sequences can be found in the literature or in public databases such as gene libraries. For example, see 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 to teach the nucleic acid and amino acid sequences of AAV.

[0032] As used herein, "rAAV vector" refers to an AAV vector containing a polynucleotide sequence of non-AAV origin (i.e., AAV is a different type of polynucleotide), and is typically an AAV vector for target sequences for genetic transformation of cells. Generally, the different type of polynucleotide is flanked by at least one, usually two, AAV reverse-terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors may be single-stranded (ssAAV) or self-complementary (scAAV).

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

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

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

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

[0037] Lentiviruses are viral vectors derived from HIV-1. “Recombinant lentiviral vectors” or “recombinant lentiviral vector particles” refer to recombinant viral particles and recombinant virus-like particles produced in host or production cells after transfection of a plasmid vector, where the plasmid vector consists of a transfer vector, an envelope vector encoding a selected envelope protein, and a packaging vector providing lentiviral proteins (e.g., lentiviral GAG and POL proteins, particularly mutant POL proteins for preventing integration) in a trans-like manner, according to methods well known in the art.

[0038] Virus-like particles are generated by an incomplete assembly of proteins present for capsid formation in recombinant lentivirus genomes, and the methods used cannot form true viral particles.

[0039] The term "polynucleotide" refers to a nucleotide polymer of any length, such as a deoxyribonucleotide or ribonucleotide, or analogues thereof. Polynucleotides may include modified nucleotides, such as methylated nucleotides or nucleotide analogues, and may also include non-nucleotide components. Where modifications to the nucleotide structure are present, the modifications may be applied before or after the assembly of the polymer. As used herein, the term polynucleotide may refer to the alternating presence of double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of a polynucleotide in this disclosure encompasses both a double-stranded form and two complementary single-stranded forms known or expected to constitute a double-stranded form.

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

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

[0042] A "gene" refers to a polynucleotide that contains at least one open reading frame capable of encoding a specific protein after transcription and translation.

[0043] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of a different combination of steps, including cloning, restriction, or ligation, and other procedures that produce a construct different from those found in nature. A recombinant virus is a viral particle containing recombinant polynucleotides. This term includes offspring of pro-polynucleotide constructs or pro-viral constructs.

[0044] A “regulatory element” or “regulatory sequence” is a nucleotide sequence involved in the functional regulation of polynucleotides, such as molecular interactions that contribute to the replication, duplication, transcription, splicing, translation, or degradation of polynucleotides. Regulation may affect the frequency, rate, or specificity of a process, and may actually be either promotion or inhibition. Regulatory elements known in the art include transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, can bind to RNA polymerase and initiate transcription of the coding region downstream (3' direction) of the promoter.

[0045] "Operatively linked" refers to the parallelism of genetic elements, indicating a relationship in which genetic elements function as expected. For example, when a promoter assists in the transcription initiation of a coding sequence, the promoter is operationally linked to the coding region. As long as this functional relationship is maintained, there may be inserted residues between the promoter and the coding region.

[0046] An "expression vector" is a vector containing a region encoding a target polypeptide, used to achieve protein expression in a given target cell. Expression vectors further include regulatory elements that are operably ligated to the coding region to promote protein expression in the target. A combination of a regulatory element that can be operably ligated for expression and a gene is sometimes referred to as 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] "Heterogeneous" refers to an entity that originates from a different genotype compared to another entity. For example, a polynucleotide introduced into a plasmid or vector from a different species using genetic engineering techniques is a heterogeneous polynucleotide. A promoter that is extracted from a natural coding sequence and operably ligated to a coding sequence that is known not to be naturally linked to that coding sequence is a heterogeneous promoter. Therefore, for example, an rAAV containing a heterogeneous nucleic acid encoding a heterogeneous gene product is typically an rAAV containing nucleic acids not found in natural wild-type AAVs, and the encoded heterogeneous gene product is typically not a gene product encoded by wild-type AAVs.

[0048] The terms “polypeptide,” “peptide,” and “protein” may be used interchangeably herein and refer to amino acid polymers of any length. These terms also encompass modified amino acid polymers, such as those resulting from disulfide bond formation, glycosylation, lipoylation, phosphorylation, or attachment to labeling components.

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

[0050] The term "conservative substitution" is also known as substitution by "homologous" amino acid residues and refers to amino acid residues that are substituted for other amino acids with similar side chains. For example, amino acids with alkaline side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with polar side chains that have no charge (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

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

[0052] As used herein, "homologous" amino acid residues refer to amino acid residues that have similar chemical properties, including hydrophobicity, charge, polarity, spatial properties, and aromaticity. Examples of homologous amino acids 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 conserved amino acid substitutions in proteins include Ser instead of Ala, Lys instead of Arg, Gln or His instead of Asn, Glu instead of Asp, Ser instead of Cys, Asn instead of Gln, Asp instead of Glu, Pro instead of Gly, Asn or Gln instead of His, Leu or Val instead of Ile, Ile or Val instead of Leu, Arg or Gln instead of Lys, Leu or Ile instead of Met, Met, Leu or Tyr instead of Phe, Thr instead of Ser, Ser instead of Thr, Tyr instead of Tyr, Trp or Phe instead of Tyr, or Ile or Leu instead of Val.

[0054] "Isolated" plasmids, nucleic acids, vectors, viruses, viral bodies, host cells, or other substances refer to preparations that do not contain at least the substance or other components of similar substances that are naturally occurring or originally prepared. Therefore, for example, purification techniques may be used to concentrate an isolated substance from the original mixture.

[0055] The term “treatment,” as used herein, means obtaining a desired pharmacological and / or physiological effect. Such effect may be a preventive effect that completely or partially prevents the disease or its symptoms, and / or a therapeutic effect that 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 (in particular human), and includes (a) preventing the onset of the disease in a subject that is susceptible to or at risk of the disease but has not yet been diagnosed with the disease, (b) suppressing the disease, i.e., preventing its onset, and (c) alleviating the disease, i.e., slowing its progression.

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

[0057] II. Modified TPK Polypeptide The inventors have found that the catalytic activity of converting the TM of mouse thiamine pyrophosphokinase (mTPK) to TDP thiamine is higher than that of human TPK (hTPK). The 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 its mouse origin. That is, when the charge characteristics of residue Xh in hTPK are different from those of the residue Xm at the corresponding position in mTPK, residue Xh in hTPK is replaced with Xm.

[0058] Therefore, a modified thiamine pyrophosphokinase (TPK) polypeptide comprising 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 as compared to the original TPK polypeptide, the modified TPK polypeptide has improved catalytic activity of converting thiamine (TM) to thiamine diphosphate (TDP) as compared to the original TPK polypeptide, a modified TPK polypeptide wherein the above positions are numbered with reference to SEQ ID NO: 1 or 8, is provided.

[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 on which 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 variant thereof. For example, if the modification is performed on the 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 variant of the polypeptide of SEQ ID NO: 1, the variant polypeptide becomes the "original TPK polypeptide" of the modified TPK polypeptide.

[0061] Depending on the embodiment, the original TPK polypeptide may be 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 herein includes amino acid substitutions 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 herein includes amino acid substitutions 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 herein includes amino acid substitutions at positions 13, 30, 31, 37, 85, 129, 158, and 181.

