Cleavage-type protein and its use

Abstract

Provided is a truncated TRIM72 protein comprising a coiled PRYSPRY domain or a PRYSPRY domain of the TRIM72 protein, or a functional fragment thereof. Also provided is the use of the truncated TRIM72 protein.

Description

Background Art

[0001] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease and motor neuron disease (MND), is a specific disease that causes the death of neurons that control voluntary muscles. Some also use the term "motor neuron disease" for the group of symptoms where ALS is most common. ALS is characterized by muscle stiffness, muscle fasciculations, and a progressively worsening weakness due to a decrease in muscle size. As a result, speaking, swallowing, and ultimately breathing become difficult. In 90% - 95% of cases, the cause is unknown. Approximately 5 - 10% of cases are inherited from a person's parents. About half of these genetic cases are due to four specific genes, SOD1, TDP - 43, FUS, and C9orf72. Currently, there is no known cure for ALS.

[0002] TRIM72 is a tripartite motif (TRIM) family protein consisting of a ring finger, B - box motif, coiled - coil region, and C - terminal PRYSPRY domain. It is involved in the fascia repair process and is related to the insulin signaling pathway. It is also involved in cardiac protection against ischemia / reperfusion (IR) injury. Full - length TRIM72 has been reported to act as a potential target for ALS by ubiquitinating mutant FUS protein. However, the delivery of full - length TRIM72 is greatly limited by the characteristics of the vector, and some vectors have a limited size of the gene to be delivered. Therefore, it is necessary to further investigate the effect of TRIM72 for protecting neurons and to further develop different TRIM72 protein products.

Summary of the Invention

[0003] The present disclosure provides a TRIM72 truncated protein and its use. The TRIM72 truncated protein has one or more of the following characteristics: (1) it can effectively protect neurons; (2) it can reduce oxidative stress; (3) it can treat, prevent, and / or alleviate nervous system diseases.

[0004] In one aspect, the present application provides a truncated TRIM72 protein comprising the PRYSPRY domain of the TRIM72 protein or a functional fragment thereof.

[0005] In some embodiments, the TRIM72 protein is a human TRIM72 protein.

[0006] In some embodiments, the TRIM72 protein comprises the amino acid sequence of SEQ ID NO: 2.

[0007] In some embodiments, the PRYSPRY domain comprises the amino acid site of 278aa - 470aa of the TRIM72 protein.

[0008] In some embodiments, the PRYSPRY domain comprises the amino acid sequence shown in SEQ ID NO: 6.

[0009] In some embodiments, the truncated TRIM72 protein further comprises the coiled - coil domain of the TRIM72 protein or a functional fragment thereof.

[0010] In some embodiments, the truncated TRIM72 protein does not comprise the coiled - coil domain of the TRIM72 protein or a functional fragment thereof.

[0011] In some embodiments, the coiled - coil domain comprises the amino acid site of 135aa - 232aa of the TRIM72 protein.

[0012] In some embodiments, the coiled - coil domain comprises the amino acid sequence shown in SEQ ID NO: 5.

[0013] In some embodiments, the truncated TRIM72 protein further comprises the B - box domain of the TRIM72 protein or a functional fragment thereof.

[0014] In some embodiments, the TRIM72 cleavage protein does not contain the B-box domain of the TRIM72 protein or a functional fragment thereof.

[0015] In some embodiments, the B-box domain contains the amino acid site of 86aa - 117aa of the TRIM72 protein.

[0016] In some embodiments, the B-box domain contains the amino acid sequence shown in SEQ ID NO: 4.

[0017] In some embodiments, the TRIM72 cleavage protein further contains the ring finger domain of the TRIM72 protein or a functional fragment thereof.

[0018] In some embodiments, the TRIM72 cleavage protein does not contain the ring finger domain of the TRIM72 protein or a functional fragment thereof.

[0019] In some embodiments, the ring finger domain contains the amino acid site of 14aa - 56aa of the TRIM72 protein.

[0020] In some embodiments, the ring finger domain contains the amino acid sequence shown in SEQ ID NO: 3.

[0021] In some embodiments, the TRIM72 cleavage protein contains the amino acid sequence shown in any one of SEQ ID NOs: 6, 7, 8, 9, and 11.

[0022] In some embodiments, the TRIM72 cleavage protein includes its variants.

[0023] In some embodiments, the TRIM72 cleavage protein does not contain an amino acid mutation at position C242.

[0024] In some embodiments, the TRIM72 cleavage protein further contains an amino acid mutation at position C14.

[0025] In some embodiments, the TRIM72 cleavage protein further comprises the amino acid mutation C14A.

[0026] In some embodiments, the TRIM72 cleavage protein is used to protect neurons by reducing oxidative stress.

[0027] In some embodiments, the TRIM72 cleavage protein is secreted via exosomes.

[0028] In some embodiments, the TRIM72 cleavage protein is used to prevent or treat a nervous system disease.

[0029] In some embodiments, the nervous system disease is a nerve injury disease induced by oxidative stress.

[0030] In some embodiments, the TRIM72 cleavage protein is used to prevent or treat ALS, PD, and / or stroke.

[0031] In another aspect, the present application provides a recombinant protein comprising a TRIM72 cleavage protein.

[0032] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding a TRIM72 cleavage protein.

[0033] In another aspect, the present application provides a vector comprising a nucleic acid molecule.

[0034] In some embodiments, the vector comprises a viral vector.

[0035] In some embodiments, the vector comprises an AAV vector.

[0036] In some embodiments, the viral vector comprises an AAV9 vector.

[0037] In another aspect, the present application provides a cell comprising a nucleic acid molecule or a vector.

[0038] In another aspect, the present application provides a fusion protein comprising a TRIM72 cleavage protein.

[0039] In another aspect, the present application provides a pharmaceutical composition comprising the TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, a cell and / or a fusion protein, and a pharmaceutically acceptable adjuvant.

