Truncated proteins and uses thereof

Abstract

Provided is a TRIM 72 truncated protein comprising a crimped-PRYSPRY domain of the TRIM 72 protein or a PRYSPRY domain of the TRIM 72 protein or a functional fragment of the crimped-PRYSPRY domain of the TRIM 72 protein. Also provided is a use of the TRIM 72 truncated 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 people also use the term "motor neuron disease" to refer to a group of conditions, the most common of which is ALS. ALS is characterized by muscle stiffness, muscle twitching, and increasing weakness due to muscle atrophy. This leads to difficulty speaking, swallowing, and eventually breathing. The cause is unknown in 90% to 95% of cases. About 5-10% of cases are inherited from a parent. About half of these inherited cases are due to four specific genes, SOD1, TDP-43, FUS, and C9orf72. There is currently no cure for ALS.

[0002] TRIM72 is a three-motif (TRIM) family protein, which consists of a ring finger, a B-box motif, a coiled-coil region, and a C-terminal PRYSPRY domain. It is involved in the sarcomere membrane repair process and is related to the insulin signaling pathway. It is also involved in protecting the heart from ischemia / reperfusion (IR) injury. Full-length TRIM72 has been reported 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 restrictions on the size of the delivered gene. Therefore, the role of TRIM72 in protecting neurons needs to be further explored, and different TRIM72 protein products need to be further developed. Summary of the invention

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

[0004] On the one hand, the present application provides a TRIM72 truncated protein, which comprises 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 amino acid positions 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 TRIM72 truncated protein further comprises a coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

[0010] In some embodiments, the TRIM72 truncated 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 amino acid positions 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 TRIM72 truncated protein further comprises the B-box domain of the TRIM72 protein or a functional fragment thereof.

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

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

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

[0017] In some embodiments, the TRIM72 truncated protein further comprises the RING finger domain of the TRIM72 protein or a functional fragment thereof.

[0018] In some embodiments, the TRIM72 truncated protein does not comprise the RING finger domain of the TRIM72 protein or a functional fragment thereof.

[0019] In some embodiments, the RING finger domain comprises amino acid positions 14aa-56aa of the TRIM72 protein.

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

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

[0022] In some embodiments, the TRIM72 truncated protein comprises a variant thereof.

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

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

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

[0026] In some embodiments, TRIM72 truncated proteins are used to protect neurons by reducing oxidative stress.

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

[0028] In some embodiments, TRIM72 truncated proteins are used to prevent or treat neurological diseases.

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

[0030] In some embodiments, TRIM72 truncated proteins are used to prevent or treat ALS, PD and / or stroke.

[0031] On the other hand, the present application provides a recombinant protein comprising a TRIM72 truncated protein.

[0032] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding a TRIM72 truncated 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] On the other hand, the present application provides a fusion protein comprising a TRIM72 truncated protein.

[0039] On the other hand, the present application provides a pharmaceutical composition comprising the TRIM72 truncated protein, recombinant protein, nucleic acid molecule, vector, cell and / or 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] On the other hand, the present application provides a method for protecting neurons in a subject, comprising administering an effective amount of a TRIM72 truncated protein, a recombinant protein, a nucleic acid molecule, a vector, the cell, a fusion protein and / or a pharmaceutical composition to a subject in need thereof.

[0042] On the other hand, the present application provides a method for preventing and / or treating nervous system diseases, which comprises administering an effective amount of TRIM72 truncated protein, recombinant protein, nucleic acid molecule, vector, the cell, fusion protein and / or pharmaceutical composition to a subject in need.

[0043] In some embodiments, the nervous system disease is a neurological damage disease induced by oxidative stress,

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

[0045] In some embodiments, the neurological disease comprises ALS, PD and / or ALS.

[0046] On the other hand, the present application provides the use of TRIM72 truncated protein, recombinant protein, nucleic acid molecule, vector, the cell, fusion protein and / or pharmaceutical composition in the manufacture of drugs for preventing and / or treating nervous system diseases.

[0047] In some embodiments, the nervous system disease is a neurological damage disease induced by oxidative stress,

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

[0049] In some embodiments, the neurological disease comprises ALS, PD, and / or stroke.