[0063] In each embodiment, the original TPK polypeptide is a mouse TPK polypeptide, and the modified TPK polypeptide herein comprises amino acid substitutions at the 11th and / or 35th positions.

[0064] In each embodiment, the original TPK polypeptide contains the amino acid sequence of SEQ ID NO: 8, or contains 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 with respect to SEQ ID NO: 8, wherein the modified TPK polypeptide, compared to the original TPK polypeptide, has, for example, amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 11. This includes amino acid substitutions at one or more positions selected from the group consisting of and 35 positions, for example, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, and 181; substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 11; substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 35; or substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35.

[0065] In each embodiment, 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide comprises an amino acid substitution at position 11 and / or position 35 with W, compared to the original TPK polypeptide.

[0066] In each embodiment, 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide comprises an amino acid substitution at position 11, W, compared to the original TPK polypeptide.

[0067] In each embodiment, the original TPK polypeptide contains the amino acid sequence of SEQ ID NO: 1, or contains 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide contains an amino acid substitution of W at position 35 compared to the original TPK polypeptide.

[0068] Depending on the embodiment, the modified TPK polypeptide may have 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] Depending on the embodiment, the modified TPK polypeptide contains the amino acid sequence of SEQ ID NO: 9, 10, or 11, or contains 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 with respect 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 contains one or more amino acid substitutions.

[0070] Depending on the embodiment, the modified TPK polypeptide includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids 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, preferably conservative substitutions.

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

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

[0073] In each embodiment, the modified TPK polypeptide has catalytic activity that converts TM to TDP, and is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, or 300% 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 may be derived from humans or non-human animals, such as non-human mammals. Examples of TPKs derived from non-human mammals include, but are not limited to, those derived from non-human primates, such as monkeys, horses, cattle, sheep, goats, pigs, dogs, cats, mice, and rats.

[0076] Depending on the embodiment, TPK is wild-type TPK. Depending on the embodiment, 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, TPK is a modified TPK comprising one or more amino acid substitutions, insertions, deletions and / or additions compared to the original TPK, wherein 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%, and 300% or more of that of the original TPK. In some embodiments, the modified TPK comprises one, two, three, four, five, six, seven, eight, nine, and ten 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%, and 99% or more identity with the original TPK.

[0079] The original TPK may be wild-type TPK or a modified TPK. Depending on the embodiment, the original TPK may be wild-type mouse TPK or wild-type human TPK.

[0080] In one embodiment, the original TPK contains the amino acid sequence of SEQ ID NO: 1. In another embodiment, the original TPK consists of the amino acid sequence of SEQ ID NO: 1.

[0081] In each embodiment, 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, compared to the original TPK polypeptide, wherein the modified TPK polypeptide exhibits improved catalytic activity in converting thiamine(TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide. The modified TPK polypeptide exhibits improved catalytic activity in converting thiamine(TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide, and the aforementioned positions are numbered with reference to Sequence 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] Depending on the embodiment, the original TPK polypeptide may be 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 herein includes amino acid substitutions 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 herein includes amino acid substitutions 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 herein includes amino acid substitutions at positions 13, 30, 31, 37, 85, 129, 158, and 181.

[0085] In each embodiment, the original TPK polypeptide is a mouse TPK polypeptide, and the modified TPK polypeptide herein comprises amino acid substitutions at the 11th and / or 35th positions.

[0086] In each embodiment, the original TPK polypeptide contains the amino acid sequence of SEQ ID NO: 8, or contains 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 with respect to SEQ ID NO: 8, wherein the modified TPK polypeptide, compared to the original TPK polypeptide, has, for example, amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 11. This includes amino acid substitutions at one or more positions selected from the group consisting of and 35 positions, for example, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, and 181; substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 11; substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 35; or substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35.

[0087] In each embodiment, 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide comprises an amino acid substitution at position 11 and / or position 35 with W, compared to the original TPK polypeptide.

[0088] In each embodiment, 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide comprises an amino acid substitution at position 11, W, compared to the original TPK polypeptide.

[0089] In each embodiment, the original TPK polypeptide contains the amino acid sequence of SEQ ID NO: 1, or contains 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide contains an amino acid substitution with a W amino acid substitution at position 35 compared to the original TPK polypeptide.

[0090] Depending on the embodiment, the modified TPK polypeptide may have 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] Depending on the embodiment, the modified TPK polypeptide contains the amino acid sequence of SEQ ID NO: 9, 10, or 11, or contains 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 with respect 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 contains one or more amino acid substitutions.

[0092] Depending on the embodiment, the modified TPK polypeptide includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids 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, preferably conservative substitutions.

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

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

[0095] In each embodiment, the modified TPK polypeptide has catalytic activity that converts TM to TDP, and is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, or 300% 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 comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect 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 with respect 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 with respect to SEQ ID NO: 15, and such nucleotide sequence encodes an amino acid sequence that, compared to SEQ ID NO: 9, comprises substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, preferably conservative substitutions, at positions other than 13, 30, 31, 37, 85, 129, 158, and 181, preferably other than 241.

[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 with respect 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 with respect to SEQ ID NO: 16, and such nucleotide sequence encodes an amino acid sequence that, compared to SEQ ID NO: 10, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than positions 11 and 241, preferably positions 13, 30, 31, 37, 85, 129, 158, and 181.

[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 with respect 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 with respect to SEQ ID NO: 17, and such nucleotide sequence encodes an amino acid sequence that, compared to SEQ ID NO: 11, 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 13, 30, 31, 37, 85, 129, 158, and 181.

[0100] Depending on the embodiment, the polynucleotide may be DNA or RNA, such as mRNA.

[0101] Also provided is an expression cassette comprising a polynucleotide according to this disclosure, which is operably linked to a promoter.

[0102] In some embodiments, the promoter is a neuron-specific promoter. In some embodiments, the promoter contains 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 contains a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect 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%, or 99% sequence identity with respect 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 includes 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 includes a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect 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%, or 99% sequence identity with respect to SEQ ID NO: 7.

[0104] The expression cassette may include additional regulatory elements, such as enhancers, introns, mRNA stabilization sequences, and polyadenylation signal sequences.

[0105] In some embodiments, the expression cassette further comprises an mRNA stabilizing sequence, such as 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%, or 99% identity with respect 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%, or 99% identity with respect to SEQ ID NO: 4.

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

[0107] In some embodiments, the expression cassette further comprises polyadenylation signal sequences, for example, but not limited to, the polyadenylation signal sequence of a human growth factor gene and the polyadenylation signal sequence of a rabbit globulin gene. In some embodiments, the expression cassette further comprises the 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%, or 99% identity with respect 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%, or 99% identity with respect to SEQ ID NO: 5.

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

[0109] The expression cassette may be part of the rAAV genome. Therefore, the expression cassette is flanked by the ITR of the AAV. Non-restrictive examples of ITRs include those derived 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 sheep AAV.

[0110] Depending on the embodiment, the ITR is the ITR of the AAV-2. Depending on the embodiment, the 5'ITR is identical to the 3'ITR. Depending on the embodiment, the 5'ITR is different from the 3'ITR.