[0040] In some embodiments, the pharmaceutically acceptable adjuvant comprises a drug, a toxin, a cytokine, a radioactive element, a carrier protein, an enzyme, a lectin, a fluorescent quantum dot, and / or a chromophore with a high absorption coefficient.

[0041] In another aspect, the present application provides a method for protecting neurons of a subject, comprising administering to the subject in need thereof an effective amount of a TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, the cell, a fusion protein, and / or a pharmaceutical composition.

[0042] In another aspect, the present application provides a method for preventing and / or treating a nervous system disease, comprising administering to the subject in need thereof an effective amount of a TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, the cell, a fusion protein, and / or a pharmaceutical composition.

[0043] In some embodiments, the nervous system disease is a nerve injury disease induced by oxidative stress.

[0044] In some embodiments, the nervous system disease is a neurodegenerative disease.

[0045] In some embodiments, the nervous system disease includes ALS, PD, and / or ALS.

[0046] In another aspect, the present application provides the use of a TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, the cell, a fusion protein, and / or a pharmaceutical composition in the manufacture of a drug for preventing and / or treating a nervous system disease.

[0047] In some embodiments, the nervous system disease is a nerve injury disease induced by oxidative stress.

[0048] In some embodiments, the nervous system disease is a neurodegenerative disease.

[0049] In some embodiments, the nervous system disease includes ALS, PD, and / or stroke.

[0050] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other different embodiments, and some of its details are capable of modification in various obvious respects without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0051] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0052] The novel features of the present invention are particularly described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are used, and the accompanying drawings (also referred to herein as "figures" and "FIGs"). BRIEF DESCRIPTION OF THE DRAWINGS

[0053]

Figure 1

[0054]

Figure 2

[0055]

Figure 3

[0056]

Figure 4

[0057]

Figure 5

[0058]

Figure 6

[0059]

Figure 7

[0060]

Figure 8

[0061]

Mode for Carrying Out the Invention

[0062] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art can make numerous variations, changes, and substitutions without departing from the present invention. It should be understood that various alternative forms to the embodiments of the present invention described herein can be used.

[0063] In this application, the term "TRIM72 protein" can be used interchangeably with the "MG53" protein and generally includes the TRIM72 protein or its variants, functional fragments, analogs, homologs. The TRIM72 protein may contain a ring finger, B-box motif, coiled-coil region, and / or C-terminal PRYSPRY domain. For example, the ring finger domain may include the amino acid site of 14aa - 56aa of the TRIM72 protein or its functional fragment. For example, the B-box domain may include the amino acid site of 86aa - 117aa of the TRIM72 protein or its functional fragment. For example, the coiled-coil domain may include the amino acid site of 135aa - 232aa of the TRIM72 protein or its functional fragment. For example, the PRYSPRY domain may include the amino acid site of 278aa - 470aa of the TRIM72 protein. This term may also include TRIM72 proteins derived from any known species having the TRIM72 protein.

[0064] In this application, the term "truncated protein" generally refers to a protein having one or more amino acid deletions compared to the full-length protein. For example, a truncated protein may contain a major functional fragment of the protein. For example, truncated proteins include, but are not limited to, their variants, functional fragments, analogs, homologs.

[0065] In the present application, "amino acid mutation Xn" refers to an amino acid mutation occurring in the amino acid residue X at position n in the amino acid sequence shown in SEQ ID NO: 2, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "amino acid mutation C14" refers to an amino acid substitution occurring in the amino acid residue C corresponding to position 14 in the amino acid sequence shown in SEQ ID NO: 2.

[0066] The amino acid mutations of the present application can be non-conservative mutations. Such non-conservative mutations can change the amino acid residues in the target protein or polypeptide in a non-conserved manner, for example, replacing an amino acid residue having a specific side chain size or a specific property (e.g., hydrophilicity) with an amino acid residue having a different side chain size or a different property (e.g., hydrophobicity).

[0067] The amino acid substitution may be a conservative substitution. Such conservative substitutions can change the amino acid residues in the target protein or polypeptide in a conserved manner, for example, replacing an amino acid residue having a specific side chain size or a specific property (e.g., hydrophilicity) with an amino acid residue having the same or a similar side chain size or the same or a similar different property (e.g., still hydrophilic). Such conservative substitutions generally do not have a significant effect on the structure or function of the resulting protein. In the present application, an amino acid sequence variant that is a mutant of a fusion protein, a fragment thereof, or a variant thereof that undergoes one or more amino acid substitutions can include conservative amino acid substitutions that do not significantly change the structure or function of the protein.

[0068] As an example, the inter-amino acid substitutions in each of the following groups can be considered conservative substitutions in the present application. Group of amino acids having non-polar side chain(s): alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. Group of uncharged amino acids with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Group of negatively charged amino acids with polar side chains: aspartic acid and glutamic acid. Group of positively charged basic amino acids: lysine, arginine, and histidine. Group of amino acids with phenyl: phenylalanine, tryptophan, and tyrosine.

[0069] In the present application, the term "fusion protein" generally refers to a composite polypeptide, i.e., a single continuous amino acid sequence consisting of two (or more) polypeptides. Fusion proteins can generally be artificially prepared by recombinant nucleic acids or chemical synthesis.

[0070] In the present application, the term "neurodegenerative disease" generally refers to various nervous system disorders characterized by a progressive and degenerative loss of nerve tissue and / or nerve tissue function. Neurodegenerative diseases are a type of nerve disorder or nerve disease, and nerve diseases are characterized by a progressive and degenerative loss of nerve tissue and / or changes in nerve function, typically a reduction in nerve function as a result of a progressive and degenerative loss of nerve tissue. For example, neurodegenerative diseases can be caused by oxidative stress.

[0071] In the present application, the term "Parkinson's disease" generally refers to a type of neurodegenerative disorder. Parkinson's disease is a chronic progressive neurological disease characterized by neurodegeneration, particularly the degeneration of dopaminergic neurons. Symptoms include a stooped posture, resting tremors, muscle weakness at rest, shuffling gait, speech impairment, movement difficulties, and ultimately delays in mental processes and / or dementia.