[0050] Other aspects and advantages of the present application will be apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present application are shown and described. As will be appreciated, the present application is capable of other and different embodiments, and its several details are capable of modification in various obvious aspects, all without departing from the present application. Therefore, the drawings and description are to be regarded as illustrative in nature, and not restrictive.

[0051] Incorporated by Reference

[0052] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The novel features of the present invention are specifically set forth in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description, which sets forth exemplary embodiments employing the principles of the present invention, and the accompanying drawings (also referred to herein as "figures"), in which:

[0054] Figure 1 It is shown that the loss of function of TRIM72 accelerates the disease progression shown in FUS-R521C knock-in mice. (A) TRIM72 is upregulated in cultured C / C mouse motor neurons (MN, 3DIV), but not in cultured + / + mouse motor neurons. RT-PCR, 3 biological replicates, Gapdh as an internal reference. (B and C) The number of ChAT-positive motor neurons in the ventral horn at 1 week of age (n≥3). ChAT-positive motor neurons in sections from 4-5 spinal cords in the lumbar region. Scale bar in B, 50μm. (D) Survival curves of the indicated genotypes. The loss of function of TRIM72 significantly shortens the lifespan of FUS-R521C knock-in mice. Mice, male, n=25 (+ / +), 19 (C / C), 17 (C / C; - / -) and 18 (- / -).

[0055] Figure 2 It is shown that loss of function of TRIM72 increases oxidative stress. (A) DHE (dihydroethidium) and MitoSOX staining of cortical neurons in the indicated genotypes are presented. DIV12-14. (B, C) Both DHE and MitoSOX staining demonstrated that oxidative stress in C / C;- / - cortical cultures was higher than that in the other three genotypes. Cells treated with AS and H2O2 were used as positive controls for oxidative stress. Values ​​are presented as mean ± SEM (n = 3-4). **p < 0.01 (ANOVA, SPSS). NS, not statistically significant (ANOVA, SPSS).

[0056] Figure 3Figure 1 illustrates that TRIM72 protects cells from oxidative stress. (A) TRIM72 domain annotation and labeling of key residues of previously reported functions of TRIM72. (B) Expression of TRIM72 with a Flag-tagged domain disruption fused to EGFP. GAPDH was used as a protein internal reference. FL, full-length TRIM72. (C) Effect of domain disruption of TRIM72 on cell viability after induced stress. Cell viability / dehydrogenase activity was measured by WST-8 assay within two hours after AS treatment. Expression of TRIM72 protects cells from AS-induced stress and increases cell viability. Disruption of the coiled-coil domain or PRYSPRY domain eliminates the protective effect of TRIM72, but disruption of the ring domain or B-box domain does not eliminate it. (D) Effect of expression of TRIM72 mutants on cell viability after induced stress. The C242A mutation eliminates the anti-stress effect of TRIM72, but the C14A mutation does not. Values ​​are presented as mean ± SEM. In C and D, the values ​​were generated from at least three independent experiments (n≥3). *p<0.05, **p<0.01, ***p<0.001 (t-test or ANOVA, SPSS). NS, not statistically significant.

[0057] Figure 4 It is shown that TRIM72 protects cells from oxidative stress in cultured neurons. (A) DHE staining of cortical neurons after TRIM72 expression by lentiviral infection. Cortical neurons were isolated from C / C;- / - mice (P0, DIV12), scale bar, 20 μm. (B) TRIM72 expression reduced the average intensity of DHE. Images were analyzed by Fiji ImageJ. Average DHE fluorescence intensity was measured, substrate background. Values ​​are presented as mean ± SEM. ***p<0.001 (t test).

[0058] Figure 5 The reduction of ROS levels by treating TRIM72 protein and its fragments is illustrated. (A) Schematic diagram showing TRIM72 protein and its fragments purified from E. coli. (B) DHE staining of cortical neurons after treatment with TRIM72 protein at concentrations of 0, 10, 20 or 40 μg / ml. Values ​​are presented as mean ± SEM. ***p<0.001 (ANOVA, SPSS). NS, not statistically significant.

[0059] Figure 6Cell viability under oxidative stress is illustrated. (A) Schematic diagram showing full-length and differently truncated TRIM72 constructs in pAAV-ITR vector. (B) Cell viability under H2O2 stress with full-length or differently truncated TRIM72 expression by scAAV9 infection. Values ​​for all data points are presented as mean ± SEM, and t-tests were performed to compare data sets to controls. **p<0.01, *p<0.05, NS, not statistically significant.