[0111] In some embodiments, the 5'ITR and 3'ITRR are both ITR130 and include, for example, 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 contain nucleotide sequences that have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99% or more identity with respect to SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each consist of nucleotide sequences that have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99% or more identity with respect to SEQ ID NO: 6.

[0112] Depending on the embodiment, the expression cassette includes SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome includes 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, from the 5' end to the 3' end.

[0113] Depending on the embodiment, the expression cassette includes SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome includes 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, from the 5' end to the 3' end.

[0114] Depending on the embodiment, the expression cassette may contain SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome may contain 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, from the 5' end to the 3' end.

[0115] Depending on the embodiment, the expression cassette includes SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome includes 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, from the 5' end to the 3' end.

[0116] Depending on the embodiment, the expression cassette may contain SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome may contain 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, from the 5' end to the 3' end.

[0117] Depending on the embodiment, the expression cassette includes SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0118] Depending on the embodiment, the expression cassette includes SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0119] Depending on the embodiment, the expression cassette includes SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0120] Depending on the embodiment, the expression cassette includes SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0121] Depending on the embodiment, the expression cassette includes SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0122] A vector comprising a polynucleotide or expression cassette in accordance with this disclosure is also provided.

[0123] Depending on the embodiment, the vector is an expression vector. Depending on the embodiment, the vector is a eukaryotic expression vector.

[0124] Depending on the embodiment, the vector is a vector that provides the rAAV genome in rAAV packaging, i.e., a transgenic plasmid.

[0125] According to an embodiment, the vector is a vector that provides the genome of a lentivirus in lentivirus packaging, that is, a transgenic plasmid.

[0126] IV. Recombinant virus There is provided a recombinant adeno-associated virus (rAAV) or a recombinant lentivirus that contains an expression cassette in the genome, the expression cassette containing a polynucleotide encoding thiamine pyrophosphokinase (TPK) operably linked to a promoter.

[0127] TPK may be a TPK derived from a human or non-human animal, such as a non-human mammal. Examples of TPKs derived from non-human mammals include, but are not limited to, those derived from non-human primates such as monkeys, horses, cows, sheep, goats, pigs, dogs, cats, mice, and rats.

[0128] According to an embodiment, TPK is a wild-type TPK. According to an embodiment, TPK is a mouse TPK or a human TPK.

[0129] According to an embodiment, TPK contains the amino acid sequence of SEQ ID NO: 1. According to an embodiment, the polynucleotide encoding TPK contains the nucleotide sequence of SEQ ID NO: 2. According to an embodiment, TPK consists of the amino acid sequence of SEQ ID NO: 1. According to an embodiment, the polynucleotide encoding TPK consists of the nucleotide sequence of SEQ ID NO: 2.

[0130] In some embodiments, TPK is a modified TPK comprising one or more amino acid substitutions, insertions, deletions and / or additions compared to the original TPK, wherein 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%, and 300% or more of that of the original TPK. In some embodiments, the modified TPK comprises one, two, three, four, five, six, seven, eight, nine, and ten 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%, and 99% or more identity with the original TPK.

[0131] The original TPK may be wild-type TPK or a modified TPK. Depending on the embodiment, the original TPK may be wild-type mouse TPK or wild-type human TPK.

[0132] In one embodiment, the original TPK contains the amino acid sequence of SEQ ID NO: 1. In another embodiment, the original TPK consists of the amino acid sequence of SEQ ID NO: 1.

[0133] In each embodiment, 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, compared to the original TPK polypeptide, wherein the modified TPK polypeptide exhibits improved catalytic activity in converting thiamine(TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide. The modified TPK polypeptide exhibits improved catalytic activity in converting thiamine(TM) to thiamine diphosphate (TDP) compared to the original TPK polypeptide, and the aforementioned positions are numbered with reference to Sequence 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] Depending on the embodiment, the original TPK polypeptide may be 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 herein includes amino acid substitutions 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 herein includes amino acid substitutions 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 herein includes amino acid substitutions at positions 13, 30, 31, 37, 85, 129, 158, and 181.

[0137] In each embodiment, the original TPK polypeptide is a mouse TPK polypeptide, and the modified TPK polypeptide herein comprises amino acid substitutions at the 11th and / or 35th positions.

[0138] In each embodiment, the original TPK polypeptide contains the amino acid sequence of SEQ ID NO: 8, or contains 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 with respect to SEQ ID NO: 8, wherein the modified TPK polypeptide, compared to the original TPK polypeptide, has, for example, amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 11. This includes amino acid substitutions at one or more positions selected from the group consisting of and 35 positions, for example, substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, and 181; substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 11; substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, and 35; or substitutions of amino acids at positions 13, 30, 31, 37, 85, 129, 158, 181, 11, and 35.

[0139] In each embodiment, 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide comprises an amino acid substitution at position 11 and / or position 35 with W, compared to the original TPK polypeptide.

[0140] In each embodiment, 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide comprises an amino acid substitution at position 11, W, compared to the original TPK polypeptide.

[0141] In each embodiment, the original TPK polypeptide contains the amino acid sequence of SEQ ID NO: 1, or contains 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 with respect to SEQ ID NO: 1, wherein the modified TPK polypeptide contains an amino acid substitution with a W amino acid substitution at position 35 compared to the original TPK polypeptide.

[0142] Depending on the embodiment, the modified TPK polypeptide may have 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] Depending on the embodiment, the modified TPK polypeptide contains the amino acid sequence of SEQ ID NO: 9, 10, or 11, or contains 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 with respect 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 contains one or more amino acid substitutions.

[0144] Depending on the embodiment, the modified TPK polypeptide includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids 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, preferably conservative substitutions.

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

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

[0147] In each embodiment, the modified TPK polypeptide has catalytic activity that converts TM to TDP, and is at least 110%, 120%, 130%, 140%, 150%, 200%, 250%, or 300% 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 comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect 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 with respect 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 with respect to SEQ ID NO: 15, and such nucleotide sequence encodes an amino acid sequence that, compared to SEQ ID NO: 9, comprises substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, preferably conservative substitutions, at positions other than 13, 30, 31, 37, 85, 129, 158, and 181, preferably other than 241.

[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 with respect 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 with respect to SEQ ID NO: 16, and such nucleotide sequence encodes an amino acid sequence that, compared to SEQ ID NO: 10, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than positions 11 and 241, preferably positions 13, 30, 31, 37, 85, 129, 158, and 181.

[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 with respect 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 with respect to SEQ ID NO: 17, and such nucleotide sequence encodes an amino acid sequence that, compared to SEQ ID NO: 11, 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 13, 30, 31, 37, 85, 129, 158, and 181.

[0152] In some embodiments, the promoter is a neuron-specific promoter. In some embodiments, the promoter contains 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 contains a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect 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%, or 99% sequence identity with respect 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 includes 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 includes a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect 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%, or 99% sequence identity with respect to SEQ ID NO: 7.

[0154] The expression cassette may include additional regulatory elements, such as enhancers, introns, mRNA stabilization sequences, and polyadenylation signal sequences.