[0072] In the present application, the term "stroke" generally refers to a condition caused by the occlusion or bleeding of one or more blood vessels supplying the brain that can lead to cell death. This term may include ischemic stroke and hemorrhagic stroke. As used herein, "ischemic stroke" generally refers to a stroke caused by the occlusion of one or more blood vessels supplying the brain. Types of ischemic stroke may include, but are not limited to, embolic stroke, cardiogenic embolic stroke, thrombotic stroke, large vessel thrombosis, lacunar infarction, arterial stroke, and cryptogenic stroke. As used herein, "hemorrhagic stroke" generally refers to a stroke caused by bleeding of one or more blood vessels supplying the brain. Types of hemorrhagic stroke include, for example, subdural stroke, intracerebral hemorrhage, epidural stroke, and subarachnoid stroke.

[0073] In the present application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof of any length, isolated from their natural environment or artificially synthesized. The nucleic acid molecules of the present application can be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification such as polymerase chain reaction (PCR) amplification, (ii) clone recombination, (iii) purification, such as fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesis, such as chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. In the present application, the nucleic acid encoding the truncated protein or a functional fragment thereof can be prepared by various methods known in the art. These methods include, but are not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. Specific procedures can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

[0074] In the present application, the term "vector" generally refers to a vector containing a recombinant polynucleotide that includes an expression control sequence efficiently linked to a nucleotide sequence to be expressed. The vector contains cis-acting elements sufficient for expression. Other elements for expression may be provided by the host cell or in an in vitro expression system. The vector may include all expression vectors known in the art that can be incorporated into a recombinant polynucleotide, including cosmids, plasmids (e.g., naked or encapsulated in liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0075] In the present application, the term "encoding" generally refers to the inherent property of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA that acts as a template for the synthesis of other multimers and macromolecules in a biological process, and the multimers and macromolecules have either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties result therefrom. Thus, when transcription and translation of mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes the protein. Both the nucleotide sequence that is identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, can be referred to as the protein or other product encoded by the gene or cDNA. In the present application, the term "coding element" generally refers to a nucleic acid (RNA or DNA molecule) that includes a nucleotide sequence encoding a protein.

[0076] In this application, the terms "host cell", "cell", and "host" are used interchangeably and generally refer to a plasmid or vector that can contain or has contained a nucleic acid molecule of this application, or can express an individual cell, cell line, or cell culture of a protein, fragment, or variant thereof of this application. The host cell can include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny cells and the original parental cells do not necessarily have to be identical in form or genome as long as they can express the protein or fragment thereof of this application. The host cell can be obtained by transfecting cells in vitro with a vector of this application. The host cell can be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell). In this application, the host cell can be a CHO cell.

[0077] In this application, the term "treat" generally refers to delaying or improving the progression, severity, and / or duration of a proliferative disorder, or improving one or more symptoms (e.g., one or more distinguishable symptoms) of a proliferative disorder as a result of the administration of one or more treatments.

[0078] In this application, the term "subject" generally refers to any human or non-human animal. The term "non-human animal" can include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, goats, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0079] In this application, the terms "peptide", "polypeptide" and "protein" can be used interchangeably and generally refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included in a protein or peptide sequence. A polypeptide can include any peptide or protein containing two or more amino acids linked to each other via peptide bonds. In this application, this term refers to two short chains, which are also commonly known as peptides, oligopeptides and oligomers in the art, for example, long chains that are commonly known as proteins in the art, and there are many types of them. "Polypeptide" includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides or combinations thereof.

[0080] In addition to the specific proteins and nucleotides referred to herein, this application may also include functional variants, derivatives, analogs, homologs and fragments thereof.

[0081] The term "functional variant" refers to a polypeptide that has an amino acid sequence substantially the same as a naturally occurring sequence or is encoded by a substantially the same nucleotide sequence and can have one or more activities of a naturally occurring sequence. In the context of this application, a variant of any given sequence refers to a sequence in which the specific sequence of residues (either amino acid residues or nucleotide residues) has been modified such that the polypeptide or polynucleotide maintains at least one endogenous function substantially. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or alteration of at least one amino acid residue and / or nucleotide residue present in a naturally occurring protein and / or polynucleotide, as long as the original functional activity is maintained. In this application, the term "derivative" generally refers to a polypeptide or polynucleotide of this application that includes any substitution, alteration, modification, replacement, deletion and / or addition from / to one (or more) amino acid residues of the sequence, as long as the resulting polypeptide or polynucleotide substantially maintains at least one of its endogenous functions.

[0082] In the present application, the term "analogue" generally refers to any mimetic of a polypeptide or polynucleotide, i.e., a compound that has at least one endogenous function of the polypeptide or polynucleotide being mimicked, with respect to the polypeptide or polynucleotide. Generally, amino acids can be substituted as long as the modified sequence substantially maintains the required activity or ability, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 or more) amino acids can be substituted. Amino acid substitutions can include the use of non-naturally occurring analogues. The proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues, and the amino acid residues may undergo silent changes, resulting in a functionally equivalent protein. Intentional amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric properties of the residues, as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids containing uncharged polar head groups with similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0083] In this application, the term "homolog" generally refers to an amino acid sequence or nucleotide sequence having a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be equivalent to "identity" of a sequence. A homologous sequence may include an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the target sequence. Generally, a homolog contains the same active site as the target amino acid sequence, etc. Homology may be considered based on similarity (i.e., amino acid residues having similar chemical properties / functions), or homology may be expressed in terms of sequence identity. In this application, a sequence having percentage identity at any of the sequence numbers of the recited amino acid sequences or nucleotide sequences refers to a sequence having percentage identity over the full length of the recited sequence number. To determine sequence identity, sequence alignment can be performed by various methods known to those skilled in the art, for example, by using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters suitable for alignment, including any algorithms necessary to achieve optimal alignment in the full-length sequences being compared.

[0084] In this application, the term "about" generally refers to varying in the range of 0.5% to 10% above or below a specified value, for example, varying in the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

[0085] In this application, the term "comprising" generally means including, containing, having, or encompassing. In some cases, it may also refer to the meaning of "being" or "consisting of".