[0060] Figure 7 The expression pattern of TRIM72 in exosomes was illustrated by Western blot analysis.

[0061] Figure 8 The secretion efficiency of full-length or differently truncated TRIM72 in exosomes is illustrated. The values ​​of all data points are expressed as mean ± SEM. One-way ANOVA was performed to compare the data sets, ***p < 0.001, NS, not statistically significant. DETAILED DESCRIPTION

[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 these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art may conceive of many variations, changes and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

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

[0064] In the present application, the term "truncated protein" generally refers to a protein having one or more amino acid deletions compared to a full-length protein. For example, a truncated protein may contain a major functional fragment of a protein. For example, a truncated protein also includes (but is not limited to) variants, functional fragments, analogs, and homologs thereof.

[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 of the amino acid sequence as shown in SEQ ID NO: 2, wherein 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 of the amino acid sequence as shown in SEQ ID NO: 2.

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

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

[0068] For example, in the present application, substitutions between the following amino acids in each group can be considered conservative substitutions:

[0069] The group of amino acids with nonpolar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0070] The group of uncharged amino acids with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0071] A group of negatively charged amino acids with polar side chains: aspartic acid and glutamic acid.

[0072] Positively charged basic amino acid group: lysine, arginine, and histidine.

[0073] Group of amino acids with a phenyl group: phenylalanine, tryptophan, and tyrosine.

[0074] In the present application, the term "fusion protein" generally refers to a composite polypeptide, that is, a single continuous amino acid sequence composed of two (or more) polypeptides. Fusion proteins can generally be artificially prepared by recombinant nucleic acid or chemical synthesis.

[0075] In this application, the term "neurodegenerative disease" generally refers to various neurological disorders characterized by a gradual, progressive loss of neural tissue and / or neural tissue function. Neurodegenerative diseases are a class of neurological disorders or diseases, and wherein the neurological disease is characterized by a gradual, progressive loss of neural tissue, and / or changes in neural function, which are generally reduced due to the gradual, progressive loss of neural tissue. For example, neurodegenerative diseases can be caused by oxidative stress.

[0076] In this application, the term "Parkinson's disease" generally refers to a neurodegenerative disorder. Parkinson's disease is a chronic progressive neurological disease characterized by neurodegeneration, especially degeneration of dopaminergic neurons. Symptoms include stooped posture, resting tremor, resting muscle weakness, unsteady gait, speech disorders, movement difficulties and eventually slowing of mental processes and / or dementia.

[0077] In the present application, the term "stroke" generally refers to a condition caused by obstruction or bleeding of one or more blood vessels supplying the brain, which may lead to cell death. The term may include ischemic stroke and hemorrhagic stroke. As used herein, "ischemic stroke" generally refers to a stroke caused by obstruction of one or more blood vessels supplying the brain. The types of ischemic stroke may include, but are not limited to, for example, embolic stroke, cardiogenic stroke, thrombotic stroke, large vessel thrombosis, lacunar infarction, arterial-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. The types of hemorrhagic stroke include, for example, subdural stroke, intraparenchymal stroke, epidural stroke, and subarachnoid stroke.

[0078] In the present application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide or ribonucleotide in an isolated form, or an analog thereof of any length isolated from its natural environment or artificially synthesized. The nucleic acid molecule of the present application can be isolated. For example, it can be produced or synthesized by: (i) in vitro amplification, such as polymerase chain reaction (PCR) amplification, (ii) cloning recombination, (iii) purification, such as by restriction enzyme digestion and gel electrophoresis fractionation, 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 its functional fragment can be prepared by a variety of methods known in the art. These methods include (but are not limited to) overlap extension PCR using restriction fragment manipulation or synthetic oligonucleotides. Specific operations 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.