[0155] In some embodiments, the expression cassette further comprises an mRNA stabilizing sequence, such as 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%, or 99% identity with respect 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%, or 99% identity with respect to SEQ ID NO: 4.

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

[0157] In some embodiments, the expression cassette further comprises polyadenylation signal sequences, for example, but not limited to, the polyadenylation signal sequence of a human growth factor gene and the polyadenylation signal sequence of a rabbit globulin gene. In some embodiments, the expression cassette further comprises the 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%, or 99% identity with respect 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%, or 99% identity with respect to SEQ ID NO: 5.

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

[0159] Non-limiting examples of ITRs in the rAAV genome include ITRs derived from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, poultry AAVs, bovine AAVs, canine AAVs, equine AAVs, primate AAVs, non-primate AAVs, and sheep AAVs. By embodiment, the ITR is the ITR of AAV-2. By embodiment, the 5'ITR is identical to the 3'ITR. By embodiment, the 5'ITR is different from the 3'ITR.

[0160] In some embodiments, the 5'ITR and 3'ITRR are both ITR130 and include, for example, 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 contain nucleotide sequences that have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99% or more identity with respect to SEQ ID NO: 6. In some embodiments, the 5'ITR and 3'ITRR each consist of nucleotide sequences that have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99% or more identity with respect to SEQ ID NO: 6.

[0161] Depending on the embodiment, the rAAV genome may include SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome may include 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, from the 5' end to the 3' end.

[0162] Depending on the embodiment, the rAAV genome may include SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome may include 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, from the 5' end to the 3' end.

[0163] Depending on the embodiment, the rAAV genome may include SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome may include 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, from the 5' end to the 3' end.

[0164] Depending on the embodiment, the rAAV genome may include SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome may include 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, from the 5' end to the 3' end.

[0165] Depending on the embodiment, the rAAV genome may include SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end. Depending on the embodiment, the rAAV genome may include 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, from the 5' end to the 3' end.

[0166] Depending on the embodiment, the recombinant lentivirus genome includes SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0167] Depending on the embodiment, the recombinant lentivirus genome includes SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0168] Depending on the embodiment, the recombinant lentivirus genome includes SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0169] Depending on the embodiment, the recombinant lentivirus genome includes SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0170] Depending on the embodiment, the recombinant lentivirus genome includes SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 4, and SEQ ID NO: 5, from the 5' end to the 3' end.

[0171] rAAV can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, poultry AAV, cattle AAV, dog AAV, horse AAV, primate AAV, non-primate AAV, and sheep AAV. By embodiment, rAAV is rAAV of serotype AAV_PHP eB.

[0172] In this industry, rAAV is known to be packaged using a system comprising three plasmids: 1) a transgenic plasmid containing the rAAV genome encoding the gene product of the target; ii) a packaging plasmid encoding the REP and / or CAP proteins; and iii) a helper plasmid (see, for example, 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 introducing the system into host cells.

[0173] Retroviral packaging systems are well known in the industry and consist of a transfer vector (transgenic plasmid), an envelope vector encoding a selected envelope protein, and a packaging vector that delivers retroviral proteins (retroviral GAG and POL proteins, particularly variant POL proteins to prevent integration) according to methods well known in the industry. Retroviral packaging involves introducing the system into host cells.

[0174] Also provided are polynucleotides, vectors, and host cells containing rAAV or recombinant lentiviruses according to this disclosure. The host cell is referred to as a “packaging cell” when used to package rAAV or recombinant lentiviral virions. In some embodiments, the host cell is stably genetically engineered with the polynucleotide or vector according to this disclosure. In other embodiments, the host cell is transiently genetically engineered with the polynucleotide or vector according to this disclosure.

[0175] Certain techniques, such as, but not limited to, electroporation, calcium phosphate precipitation, and liposome-mediated transfection, are used to stably or transiently introduce polynucleotides or vectors according to this disclosure into host cells.

[0176] For stable transformations, the polynucleotides or vectors according to this disclosure typically further include a selection marker, such as one of several well-known selection markers, e.g., neomycin resistance.

[0177] The host cells used in accordance with this 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, and the like. Non-specific examples of host cells include, for example, HeLa cells (e.g., ATCC number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, N1H3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCL1.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, and others.

[0178] V. Pharmaceutical Compositions Provided are pharmaceutical compositions comprising polynucleotides, vectors, rAAV or recombinant lentiviruses, and pharmaceutically acceptable carriers, diluents, excipients, or buffers in accordance with this disclosure. Depending on the embodiment, the pharmaceutically acceptable carriers, diluents, excipients, or buffers are suitable for use in humans.

[0179] Such excipients, carriers, diluents, and buffers include any pharmaceuticals that can be applied without unusual toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol, and may include pharmaceutically acceptable salts, e.g., mineral salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.; and organic salts, e.g., acetate, propionate, malonate, benzoate, etc. Furthermore, auxiliary substances such as wetting agents, emulsifiers, and pH buffers may be present in such carriers. Various pharmaceutically acceptable excipients are known in the art and require no further explanation. Pharmaceutical excipients are described in detail in various publications, such as A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th Edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) Edited by H.C. Ansel et al., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., 3rd edition, American Pharmaceutical Association.

[0180] VI. Prevention and Treatment of Diseases A method is provided for promoting glucose metabolism, or for preventing or treating glucose metabolic disorders, preferably cerebral glucose metabolic disorders, comprising administering a polynucleotide, vector, rAAV, recombinant lentivirus, or pharmaceutical composition according to this disclosure to a subject in need. A method is also provided for treating or preventing Alzheimer's disease, comprising administering a polynucleotide, vector, rAAV, recombinant lentivirus, or pharmaceutical composition according to this disclosure to a subject or patient in need. Depending on the embodiment, the polynucleotide, vector, rAAV, recombinant lentivirus, or pharmaceutical composition is administered intravenously, intracerebrally, or intraspinally. Depending on the embodiment, the subject or patient is human.

[0181] The use of polynucleotides, vectors, rAAVs or recombinant lentiviruses or pharmaceutical compositions according to this disclosure in the manufacture of pharmaceuticals for promoting glucose metabolism or for preventing or treating glucose metabolic disorders, preferably cerebral glucose metabolic disorders. The use of polynucleotides, vectors, rAAVs, recombinant lentiviruses or pharmaceutical compositions according to this disclosure in the manufacture of pharmaceuticals for treating or preventing Alzheimer's disease. Depending on the embodiment, such pharmaceuticals are administered intravenously, intracerebrally, or intrathecally. Depending on the embodiment, such pharmaceuticals are administered to humans.

[0182] Furthermore, polynucleotides, vectors, rAAVs, recombinant lentiviruses, or pharmaceutical compositions according to this disclosure are provided for use in promoting glucose metabolism or in preventing or treating glucose metabolic disorders, preferably cerebral glucose metabolic disorders. Also provided are polynucleotides, vectors, rAAVs, recombinant lentiviruses, or pharmaceutical compositions for use in treating or preventing Alzheimer's disease. Depending on the embodiment, the polynucleotides, vectors, rAAVs, recombinant lentiviruses, or pharmaceutical compositions are administered intravenously, intracerebrally, or intraspinally. Depending on the embodiment, the polynucleotides, vectors, rAAVs, recombinant lentiviruses, or pharmaceutical compositions are administered to humans.