[0086] In this application, the term "does not comprise" generally refers to excluding a specific behavior, structure, or structural possibility. For example, "A does not comprise B" generally means excluding the possibility of B occurring in A.

[0087] TRIM72 cleavage protein

[0088] In one aspect, this application provides a TRIM72 cleavage protein comprising a PRYSPRY domain or a functional fragment thereof. For example, the TRIM72 cleavage protein may include the amino acid site of 278aa - 470aa of the TRIM72 protein. For example, the TRIM72 cleavage protein may include the amino acid sequence shown in SEQ ID NO: 2.

[0089] In this application, the TRIM72 cleavage protein may further include other domains of the TRIM72 protein.

[0090] For example, the TRIM72 cleavage protein may include a PRYSPRY domain and a coiled - coil domain. For example, the TRIM72 protein may include the amino acid sequence shown in SEQ ID NO: 11. For example, the TRIM72 cleavage protein may include deletions of the B - box domain and the ring - finger domain of the TRIM72 protein. For example, the TRIM72 protein may include the amino acid sequence shown in SEQ ID NO: 11.

[0091] Exosomes are small extracellular biological vesicles that are released into the surrounding body fluid by the fusion of multivesicular bodies containing proteins, nucleic acids, lipids, and other bioactive substances with the plasma membrane. For example, the TRIM72 truncated protein can be secreted via exosomes. For example, the TRIM72 truncated protein can be packaged into exosomes. For example, exosomes can contain nucleic acids encoding the TRIM72 truncated protein. For example, exosomes can be marked by TSG101. When using exosomes to secrete the TRIM72 truncated protein, a coiled-coil domain and a PRYSPRY domain are required.

[0092] For example, the TRIM72 truncated protein can contain a PRYSPRY domain and a B-box domain. For example, the TRIM72 truncated protein can contain deletions of the coiled-coil domain and the ring finger domain of the TRIM72 protein.

[0093] For example, the TRIM72 truncated protein can contain a PRYSPRY domain and a ring finger domain. For example, the TRIM72 truncated protein can contain deletions of the B-box domain and the coiled-coil domain of the TRIM72 protein.

[0094] For example, the TRIM72 truncated protein can contain a PRYSPRY domain, a coiled-coil domain, and a ring finger domain. For example, the TRIM72 truncated protein can contain a deletion of the B-box domain of the TRIM72 protein.

[0095] For example, the TRIM72 truncated protein can contain a PRYSPRY domain, a coiled-coil domain, and a B-box domain. For example, the TRIM72 truncated protein can contain a deletion of the ring finger domain of the TRIM72 protein.

[0096] For example, the TRIM72 cleavage protein may include a PRYSPRY domain, a ring finger domain, and a B box domain. For example, the TRIM72 cleavage protein may include a deletion of the coiled coil domain of the TRIM72 protein.

[0097] In this application, the TRIM72 cleavage protein may include its variants. For example, the TRIM72 cleavage protein may include one or more amino acid mutations as compared to the corresponding wild-type sequence. For example, the TRIM72 cleavage protein may include an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% the same as the corresponding wild-type sequence.

[0098] For example, the TRIM72 cleavage protein may include the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9 or 11, or an amino acid sequence having at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% similarity to SEQ ID NO: 6, 7, 8, 9 or 11.

[0099] In this application, the TRIM72 cleavage protein may include an amino acid mutation at position C14. For example, the amino acid mutation may be C14A.

[0100] According to the applicant's research, the amino acid cysteine at position 242 is important for the oligomer formation of the TRIM72 protein. Substitution of the amino acid C242 may block the protective function of neurons. Therefore, the amino acid substitution at position C242 (for example, C242A) may not be included in the TRIM72 cleavage protein in this application.

[0101] In this application, the truncated TRIM72 protein can be used to protect neurons by reducing oxidative stress. In this application, the truncated TRIM72 protein can be used to prevent or treat nervous system diseases associated with oxidative stress, such as nerve injury diseases or neurodegenerative diseases.

[0102] In this application, the truncated TRIM72 protein can be used to prevent or treat nervous system diseases. For example, this application can be used to prevent and / or treat ALS, PD and / or stroke.

[0103] Recombinant protein, nucleic acid, vector, cell, fusion protein, pharmaceutical composition

[0104] In another aspect, this application provides a recombinant protein comprising a TRIM72 truncated protein.

[0105] In another aspect, this application provides one or more nucleic acid molecules capable of encoding the TRIM72 truncated protein of this application.

[0106] In some embodiments, the nucleic acid molecule can completely encode the TRIM72 truncated protein or fusion protein of this application. For example, the TRIM72 truncated protein or fusion protein can be obtained by using only one type of nucleic acid molecule. In some embodiments, the nucleic acid molecule can encode a part of the TRIM72 truncated protein or fusion protein of this application. For example, the fusion protein can be obtained by using more than two different such nucleic acid molecules.

[0107] In another aspect, this application provides one or more vectors capable of containing one or more nucleic acid molecules of this application. In another aspect, this application provides cells (e.g., host cells) that may contain the nucleic acid molecule or vector of this application.

[0108] In the present application, a vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product. In the present application, the vector can contain one or more of the nucleic acid molecules. For example, the vector can contain all the nucleic acid molecules necessary to encode the TRIM72 cleavage protein.

[0109] Furthermore, the vector can also contain other genes such as a marker gene that enables selection of the vector under appropriate conditions in a suitable host cell. In addition, the vector can also contain expression control elements that enable the coding region to be appropriately expressed in a suitable host. Such control elements are well known to those skilled in the art. For example, they can include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatory element. The specific structure of the expression control sequence can vary depending on the species or cell type function, but usually includes 5' non-transcribed sequences involved in transcription and translation initiation as well as 5' and 3' untranslated sequences, such as TATA box, cap sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence can include a promoter region, and the promoter region can include a promoter sequence for transcriptional control of the functionally linked nucleic acid.