[0079] In this application, the term "vector" generally refers to a vector containing a recombinant polynucleotide, wherein the recombinant polynucleotide includes an expression control sequence operably linked to the nucleotide sequence to be expressed. The vector includes cis-acting elements sufficient for expression; other elements for expression can be provided by the host cell or can be provided in an in vitro expression system. The vector can include all expression vectors known in the art that can be incorporated into a recombinant polynucleotide, including cosmids, plasmids (e.g., naked plasmids or plasmids encapsulated in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0080] In the present application, the term "coding" generally refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) as a template for synthesizing other polymers and macromolecules in bioengineering, and the polymers and macromolecules have a specified nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a specified amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes the protein. The coding strands that the nucleotide sequence is identical to the mRNA sequence and that are usually provided in the sequence table and the non-coding strands that are used as gene or cDNA transcription templates can all be referred to as proteins or other products of coding genes or cDNAs. In the present application, the term "coding element" generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein.

[0081] In the present application, the terms "host cell", "cell" and "host" are used interchangeably, and generally refer to a plasmid or vector that may include or have included a nucleic acid molecule of the present application, or a single cell, cell line or cell culture that may express a protein, a fragment thereof or a variant thereof of the present application. The host cell may include progeny of a single host cell. Due to natural, unexpected or deliberate mutations, progeny cells and original parent cells do not have to be identical in morphology or genome, as long as they can express a protein or a fragment thereof of the present application. The host cell may be obtained by transfecting cells in vitro with a vector of the present application. The host cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell). In the present application, the host cell may be a CHO cell.

[0082] As used herein, the term "treating" generally refers to slowing or ameliorating the progression, severity and / or duration of a proliferative condition, or ameliorating one or more symptoms (eg, one or more identifiable symptoms) of a proliferative condition by administering one or more therapies.

[0083] In this application, the term "subject" generally refers to any human or non-human animal. The term "non-human animal" can include all vertebrates, such as mammals and non-mammals, for example non-human primates, goats, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0084] In this application, the terms "peptide", "polypeptide" and "protein" are used interchangeably and generally refer to compounds consisting 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 on the maximum number of amino acids that can be included in a protein or peptide sequence. A polypeptide may 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, also commonly referred to as peptides, oligopeptides and oligomers in the art, such as long chains, which are commonly referred to as proteins in the art, and there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0085] In addition to the specific proteins and nucleotides mentioned herein, the present application may also include functional variants, derivatives, analogs, homologues and fragments thereof.

[0086] The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a naturally occurring sequence or being encoded by a nucleotide sequence substantially identical to a naturally occurring sequence and being capable of having one or more activities of a naturally occurring sequence. In the context of the present application, a variant of any given sequence refers to a sequence in which a specific residue (amino acid or nucleotide residue) sequence has been modified so that the polypeptide or polynucleotide substantially maintains a sequence of at least one endogenous function. The variant sequence can be obtained by adding, deleting, replacing, modifying, displacing and / or changing 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 the present application, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application comprising any substitution, change, modification, displacement, deletion and / or addition of one (or more) amino acid residues of a sequence, provided that the resulting polypeptide or polynucleotide substantially maintains at least one function of its endogenous function.

[0087] In the present application, the term "analog" about a polypeptide or polynucleotide generally includes any mimetics of a polypeptide or polynucleotide, i.e., a compound having at least one endogenous function of a polypeptide or polynucleotide simulated by a mimetics. Generally speaking, an amino acid can be substituted, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 or more) amino acid can be substituted, provided that the modified sequence substantially maintains the desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs. The protein or polypeptide used in the present application can also have a deletion, insertion or substitution of an amino acid residue, wherein the amino acid residue undergoes silent changes and produces 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 residue, 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; and amino acids containing uncharged polar head groups and having similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0088] In the present application, the term "homologue" generally refers to an amino acid sequence or nucleotide sequence with a specific homology to a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be equivalent to the "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. Typically, a homologue contains an active site identical to the target amino acid sequence, etc. Homology may be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions), or homology may be represented by sequence identity. In the present application, a sequence having a certain identity percentage with any SEQ ID NO of the amino acid sequence or nucleotide sequence mentioned refers to a sequence having the identity percentage over the entire length of the SEQ ID NO mentioned. To determine sequence identity, sequence alignment can be performed by a variety of 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 for alignment, including any algorithms required to achieve optimal alignment over the full length of the sequences being compared.

[0089] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, a variation within a 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 a specified value.

[0090] In this application, the term "comprising" generally means including, containing, having or covering. In some cases, it also means "being" or "consisting of...".