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

[0184] Alzheimer's disease involves damage to numerous brain regions, and viruses according to this disclosure can selectively and efficiently penetrate the blood-brain barrier, affecting a wide range of brain regions. In clinical applications, rAAV according to this disclosure can be administered by direct injection across the blood-brain barrier (e.g., intracerebral or intrathecal injection) or by peripheral intravenous injection, with peripheral intravenous injection achieving the same effect as direct injection into the brain. Furthermore, a key difference between this disclosure and other gene therapies is that it only mediates the compensatory expression of TPK exogenously, without involving endogenous TPK, and certainly without editing other genes, thus significantly reducing other risks. More importantly, this disclosure introduces highly active exogenous TPK to achieve beneficial therapeutic effects.

[0185] GSK3β is a serine / threonine kinase that is highly conserved during evolution. In addition to regulating glycogen synthase activity, it acts on other pathways and can regulate cell differentiation, proliferation, survival, and apoptosis. Activation of GSK3β has been reported to potentially contribute to the development of Alzheimer's disease by promoting tau protein phosphorylation, increasing Aβ deposition, activating microglia, damaging neuronal cell generation, and inhibiting LTP (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, rAAVs conforming to this disclosure express TPK in vivo (e.g., in the brain), which mediates 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, inhibiting GSK3β activity, and resulting in beneficial therapeutic effects for AD. [Examples]

[0186] Those skilled in the art will gain a clearer understanding of the Disclosure through the following embodiments. It should be understood that the following embodiments are intended to illustrate exemplary embodiments of the Disclosure and are not intended to limit its scope. Unless otherwise noted, the methods used herein are common in the industry, and the experimental materials used are commercially available.

[0187] Example 1: Preparation of rAAV and recombinant lentivirus Wuhan's BrainVTA, a CRO company, was commissioned to prepare a laboratory-level rAAV encoding TPK (serotype AAV_PHP eB), designated AAV-TPK, whose genome contains a nucleotide sequence encoding i) a neuron-specific promoter (SEQ ID NO: 3), ii) mouse TPK (SEQ ID NO: 2) and the WPRE element (SEQ ID NO: 4). A plasmid containing the genome of such rAAV is shown in Figure 1A.

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

[0189] Example 2: Overexpression of TPK and increased TPD content in nerve 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 neonatal mice (purchased from Zhejiang Charlesriver) was treated with trypsin (Thermofisher / Gibco, catalog number: 25200072) to collect primary neurons, which were then placed in a 6-well plate (1.5x10 6Cells were cultured for 4 days in (10% FBS + 10% F12 + 1% Glutemax + 78% DMEM + 1% 100X penicillin-streptomycin biantibody (5000 U / mL) on day 1, followed by Neurobasal medium + 2% B27 + 1% Glutemax + 1% 100X penicillin-streptomycin biantibody (5000 U / mL), where the above reagents were prepared in volume ratio and all purchased from Therofisher / Gibco). The medium was removed, and the lentivirus suspension prepared in Example 1 (MOI 5, 1.5 mL / well) was added and infected for 8 hours, where a lentivirus encoding GFP without the TPK coding sequence was used as a control. The liquid was removed, and fresh culture medium was added for 7 days of culture. GFP expression was observed by fluorescence microscopy. As shown in Figure 2, a large number of infected cells expressed GFP, indicating a high viral infection rate.

[0191] The culture medium was removed, and the cells were washed three times with pre-chilled PBS for 5 minutes each time. 100 μL of cell lysate was added to each well, and RIPA (purchased from Beyotime), a phosphatase inhibitor (purchased from Bimake), and a 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 lysate was added to each well, the bottom of the well was rinsed, and the cells were transferred to the same centrifuge tube. Two grinding steel balls were added to each tube for grinding (60 Hz, 60 seconds, -10°C), and the cells were centrifuged at 13000 rpm at 4°C for 15 minutes. The supernatant was stored on ice.

[0192] The total protein concentration in the supernatant was determined by the BCA method. After denaturing the extracted total protein at 95 °C, the TPK level in the sample was analyzed by Western blotting. Briefly, after denaturation, the sample was loaded into the wells of a polyacrylamide gel (the test sample was 15 μg of protein per well, and the protein size indicator (marker) was 5 μL), and after electrophoresis, the membrane was transferred. The membrane corresponding to the region of the molecular weight 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 a blocking solution (purchased from Takara. Catalog number: T7131A) at room temperature for 1 hour, 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) were incubated overnight at 4 °C. The primary antibody was recovered, and the membrane was washed 3 times with 1XTBST for 5 minutes each. As the corresponding secondary antibodies, horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (purchased from Beyotime. Catalog number: A0208) and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (purchased from Beyotime. Catalog number: A0216) were added, incubated at room temperature for 2 hours, then washed 3 times with 1xTBST, and developed with a horseradish peroxidase kit (Catalog number: 34095, purchased from Thermo Fisher). As shown in Figure 3A, in cells infected with the TPK retrovirus, TPK was significantly overexpressed.

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

[0194]

Table 1

[0195] As shown in Figure 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. On the other hand, the TM content in AAV-TPK cells was significantly lower than that in uninfected cells and cells infected with the control virus (Figure 3C). From these results, it was shown that overexpression of TPK significantly promoted the conversion from 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 brains of C57BL / 6J mice, increase the TM / TDP conversion rate in the mouse brains, and thereby improve glucose metabolism by injecting AAV into the tail vein of the 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 (the same as TPK-AAV except for the coding sequence of TPK) were administered to each group of mice by tail vein injection at a titer of 5.0E+12 vg / mL (prepared with physiological saline), 200 μL each. Specifically, the mice were fixed in a tail vein injection fixator, and the tail was disinfected with an alcohol cotton ball. Using a 1 ml syringe, 200 μL of the virus suspension was injected into the tail vein of the mice, and after injection, the injection site was pressed with a dry cotton ball for 30 seconds to prevent virus leakage. The mice were bred in the experimental area for 4 weeks, and the body weight and food intake were recorded weekly.

[0198] As shown in Figure 4, after injecting AAV-TPK into the tail vein of adult mice, the weight gain was significantly smaller compared to the control group, and the food intake also showed a decreasing trend compared to the control group.

[0199] After four weeks of rearing, whole blood was collected from mice using a 3 mL EDTA anticoagulant tube and immediately placed on ice. The following procedure was performed on ice. After 10 minutes, 0.3 mL of the whole blood sample was transferred to a 2 mL centrifuge tube, 30 μL of pure water was added, and then 0.3 mL of pre-chilled PCA (5.7%) was added to precipitate the protein. After an ice bath of approximately 30 minutes, the sample was centrifuged at 4°C and 12000 rpm for 10 minutes. The supernatant was collected and the TDP and TM content was analyzed by HPLC using the method described in Example 2.

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

[0201] The mice were euthanized, followed by cardiac perfusion with pre-chilled PBS. Brain tissue was collected from the mice (cerebellum, brainstem, and olfactory bulb were removed, and the left and right hemispheres of the brain were separated).