[0110] In another aspect, the present application provides a pharmaceutical composition comprising the TRIM72 cleavage protein, recombinant protein, nucleic acid molecule, vector, cell and / or fusion protein, and a pharmaceutically acceptable adjuvant.

[0111] In some embodiments, the pharmaceutically acceptable adjuvant can include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and / or nonionic surfactants, etc.

[0112] In some embodiments, the pharmaceutically acceptable adjuvant can include a drug, a toxin, a cytokine, a radioactive element, a carrier protein, an enzyme, a lectin, a fluorescent quantum dot, and / or a chromophore with a high absorption coefficient.

[0113] In the present application, the pharmaceutical composition can be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional technical means in the art, for example, according to the procedures of Remington: The Science and Practice of Pharmacy, 19th Edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995.

[0114] In the present application, the composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or the drug is administered via a subcutaneous depot.

[0115] In the present application, the pharmaceutical composition can be used to protect neurons. For example, the composition of the present application can inhibit or delay the onset or progression of a nervous system disease (such as ALS, PD or stroke), and / or can reduce and / or stabilize the disease state.

[0116] The pharmaceutical composition of the present application can include a therapeutically effective amount of the TRIM72 cleavage protein. The therapeutically effective amount is the dose required to prevent and / or treat (at least partially treat) the disease (such as ALS, PD or stroke) and / or any complications thereof in a subject having or at risk of having the disease.

[0117] Preparation, methods and uses

[0118] In another aspect, the present application provides a method for protecting neurons of a subject, comprising administering to the subject in need thereof an effective amount of a TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, a cell, a fusion protein, and / or a pharmaceutical composition.

[0119] In another aspect, the present application provides a method for preventing and / or treating a nervous system disease, comprising administering to the subject in need thereof an effective amount of a TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, a cell, a fusion protein, and / or a pharmaceutical composition.

[0120] In another aspect, the present application provides the use of a TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, a cell, a fusion protein, and / or a pharmaceutical composition in the manufacture of a drug for preventing and / or treating a nervous system disease.

[0121] In another aspect, the present application provides a TRIM72 cleavage protein, a recombinant protein, a nucleic acid molecule, a vector, a cell, a fusion protein, and / or a pharmaceutical composition for use in preventing and / or treating a nervous system disease.

[0122] In the present application, the nervous system disease includes ALS.

[0123]

Examples

[0124] The following examples are set forth to provide a complete disclosure and description of how to make and use the invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise noted, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations such as bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); i.m., intramuscular (into muscle); i.p., intraperitoneal (into the peritoneal cavity); s.c., subcutaneous (under the skin); rpm, revolutions per minute; etc. may be used.

[0125] Method

[0126] Preparation of Recombinant TRIM72 Protein in Escherichia coli (E. coli)

[0127] Codon optimization and gene synthesis were performed based on the amino acid sequence of mouse TRIM72 (Sequence 1). Full-length and truncated TRIM72 were constructed in a pET21b vector with a 6xHis tag at the N-terminus. These proteins were expressed and purified from Escherichia coli (E. coli) BL21(DE3) cells (Millipore) and purified under native conditions, unless otherwise specified. E. coli was grown to an OD600 of 0.8 and induced overnight at 16 °C with 0.6 mM IPTG. The pelleted cells were resuspended in lysis buffer (250 mM NaCl, 50 mM HEPES 7.5, 1 mM DTT, protease inhibitor). After sonication, the lysate was pelleted at 30,000 x g for 30 min at 4 °C. The supernatant was applied to a Ni column containing 10 mL beads (GE) pre-washed with lysis buffer at room temperature. The protein was eluted with elution buffer (25 mmol / L Tris pH 8.0, 300 mmol / L NaCl, 200 mmol / L imidazole). The protein was further treated with 0.1 mg / ml RNaseA (Thermo Fisher) to remove RNA and then purified by a Superdex 200 16 / 200 column (GE) equilibrated with SEC buffer (400 mM NaCl, 50 mM HEPES 7.5, 1 mM DTT). Fractions were analyzed by SDS-PAGE, pooled, concentrated, filtered, snap-frozen in liquid nitrogen, and stored at -80 °C.

[0128] Preparation of recombinant TRIM72 protein and its truncated fragments

[0129] Codon optimization and gene synthesis were performed based on the amino acid sequence of human TRIM72 (SEQ ID NO: 2). Using the synthetic TRIM72 gene with a 6×His tag added to the amino terminus as a template, pcDNA3.1-6His-TRIM72, pcDNA3.1-6His-coiled coil, pcDNA3.1-6His-PRYSPRY, and pcDNA3.1-6His-coiled-PRYSPRY plasmids were constructed by gene amplification, respectively (Figure 1). High-quality endotoxin-free plasmids were obtained by plasmid amplification using Escherichia coli (E. coli). HEK293F cells were transfected, collected, lysed, and the protein lysates were collected. After purification of the lysates using a Ni affinity column, TRIM72 (full-length) protein and its cleavage fragments (coiled coil (SEQ ID NO: 5), PRYSPRY (SEQ ID NO: 6), coiled-PRYSPRY (SEQ ID NO: 11)) were prepared.

[0130] Plasmids and lentiviral vectors

[0131] A DNA fragment corresponding to full-length TRIM72 was amplified from a mouse cDNA library by PCR and inserted into the pCMV-N-3×Flag expression vector between the SalI and XhoI sites using a seamless cloning kit (Beyotime) to generate Flag-tagged TRIM72. A series of Flag-tagged TRIM72 mutants: C14A (substitution of cysteine at position 14 with alanine); C242A (substitution of cysteine at position 242 with alanine) were generated from the wild-type TRIM72 construct by site-directed mutagenesis. A series of Flag-tagged TRIM72 domain deletions: ΔRing domain (deletion of domains 14 - 69, SEQ ID NO: 7); ΔB box domain (deletion of 81 - 122aa, SEQ ID NO: 8); Δcoiled-coil domain (deletion of 135 - 232aa, SEQ ID NO: 9); ΔPRYSPRY domain (deletion of 278 - 470aa, SEQ ID NO: 10) constructs were generated from the full-length TRIM72 expression vector. Flag-tagged TRIM72 coiled-coil domain (135 - 232aa) or PRYSPRY domain (278 - 470aa) or coiled-coil domain + PRYSPRY domain (135 - 470aa) constructs were generated from the full-length TRIM72 expression vector.