[0091] In this application, the term "does not include" generally refers to excluding the possibility of a certain behavior, structure or structure. For example, "A does not include B" generally means excluding the possibility of B appearing in A.

[0092] TRIM72 truncated proteins

[0093] In one aspect, the present application provides a TRIM72 truncated protein or a functional fragment thereof comprising a PRYSPRY domain. For example, the TRIM72 truncated protein may comprise the amino acid sites 278aa-470aa of the TRIM72 protein. For example, the TRIM72 truncated protein may comprise the amino acid sequence shown in SEQ ID NO: 2.

[0094] In the present application, the TRIM72 truncated protein may also contain other domains of the TRIM72 protein.

[0095] For example, the TRIM72 truncated protein may include a PRYSPRY domain and a coiled-coil domain. For example, the TRIM72 protein may include an amino acid sequence as shown in SEQ ID NO: 11. For example, the TRIM 72 truncated protein may include a deletion of the B-box domain and the RING finger domain of the TRIM72 protein. For example, the TRIM72 protein may include an amino acid sequence as shown in SEQ ID NO: 11.

[0096] Exosomes are small extracellular biological vesicles that are released into the surrounding body fluids via fusion of multivesicular bodies and plasma membranes, containing proteins, nucleic acids, lipids, and other biologically active substances. For example, TRIM72 truncated proteins can be secreted via exosomes. For example, TRIM72 truncated proteins can be packaged in exosomes. For example, exosomes can contain nucleic acids encoding TRIM72 truncated proteins. For example, exosomes can be labeled with TSG101. When exosomes are used to secrete TRIM72 truncated proteins, the coiled-coil domain and the PRYSPRY domain are required.

[0097] For example, the TRIM72 truncated protein may comprise a PRYSPRY domain and a B-box domain. For example, the TRIM72 truncated protein may comprise a deletion of the coiled-coil domain and the RING finger domain of the TRIM72 protein.

[0098] For example, the TRIM72 truncated protein may comprise a PRYSPRY domain and a RING finger domain. For example, the TRIM72 truncated protein may comprise a deletion of the B-box domain and the coiled-coil domain of the TRIM72 protein.

[0099] For example, the TRIM72 truncated protein may comprise a PRYSPRY domain, a coiled-coil domain, and a RING finger domain. For example, the TRIM 72 truncated protein may comprise a deletion of the B-box domain of the TRIM72 protein.

[0100] For example, the TRIM72 truncated protein may comprise a PRYSPRY domain, a coiled-coil domain, and a B-box domain. For example, the TRIM72 truncated protein may comprise a deletion of the RING finger domain of the TRIM72 protein.

[0101] For example, the TRIM72 truncated protein may comprise a PRYSPRY domain, a RING finger domain, and a B-box domain. For example, the TRIM72 truncated protein may comprise a deletion of the coiled-coil domain of the TRIM72 protein.

[0102] In the present application, the TRIM72 truncated protein may include a variant thereof. For example, compared to the corresponding wild-type sequence, the TRIM72 truncated protein may include one or more amino acid mutations. For example, the TRIM72 truncated 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% identical to the corresponding wild-type sequence.

[0103] For example, the TRIM72 truncated protein may comprise an amino acid sequence as 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.

[0104] In the present application, the TRIM72 truncated protein may include an amino acid mutation at position C14. For example, the amino acid mutation may be C14A.

[0105] According to the applicant's research, the amino acid cysteine ​​at position 242 is important for the oligomer formation of TRIM72 protein. The substitution of amino acid C242 will block the protective function of neurons. Therefore, the amino acid substitution at position C242 (e.g., C242A) may not be included in the TRIM72 truncated protein of the present application.

[0106] In the present application, the truncated TRIM72 protein can be used to protect neurons by reducing oxidative stress. In the present application, the truncated TRIM72 protein can be used to prevent or treat nervous system diseases associated with oxidative stress, such as nerve damage diseases or neurodegenerative diseases.

[0107] In the present application, the truncated TRIM72 protein can be used to prevent or treat nervous system diseases. For example, the present application can be used to prevent and / or treat ALS, PD and / or stroke.

[0108] Recombinant protein, nucleic acid, vector, cell, fusion protein, pharmaceutical composition

[0109] On the other hand, the present application provides a recombinant protein comprising a TRIM72 truncated protein.