[0202] The right brain was fixed with 4% paraformaldehyde (PFA) and immunofluorescence staining was performed. Specifically, after fixing with 4% PFA for 12-24 hours, the right brain was dehydrated and precipitated with 30% sucrose for 2 days. After precipitation, the right brain was embedded in frozen section embedding medium (OCT) and frozen at -80°C. The embedded right brain was sliced ​​into thin sections (30 μm) along the long axis (coronal) 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 at room temperature for 30 minutes to disrupt the membrane, then replaced with 3% BSA (bovine serum albumin) and blocked at room temperature for 1 hour. Primary antibody (anti-GFP antibody, purchased from Avessellabs, catalog number GFP-1020) was added and incubated overnight at 4°C. The primary antibody was collected and washed three times with 1×PBS for 5 minutes each time. As the secondary antibody, goat anti-rabbit IgG(H+L) cross-adsorbed secondary antibody and Alexa Fluor 488 (purchased from Invitrogen, catalog number A11008) were added and incubated in the dark for 2 hours. Finally, the thin sections were spread on glass slides, allowed to dry slightly, and then mounted with mounting medium. Images were taken using a Nikon Ti2E inverted microscope.

[0203] As shown in Figure 6, four weeks after injecting adult mice with AAV-TPK via the tail vein, the polypeptide encoded by the rAAV virus was widely expressed in brain tissue.

[0204] Left brain or liver tissue (left upper lobe) was placed in 1.5 ml of cell lysate (see Example 2), thawed using a freeze grinder (70 Hz, 60 seconds, twice, 20-second intervals), centrifuged at 13000 rpm, 4°C for 15 minutes, and the supernatant was collected. Western blotting and HPLC analysis were performed as described in Example 2 to detect TPK expression and TDP and TM content in the brain.

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

[0206] As shown in Figure 8, TM and TDP levels in the liver did not show significant changes, indicating that the virus possesses good brain specificity and specifically targets brain nerve cells without affecting peripheral tissues. Viral side effects 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 objective of this embodiment is to verify whether injection of AAV-TPK virus into the lateral ventricle of mice significantly increases TPK protein expression in the brain of C57BL / 6J mice, thereby increasing the TM / TDP conversion rate in the mouse brain and improving glucose metabolism.

[0208] After three 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=5) for stereotactic injection of AAV into the lateral ventricles of the brain.

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

[0210] Adult mice were anesthetized with Zoletil 50, the hair between their ears and eyes was shaved, and they were fixed to a stereotactic device. The skin of the mouse's brain region was wiped and disinfected with an alcohol swab. The skin was opened with scissors to expose the skull. The surface of the skull was wiped 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 a 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, with the skull surface as the 0 point of the Z axis. Injection rate: 0.2 μL / min. The injection was paused for 5 minutes, 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 an experimental area of ​​a feeding cage for four weeks after awakening. Body weight and food intake were recorded weekly.

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

[0212] After four weeks of rearing, samples were collected and the levels of TDP and TM in the blood, the expression of rAAV virus-encoded polypeptides in the brain, and the expression levels of TPK and TDP and TM in brain and liver tissue were detected using the method described in Example 3.

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

[0214] As shown in Figure 11, four weeks after injecting AAV into the lateral ventricle of adult mice, the gene product encoded by the rAAV virus was strongly expressed around the lateral ventricle and also expressed in several other brain regions.

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

[0216] As shown in Figure 13, TM and TDP levels in the liver were not significantly altered, indicating that the virus is brain-specific and specifically targets brain nerve cells without affecting peripheral tissues. Viral side effects were completely controllable.

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

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

[0219] The mice were deeply anesthetized and placed with their abdomens facing upwards. The abdomens of the pregnant mice were wiped with tissues soaked in 75% alcohol. The hair in the center of the abdomen was trimmed, and the abdomens of the pregnant mice were wiped with tissues soaked in PBS to remove residual alcohol and loose hair.

[0220] The surgical instruments were wiped with 75% alcohol for disinfection. An incision of approximately 1.5 cm was made in the skin along the center of the pregnant mouse's abdomen with scissors, and the peritoneum of the pregnant mouse was incised along the linea alba of the abdomen to expose the abdominal cavity. Using forceps, the embryo was carefully removed from the abdominal cavity of the pregnant mouse and placed back in the abdominal cavity (the abdominal cavity and embryo surface were kept moist with PBS throughout the following surgical procedure). A glass electrode soaked in rAAV virus suspension was gently inserted into the brain of the embryo, and air was blown in to move the virus suspension into the lateral ventricles of the embryo. 0.5–1 μL (5.0E+9–1.0E+10 vg) of virus suspension was injected into the lateral ventricles of each embryo. The embryo was returned to the abdominal cavity of the pregnant mouse, and the mouse's peritoneum and skin were sutured in sequence. After suturing, lidocaine-lincomycin ointment was applied to the wound to prevent inflammation and infection, and the mouse was placed on a warming pad until it woke up. After that, it was returned to its cage and reared.

[0221] After birth, the pups were tested for viral injection by irradiating their heads with 480 nm excitation light, and mice that were successfully injected were kept. After 21 days of rearing, three mice were treated using the method described in Example 3, and samples were taken to detect TDP and TM levels in the blood, expression of rAAV virus-encoded polypeptides in the brain, and TPK, TDP, and TM expression levels in brain and liver tissue. The other 12 mice were weaned, and their weight was recorded. They were reared in separate cages for two weeks, and their weight was recorded again.

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

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

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

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

[0226] As shown in Figure 18, TM and TDP levels in the liver did not show significant changes, indicating that the virus is highly brain-specific, specifically targeting brain neurons without affecting peripheral tissues. Viral side effects were completely controllable.

[0227] Example 6: Overexpression of TPK and increased TM / TDP conversion rate in the brains of neonatal mice injected with AAV-TPK in a stereotactic manner. The objective of this embodiment is to verify whether direct delivery of AAV-TPK to the lateral ventricles (intraventricular, ICV) and (intraparenchymal, IP) of neonatal mice (P0 mice, 0 days old) significantly increases TPK expression in brain neurons of C57BL6 / J mice, improves the TM / TDP conversion rate in neurons, and thereby enhances glucose metabolism.

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

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

[0230] Hippocampal injection site (1): 0.7 mm anterior to the lambda, 1.2 mm lateral to the left and right, 0.9 mm deep. Hippocampal injection site (2): 0.1 mm anterior to the lambda, 2.2 mm lateral to the left and right, 1.4 mm deep. 200 nL of viral suspension (1.0E + 13vg / mL) was injected into two locations on each side of the hippocampus, for a total of four locations.

[0231] After filling the glass electrode with paraffin oil, it was fixed 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 then fixed to an automatic injection pump.

[0232] The bodies of newborn mice were buried in ice for approximately 3 minutes, without covering their noses to allow them to breathe. After the P0 mice did not struggle and changed from a purplish-pink to a slightly whitish color, they were removed from the ice and placed back on the ice in preparation for the next experiment.