[0132] For the construction of TRIM72 stable expression cell lines, a series of DNA fragments of Flag-tagged TRIM72 mutants or domain deletions or single domains were amplified from the corresponding TRIM72 expression vectors by PCR and inserted into the pLJM1-EGFP lentiviral vector between the BsrGI and EcoRI sites to generate a series of Flag-tagged TRIM72 mutants or domain deletions or single domains fused with EGFP in the lentiviral vector.

[0133] Lentiviruses expressing the Flag-tagged TRIM72 constructs were generated from the corresponding EGFP fused with the Flag-tagged TRIM72 vector.

[0134] Cell culture, lentivirus packaging and lentivirus infection

[0135] HEK293FT cells were maintained in DMEM (Invitrogen) containing 10% fetal bovine serum (Gemini) in a cell incubator (37 °C, 5% CO2). For lentivirus packaging, HEK293FT cells were seeded into the growth medium of three 10-cm culture dishes. When reaching approximately 90% confluence, the cells were co-transfected with VSVG (10 μg), pxPAX2 (15 μg), and pLJM1-EGFP lentiviral vector or pLentiCRISPRv2 (Addgene) or pLenticas9-Blast (Addgene) (20 μg) using PEI (Sigma) according to the manufacturer's instructions, and the medium was replaced with fresh growth medium 5 - 6 hours after transfection. The medium was collected 72 hours after transfection and centrifuged at 20,000 rpm at 4 °C for 2 hours. After centrifugation, the lentivirus was concentrated into a pellet. The lentivirus was resuspended in 100 μl of DPBS and stored at -80 °C.

[0136] HEK293FT cells or Hela cells were infected with the indicated lentivirus. Three days after infection, the infected cells were selected with 2 μg / ml puromycin or 10 μg / ml blasticidin according to the plasmid containing resistance for at least one week. Puromycin or blasticidin-selected cells were applied for further analysis.

[0137] Cell viability assay

[0138] Cell viability was evaluated using CCK-8. The cells were seeded at 1.5×10 3 cells / well for arsenite treatment, or 7×10 3Cells were seeded in a 96-well plate at a density of cells / well. Arsenite (Sigma) was added to each well at a concentration of 0.125 mM, 0.25 mM, or 0.5 mM, and after incubation at 37 °C for 2 hours, the cells were washed. For the H2O2 treatment experiment, 24 hours of scAAV9 infection (estimated multiplicity of infection (MOI): 10,000 vg / cell) was performed 17 hours after cell seeding. Then, H2O2 was added to each well at a concentration of 300 μM, and after incubation at 37 °C for 1 hour, the cells were washed. After the cells were treated with arsenite or H2O2, a total of 10 μL of CCK-8 solution (Yeasen) was added to each well. After further incubation at 37 °C for 2 hours, the optical density (OD) value of each well was measured using a microplate reader with an excitation wavelength of 450 nm. The cell viability of 293FT was calculated. The experiment was repeated at least 3 times to obtain the average value.

[0139] Exosome purification

[0140] The protocol for purifying exosomes from 100 ml of the supernatant of 293FT cells included two steps: ultrafiltration and polyethylene glycol (PEG) precipitation. First, the collected supernatant was poured into a centrifuge tube and centrifuged at 3000 x g for 20 minutes to remove cell debris. Then, the supernatant was filtered once through a 0.45 μm filter. Then, take a new Amicon ULTRA-15 ultrafiltration tube washed with PBS or autoclaved water. Next, immediately add the supernatant to the ultrafiltration tube and centrifuge at 3000 × g for 5 - 10 minutes. Discard the filtrate, continue to add the supernatant, and centrifuge at 3000 × g for 5 - 10 minutes until all the supernatant is introduced into the ultrafiltration tube. Then, transfer the concentrate to a 50 mL centrifuge tube. Add the isolation reagent to the concentrate and mix the sample thoroughly by vortexing or pipetting. Finally, leave the sample at 2 - 8 °C overnight. The next day, the sample was centrifuged at 10,000 x g for 1 hour at 4 °C. After discarding the supernatant, the exosomes were in the pellet.

[0141] Western blot analysis

[0142] The total protein content in tissues and cells was extracted using RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) supplemented with phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail (Bimake), and the lysates were incubated on ice for 30 minutes. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was extracted, mixed with SDS loading buffer, and then incubated at 95°C for 10 minutes. Next, the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, the membrane was blocked with 5% skim milk at room temperature for 1 hour. Then, it was incubated overnight at 4°C with diluted primary antibodies (including GAPDH (Ameribio), TUBULIN (Ameribio), TRIM72 antibody (kindly provided by Dr. Jianjie Ma), CCDC22 (Sigma), GFP (Abcam), Myc (Abmart), TSG101 (Abcam), ITGAV (Abcam), H3 (Abcam), and Flag (Abmart)). Next, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 hour. Images were analyzed using Fiji ImageJ to obtain the integrated intensity.

[0143] Gastric administration of arsenite

[0144] Arsenite was prepared at 1 mg / ml in ddH2O and then injected i.g. into mice at 6 mg / kg body weight as described in the experiment (Zhang et al., 2020).

[0145] ROS measurement

[0146] ROS levels were measured using dihydroethidium (DHE, Invitrogen) and MitoSOX (Invitrogen). Cortical neurons were seeded onto 24-well chamber slides and cultured until DIV12-14. Neurons were then incubated with DHE (20 μM in medium) or MitoSOX (5 μM in medium) at 37 °C for 20 min. After incubation, neurons were fixed with 4% PFA for 10 min at room temperature. Images were captured by a Nikon A1 confocal microscope. DHE fluorescence was excited at 535 nm and emission was collected at 610 nm. MitoSOX fluorescence was excited at 510 nm and emission was collected at 580 nm. The mean intensity of DHE or MitoSOX fluorescence was accessed by FiJi ImageJ software. Also, ROS levels were represented by the relative mean intensity of DHE or MitoSOX fluorescence in different groups. RMI = mean intensity of DHE or MitoSOX fluorescence divided by the mean intensity of the background.

[0147] For lentivirus infection, cortical neurons were seeded onto 24-well chamber slides. Then, they were cultured until DIV3-5 and 10 μl of purified lentivirus was added to each well for further culture. When cultured until DIV12-14, DHE staining of neurons was performed.

[0148] Cortical neuron culture

[0149] Cortex was dissected from postnatal day 0 pups in HBSS (Invitrogen) and incubated with trypsin at 37°C for 15 minutes in Neurobasal (trademark) (Invitrogen). During digestion, DNAse I (Sigma, 10 μg / ml) was added during the last 10 minutes. The trypsinization process was stopped by adding Neurobasal medium containing 10% FBS. After digestion, the tissue was dissociated by gentle pipetting to obtain a single cell suspension. The suspension was passed through a 70 μm strainer. After centrifugation at 800 g for 10 minutes, the cells were resuspended in Neurobal (trademark) containing 10% FBS, GlutaMAX (trademark) (Invitrogen), and seeded into 24-well plates at a density of 500 μl per well at 2×10 5 cells / ml. After 5 - 6 hours of incubation (5% CO2, 37°C), the medium was replaced with Neurobal (trademark) containing B27 (Invitrogen), GlutaMAX (trademark) (Invitrogen). Cultured neurons (12 - 14 days in vitro) were stained with DHE or MitoSOX.

[0150] Example 1 TRIM72 knockout accelerates disease progression shown in FUS - R521C KI mice

[0151] TRIM72, a protein with undetectable levels in the central nervous system, is significantly upregulated in FUS-R521C KI mice (Figure 1A). To investigate the biological consequences of TRIM72 upregulation in FUS-R521C KI mice, we analyzed motor neurons and lifespan in wild-type (+ / +), FUS-R521C (C / C), FUS-R521C with TRIM72 knockout (C / C; - / -), and TRIM72 knockout alone (- / -) animals. Significantly more motor neuron loss in the spinal cord was observed in C / C; - / - animals compared to 1-year-old C / C mutants (Figures 1B, C). However, TRIM72 knockout mice did not show motor neuron loss compared to + / + controls (Figure 1C). The survival curves of these four genotypes were consistent in that the lifespan of C / C; - / - animals was shorter compared to the other three genotypes (about 40% of C / C; - / - animals died at 1 year) (Figure 1D).

[0152] Example 2 TRIM72 knockout increases oxidative stress in FUS-R521C KI mice

[0153] To study the mechanism by which loss of function of TRIM72 accelerates disease progression in FUS-R521C KI mice, we cultured cortical neurons from these four genotypes of animals and measured ROS by dihydroethidium (DHE) and MitoSOX staining (both are superoxide indicators). As positive controls, wild-type cultures treated with arsenite (AS, an oxidative stress inducer) and H2O2 showed higher relative mean intensities (RMIs) of DHE and MitoSOX staining (Figures 2A - C). The RMI values were comparable among + / +, - / -, and C / C cortical neurons, but were significantly higher in cortical neurons from C / C; - / - animals (Figures 4G, 4H), indicating that loss of function of TRIM72 actually increases ROS accumulation in FUS-R521C mice.

[0154] Example 3 Overexpression of TRIM72 protects cells from oxidative stress

[0155] The TRIM72 protein contains a RING finger motif, a B box domain, a coiled-coil domain, and a PRYSPRY domain (Figure 3A). To investigate which domain of TRIM72 plays a role in protection against oxidative stress, full-length TRIM72 or domain-disrupted TRIM72 mutants overexpressing the 293FT cell line were constructed by lentiviral infection and puromycin selection (Figure 3B). CCK-8 was used to measure cell viability after arsenite (AS, an oxidative stress inducer) treatment. Indeed, it was observed that the decrease in cell viability due to AS treatment and overexpression of TRIM72 increased cell viability (Figure 3C). TRIM72 mutants disrupted in the coiled-coil domain and PRYSPRY domain abolished the protective effect of TRIM72, but it was found that TRIM72 mutants disrupted in the RING domain or B box domain retained a protective effect equivalent to that of the wild type (Figure 3C).

[0156] Previous studies determined that TRIM72 senses changes in the oxidative environment and forms an oligomeric complex to complete membrane repair. The cysteine residue (C242) plays an important role in TRIM72 oligomer formation. Furthermore, the cysteine residue (C14) is important for TRIM72 E3 ligase activity. Mutation of C424 to alanine (C242A) blocked the protective effect of TRIM72, but its E3 ligase-inactive mutant (C14A) retained a protective effect equivalent to that of the wild type (Figure 3D). Therefore, it can be concluded that TRIM72 protects cells from oxidative stress and mainly depends on its oligomerization rather than its E3 ligase activity.

[0157] To investigate whether TRIM72 protects neurons from oxidative stress, lentivirus was used to restore TRIM72 expression in C / C;- / - cortical neurons. More than 95% of the neurons expressed TRIM72, and a reduction in DHE staining in TRIM72-expressing neurons could be observed (Figures 4A, B).

[0158] Example 4 Recombinant TRIM72 protein protects cortical neurons from oxidative stress.

[0159] Next, to examine whether recombinant TRIM72 protein can induce a survival function in culture, TRIM72 protein purified from Escherichia coli (E. coli) was used. The inventors found that TRIM72 at concentrations above 20 μg / ml could significantly reduce ROS levels and showed a dose-dependent effect in cultured cortical neurons (Figure 5). Furthermore, it was also demonstrated that the C-terminal fragment, rather than the N-terminal of the TRIM protein, mediates the effect of reducing ROS (Figure 5), which means that the PRYSPRY single domain exhibits an endogenous antioxidant capacity, can significantly reduce ROS levels, and increase cell viability. Deletion of the coiled-coil domain from the TRIM72 protein does not abrogate the protective effect, which is different from the gene expression results in Figure 3C.