[0233] The virus suspension was drawn into a glass electrode using an injection pump. Newborn mice, deeply anesthetized by ice treatment, were secured to a horizontal container with medical tape, keeping their heads horizontal.

[0234] Under a stereomicroscope, the syringe was moved to a designated position using a manipulative arm, starting from the occipital fontanelle (lambda) of a newborn mouse, and the vertical position of the syringe was adjusted using a vertical manipulative arm (Z-axis). After the needle touched the head, the position where the needle was slightly depressed was defined as the zero point, and the needle was lowered to the designated position. Finally, the virus was automatically injected into the brain of the newborn mouse by an injection pump controller. After the injection was complete, the needle remained in the brain of the newborn mouse for approximately 3 minutes, after which it was slowly withdrawn.

[0235] After the injection, the newborn mice were promptly transferred to a warming pad. Once the newborn mice's body temperature recovered and they began to move, they were returned to their mother's cage, covered with a cage pad, and left there for a certain period of time until the scent permeated the pad. The positional fixation experiment ended when the mother mouse 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 the blood, expression of rAAV virus-encoded polypeptides in the 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 weight was recorded. The animals were then separated into cages, fed for two weeks, and their weight was recorded again.

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

[0238] As shown in Figure 24, there were no significant changes in the TDP and TM content in peripheral blood three weeks after injection of AAV-TPK into the hippocampus of P0 mice.

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

[0240] As shown in Figure 22, three weeks after rAAV was administered to the lateral ventricles of P0 mice, TPK expression and TDP content significantly increased in brain tissue compared to the control group, while TM content decreased, indicating an increased TM / TDP conversion rate.

[0241] As shown in Figure 26, three weeks after injection of AAV-TPK into the hippocampus of P0 mice, the expression level of TPK in brain tissue was significantly increased compared to the control group.

[0242] As shown in Figure 27, TM and TDP in the liver did not change significantly, indicating that the virus is highly brain-specific and specifically targets brain nerve cells without affecting peripheral tissues. Viral side effects were completely controllable.

[0243] Example 7: Screening of highly active TPK variants In this example, mutants exhibiting higher activity than the wild-type TPK polypeptide were screened by introducing amino acid substitutions into the TPK polypeptide.

[0244] The inventors selected amino acids that could significantly affect TPK activity by comparing the structures of human TPK and mouse TPK proteins, and designed and prepared the TPKs and their variants shown in Table 2 using single and complex mutations in these amino acids.

[0245] [Table 2]

[0246] Specifically, the nucleotide sequences encoding the TPK (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 on LB agar medium (containing 50 mg / L kanamycin) and cultured overnight at 37°C. Subsequently, single colonies were transferred to LB liquid medium (containing 50 mg / L kanamycin) for sequence confirmation.

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

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

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

[0250]

Table 3

[0251] Equal volumes of 50 mM TPK enzyme solution were mixed with 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, followed by 30 μL of thiamine solution (20 μM), and the mixture was incubated at 37°C for 0.5 hours. Then, 270 μL of 5% PCA quenching solution was added and the reaction was stopped by thorough mixing. 150 μL of the above mixture was transferred to a 1.5 mL centrifuge tube, 30 μL of 10 mM potassium ferricyanide derivatization reagent was added for sample derivatization, and finally, 15 μL of 1 M phosphate neutralizing 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, mutant hTPK 8M showed significantly increased catalytic activity in the conversion of TM to TDP compared to human TPK (hTPK) (p<0.05), but was still lower than the catalytic activity of wild-type mouse TPK (mTPK) (p<0.05). As shown in Figure 29, mutant mTPK 11W and mTPK 35W showed higher catalytic activity for the conversion of TM to TDP compared to wild-type mTPK, with mutant mTPK 11W showing the highest activity (p<0.01 vs. mTPK), although the introduction of the 241W substitution reduced the activity.

[0253] Example 8: Effects of rAAV encoding a TPK mutant on TPK activity in vivo In this study, we investigated the therapeutic effects of rAAV encoding a TPK mutant with increased activity in neonatal mice and APP / PS1 mice.

[0254] An rAAV encoding an hTPK, referred to as AAV hTPK, was prepared by the method described in Example 1, wherein the genome comprises i) a neuron-specific promoter (SEQ ID NO: 3), ii) a nucleotide sequence encoding an hTPK (SEQ ID NO: 14), and a WPRE element (SEQ ID NO: 4).

[0255] 8.1. Overexpression of TPK and increased TM / TDP conversion rate in the brains of neonatal mice injected intraventricularly with AAV-hTPK. The objective of this embodiment is to verify whether direct delivery of AAV-hTPK to the lateral ventricle (intraventricular cavity, ICV) of neonatal mice (P0 mice, 0 days old) significantly increases TPK expression in brain neurons of C57BL6 / J mice, improves the TM / TDP conversion rate in neurons, and thereby enhances glucose metabolism.

[0256] Eleven C57BL6 / J mice (embryonic day 18.5, E18.5) were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. The total volume of the 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. Experiments were conducted after the birth of newborn mice.

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

[0258] After filling the glass electrode with paraffin oil, it was fixed 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 then fixed to an automatic injection pump.

[0259] The bodies of newborn mice were buried in ice for approximately 3 minutes, without covering their noses to allow them to breathe. After the P0 mice did not struggle and changed from a purplish-pink to a slightly whitish color, they were removed from the ice and placed back on the ice in preparation for the next experiment.

[0260] The virus suspension was drawn into a glass electrode using an injection pump. Newborn mice, deeply anesthetized by ice treatment, were secured to a horizontal container with medical tape, keeping their heads horizontal.

[0261] Under a stereomicroscope, the syringe was moved to a designated position using a manipulative arm, starting from the occipital fontanelle (lambda) of a newborn mouse, and the vertical position of the syringe was adjusted using a vertical manipulative arm (Z-axis). After the needle touched the head, the position where the needle was slightly depressed was defined as the zero point, and the needle was lowered to the designated position. Finally, the virus was automatically injected into the brain of the newborn mouse by an injection pump controller. After the injection was complete, the needle remained in the brain of the newborn mouse for approximately 3 minutes, after which it was slowly withdrawn.

[0262] After the injection, the newborn mice were promptly transferred to a warming pad. Once the newborn mice's body temperature recovered and they began to move, they were returned to their mother's cage, covered with a cage pad, and left there for a certain period of time until the scent permeated the pad. The positional fixation experiment ended when the mother mouse spontaneously returned the newborn mice to the nest.

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

[0264] As shown in Figure 30, TPK protein expression in brain tissue 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) resulted in a dose-dependent decrease in TDP content in brain tissue, similar to viral dilution (Figure 31).