[0160] Example 5 AAV-TRIM72 packaging and scAAV-TRIM72 protect N2a cells from oxidative stress

[0161] The AAV packaging system is a commonly used triple plasmid system. By simultaneously transfecting three plasmids into mammalian cells (e.g., HEK293), all the components necessary for AAV packaging can be expressed in this cell and assembled into virus particles. Here, the inventors used a modified triple plasmid system from PackGene (Guangzhou PackGene Biotech Co., Ltd). This system consists of three plasmids: pAAV-ITR containing the target gene, the serotype vector pRepCapX, and the helper vector pADHelper. The target vector pAAV-ITR contains a eukaryotic promoter and other components required for high-level gene expression in mammalian cells when cloning foreign sequences into the polylinker site (MCS). The vector also contains the AAV inverted terminal repeat sequence (ITR) that guides virus replication and packaging. The vector pRepCapX contains the AAV rep and CAP genes encoding the replication protein and the virus capsid protein. Stabilization of the rep and CAP gene expression levels is an important step to obtain the desired high-titer virus product. The vector pADHelper contains a collection of adenovirus genes VA, E2A, and E4 that are essential for the cellular production of high-titer virus. Figure 6 shows the construction of the pAAV-ITR vector for scAAV.

[0162] To further investigate which major domains of the TRIM72 protein are necessary to protect neurons, different domain-deleted TRIM72s were constructed and overexpressed in the N2a cell line by scAAV9 infection (Figure 6A). CCK-8 was used to measure cell viability after H2O2 treatment. Indeed, cell viability decreased after H2O2 treatment (Figure 6B). Notably, overexpression of the coiled-coil-PRYSPRY or PRYSPRY single domain was sufficient to protect cells from oxidative stress, while the coiled-coil single domain was not, suggesting that only the coiled-coil-PRYSPRY construct or PRYSPRY can induce a protective effect (Figure 6B).

[0163] Example 6. TRIM72 can be secreted via exosomes

[0164] Exosomes are small extracellular biological vesicles released into the surrounding body fluids by the fusion of multivesicular bodies containing proteins, nucleic acids, lipids and other bioactive substances with the plasma membrane. Exosomes play an important role in cell-to-cell information exchange by releasing bioactive substances that fuse with the recipient cell membrane or bind to cell surface receptors. Full-length TRIM72 or different domain-disrupted TRIM72 mutants were constructed and stably overexpressed in the 293FT cell line by lentiviral infection. The inventors found that TRIM72 was enriched in TSG101-labeled exosomes, which means that TRIM72 can affect the biological processes of surrounding cells via the exosome secretion pathway (Figure 7). Further analysis showed that the coiled-coil domain and the PRYSPRY domain are required for their exosome secretion, and deletion of either domain abrogates the secretion of TRIM72 via exosomes (Figure 6). Comparatively, removal of the RING domain or the B-box domain has only a limited effect on the secretion efficiency of TRIM72 (Figure 8).

[0165] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided within this specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Without departing from the present invention, numerous variations, modifications, and substitutions will occur to those skilled in the art. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein that depend on various conditions and variables. It should be understood that various alternative forms of the embodiments of the present invention described herein may be used in practicing the present invention. Accordingly, the present invention is considered to encompass any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within these claims and their equivalents be covered thereby.

Claims

1. A TRIM72 cleavage protein containing the PRYSPRY domain of the TRIM72 protein or a functional fragment thereof.

2. The TRIM72 cleavage protein according to claim 1, wherein the TRIM72 protein is human TRIM72 protein.

3. The TRIM72 cleavage protein according to claim 1, wherein the PRYSPRY domain comprises the amino acid sequence shown in SEQ ID NO:

6.

4. The TRIM72 cleavage protein according to claim 1, further comprising the coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

5. The TRIM72 cleavage protein according to claim 4, wherein the coiled-coil domain comprises the amino acid sequence shown in Sequence ID No.

5.

6. The TRIM72 cleavage protein according to claim 1, further comprising the B-box domain of the TRIM72 protein or a functional fragment thereof.

7. The TRIM72 cleavage protein according to claim 6, wherein the B-box domain comprises the amino acid sequence shown in SEQ ID NO:

4.

8. The TRIM72 cleavage protein according to claim 1, further comprising the ring finger domain of the TRIM72 protein or a functional fragment thereof.

9. The TRIM72 cleavage protein according to claim 8, wherein the ring finger domain comprises the amino acid sequence shown in SEQ ID NO:

3.

10. A TRIM72 cleavage protein according to claim 1, comprising the amino acid sequence described in any one of SEQ ID NOs: 6, 7, 8, 9, and 11.

11. The TRIM72 cleavage protein according to claim 1, comprising the variant.

12. A TRIM72 cleavage protein according to claim 1, which is secreted via exosomes.

13. A recombinant protein comprising the TRIM72 cleavage type protein described in claim 1.

14. One or more isolated nucleic acid molecules encoding the TRIM72 cleavage protein according to claim 1.

15. A vector comprising the nucleic acid molecule according to claim 14.

16. The vector according to claim 15, comprising an AAV vector.

17. A cell comprising the nucleic acid molecule described in claim 14.

18. A fusion protein comprising the TRIM72 cleavage type protein described in claim 1.

19. A pharmaceutical composition comprising the TRIM72 cleavage protein described in claim 1, and a pharmaceutically acceptable adjuvant.

20. The pharmaceutical composition according to claim 19, for use in a method for preventing and / or treating a neurological disorder, comprising administering an effective amount of the TRIM72 cleaved protein according to claim 1 to a subject in need thereof.

21. The pharmaceutical composition according to claim 20, wherein the neurological disease includes ALS, PD and / or ALS.

Images