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

[0266] After three days of adaptive feeding, adult APP / PS1 male mice (13 months old) (purchased from Cavens Biogle (Suzhou) Model Animal Research Co. Ltd.) were randomly divided into two groups (TPK group and control group, n=12), and stereotactic injection of AAV into the lateral ventricles was performed. The concentration of the viral concentrate used was 1.0E+13vg / 1mL, and the injection volume into the lateral ventricles was 4.0E+10vg / 4μL per mouse (2μL each side). Adult mice were anesthetized with isoflurane, the hair between the ears and eyes was shaved, and they were fixed to a stereotactic device. The skin of the mice's brains was wiped and disinfected with an alcohol swab. The skin was cut open with scissors to expose the skull. The surface of the skull was wiped with a dry cotton swab until dry, 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, with the skull surface as the 0 point of the Z axis. The injection rate was 0.2 μL / min, the injection was paused for 5 minutes, and the needle was slowly removed. After suturing the skin, the mice were placed in a cage equipped with a heated pad and fed in the experimental area of ​​the feeding cage for at least 8 weeks after awakening, while waiting for AAV expression. Body weight, food intake, and blood glucose levels were recorded weekly (at specific time points).

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

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

[0269] As shown in FIG. 33, the expression level of TPK in the brain tissue of mice injected with AAV-TPK was high, while TPK was hardly expressed 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 the same as that of mice injected with control AAV, indicating that the AAV according to the present disclosure does not show an off-target effect.

[0270] As shown in FIG. 34, the TDP content in the brain tissue of APP / PS1 mice injected with AAV-TPK was significantly higher than that of APP / PS1 mice injected with control AAV, but the TDP content in the blood and liver tissues of APP / PS1 mice injected with AAV-TPK was the same as that of APP / PS1 mice injected with control AAV, indicating that the AAV according to the present disclosure does not show an off-target effect.

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

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

Sequence Listing Free-Text

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

[0274] Sequence ID 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] Sequence ID 7 Eukaryotic promoter GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCCCCCATTGACGTCAATGGGAGTTTGTTTT GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTTACGTGGGAGGTCTATATAAGCAGAGCT

[0280] Sequence ID 8: Amino acid sequence of human TPK MEHAFTPLEPLLSTGNLKYCLVILNQPLDNYFRHLWNKALLRACADGGANRLYDITEGERESFLPEFINGDFDSIRPEVREYYATKGCELISTPDQDHTDFTKCLKMLQKKIEEKDLKVDVIVT LGGLAGRFDQIMASVNTLFQATHITPFPIIIIQEESLIYLLQPGKHRLHVDTGMEGGDWCGLIPVGQPCMQVTTTGLKWNLTNDVLAFGTLVSTSNTYDGSGVVTVETDHPLLWTMAIKS

[0281] Sequence ID 9: Human TPK 8M amino acid sequence MEHAFTPLEPLLPTGNLKYCLVILNQPLDARFRHLWKKALLRACADGGANRLYDITEGERESFLPEFINGDFDSIRPEVREYYAKKGCELISTPDQDHTDFTKCLKMLQKKIEEKDLKVDVIVTLG GLGGRFDQIMASVNTLFQATHITPFPIIIIQKESLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCMQVTTTGLKWNLTNDVLAFGTLVSTSNTYDGSGVVTVETDHPLLWTMAIKS

[0282] Sequence ID No. 10: Mouse TPK 11W amino acid sequence MEHAFTPLEPWLPTGNLKYCLVVLNQPLDARFRHLWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAIKS

[0283] Sequence ID 11: Mouse TPK 35W amino acid sequence MEHAFTPLEPLLPTGNLKYCLVVLNQPLDARFRHWWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAIKS

[0284] Sequence ID 12: Mouse TPK 241W amino acid sequence MEHAFTPLEPLLPTGNLKYCLVVLNQPLDARFRHLWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAWKS

[0285] Sequence ID 13: Mouse TPK 11W+241W amino acid sequence MEHAFTPLEPWLPTGNLKYCLVVLNQPLDARFRHLWKKALLRACADGGANHLYDLTEGERESFLPEFVSGDFDSIRPEVKEYYTKKGCDLISTPDQDHTDFTKCLQVLQRKIEEKELQVDVIVTLG GLGGRFDQIMASVNTLFQATHITPVPIIIIQKDSLIYLLQPGKHRLHVDTGMEGSWCGLIPVGQPCNQVTTTGLKWNLTNDVLGFGTLVSTSNTYDGSGLVTVETDHPLLWTMAWKS

[0286] Sequence ID 14: Human TPK nucleotide sequence ATGGAGCATGCCTTTACCCCGTTGGAGCCCCTGCTTTCCACTGGGAATTTGAAGTACTGCCTTGTAATTCTTAATCAGCCTTTGGACAACTATTTTCGTCATCTTTGGAACAAAGCTCTTTTAAGAGCCTGTGCCGATGGAGGTGCCAACCGCTTATATGATATCACCGAAGGAGAGAG AGAAAGCTTTTTGCCTGAATTCATCAATGGAGACTTTGATTCTATTAGGCCTGAAGTCAGAGAATACTATGCTACTAAGGGATGTGAGCTCATTTCAACTCCTGATCAAGACCACACTGACTTTACTAAGTGCCTTAAAATGCTCCAAAAGAAGATAGAAGAAAAAGACTTAAAGGTTGATGTGATCGT GACACTGGGAGGCCTTGCTGGGCGTTTTGACCAGATTATGGCATCTGTGAATACCTTGTTCCAAGCGACTCACATCACTCCTTTTCCAATTATAATAATCCAAGAGGAATCGCTGATCTACCTGCTCCAACCAGGAAAGCACAGGTTGCATGTAGACACTGGAATGGAGGGTGATTGGTGTGGCC TTATTCCTGTTGGACAGCCTTGTATGCAGGTTACAACCACAGGCCTCAAGTGGAACCTCACAAATGATGTGCTTGCTTTTGGAACATTGGTCAGTACTTCCAATACCTACGACGGGTCTGGTGTTGTGACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCATCAAAAGCTAA

[0287] SEQ ID NO: 15 Nucleotide sequence of human TPK8M 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. Recombinant adeno-associated virus (rAAV) or recombinant lentivirus having an expression cassette in its genome comprising a polynucleotide encoding a thiamine pyrophosphokinase (TPK) operably linked to a promoter, wherein the TPK comprises the amino acid sequence of SEQ ID NOs: 1, 9, 10, or 11.

2. The rAAV or recombinant lentivirus according to claim 1, wherein the promoter is a neuron-specific promoter.

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

3.

4. The rAAV or recombinant lentivirus according to claim 1, wherein the expression cassette further comprises a WPRE element.

5. The rAAV or recombinant lentivirus according to claim 4, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO:

4.

6. The rAAV according to claim 1, which is an rAAV of serotype AAV_PHP eB.

7. A pharmaceutical composition comprising rAAV or recombinant lentivirus as described in claim 1 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, formulated for intravenous, intracerebral, or intrathecal administration.

9. A pharmaceutical composition comprising the rAAV or recombinant lentivirus according to claim 1 for the prevention or treatment of Alzheimer's disease.

10. The pharmaceutical composition according to claim 9, wherein the rAAV or recombinant lentivirus, or the pharmaceutical composition, is administered intravenously, intracerebrally, or intrathecally.

11. Use of the rAAV or recombinant lentivirus according to claim 1, or the pharmaceutical composition according to claim 7, in the manufacture of a pharmaceutical product for preventing or treating Alzheimer's disease.

12. The use according to claim 11, wherein the pharmaceutical product is administered intravenously, intracerebrally, or intrathecally.

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