[0110] On the other hand, the present application provides one or more nucleic acid molecules that can encode the TRIM72 truncated protein of the present application.

[0111] In some embodiments, the nucleic acid molecule may completely encode the TRIM72 truncated protein or fusion protein of the present 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 may encode a portion of the TRIM72 truncated protein or fusion protein of the present application. For example, the fusion protein can be obtained by using more than two types of different nucleic acid molecules.

[0112] On the other hand, the present application provides one or more vectors, which may contain one or more nucleic acid molecules of the present application. On the other hand, the present application provides a cell (eg, a host cell), which may contain a nucleic acid molecule of the present application or a vector of the present application.

[0113] In the present application, the vector can be a polynucleotide, which can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector. In the present application, the vector can include one or more of the nucleic acid molecules. For example, the vector may include all nucleic acid molecules required to encode the TRIM72 truncated protein.

[0114] In addition, the vector may also include other genes, such as marker genes that allow the vector to be selected in a suitable host cell and under suitable conditions. In addition, the vector may also include expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art. For example, it may 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 may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, a 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control of a functionally connected nucleic acid.

[0115] On the other hand, the present application provides a pharmaceutical composition comprising the TRIM72 truncated protein, recombinant protein, nucleic acid molecule, vector, cell and / or fusion protein, and a pharmaceutically acceptable adjuvant.

[0116] In some embodiments, pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or nonionic surfactants, and the like.

[0117] In some embodiments, the pharmaceutically acceptable adjuvant may 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.

[0118] 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 techniques in the art, such as following the procedures in Remington: The Science and Practice of Pharmacy, 19th edition, Gennaro, ed., Mack Publishing Co., Easton, PA, 1995.

[0119] In the present application, the composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration of the drug via a subcutaneous reservoir.

[0120] 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 development or progression of a neurological disease (such as ALS, PD or stroke), and / or can alleviate and / or stabilize the disease state.

[0121] The pharmaceutical composition of the present application may include a therapeutically effective amount of the TRIM72 truncated protein. The therapeutically effective amount is a dose required to prevent and / or treat (at least partially treat) a disease and / or any complications thereof in a subject suffering from a disease (e.g., ALS, PD, or stroke) or at risk of suffering from the disease.

[0122] Preparation, method and use

[0123] On the other hand, the present application provides a method for protecting neurons in a subject, comprising administering an effective amount of a TRIM72 truncated protein, a recombinant protein, a nucleic acid molecule, a vector, a cell, a fusion protein and / or a pharmaceutical composition to a subject in need thereof.

[0124] On the other hand, the present application provides a method for preventing and / or treating nervous system diseases, comprising administering an effective amount of TRIM72 truncated protein, recombinant protein, nucleic acid molecule, vector, cell, fusion protein and / or pharmaceutical composition to a subject in need thereof.

[0125] On the other hand, the present application provides the use of TRIM72 truncated proteins, recombinant proteins, nucleic acid molecules, vectors, cells, fusion proteins and / or pharmaceutical compositions in the manufacture of drugs for preventing and / or treating nervous system diseases.

[0126] On the other hand, the present application provides TRIM72 truncated proteins, recombinant proteins, nucleic acid molecules, vectors, cells, fusion proteins and / or pharmaceutical compositions for preventing and / or treating nervous system diseases.

[0127] In the present application, nervous system diseases include ALS.

[0128] Example

[0129] The following examples are set forth in order to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; im, intramuscular (ly); ip, intraperitoneal; sc, subcutaneous; rpm, revolutions per minute; and the like.

[0130] method

[0131] Production of recombinant TRIM72 protein in E. coli

[0132] Codon optimization and gene synthesis were performed based on the amino acid sequence of mouse TRIM72 (SEQ ID NO: 1). Full-length and truncated TRIM72 were constructed in the pET21b vector with a 6×His tag at the N-terminus. These proteins were expressed and purified from Escherichia coli BL21 (DE3) cells (Millipore), and purified under native conditions unless otherwise stated. Escherichia coli was grown to an OD600 of 0.8 and induced overnight with 0.6mM IPTG at 16°C. The cell pellet was resuspended in lysis buffer (250mM NaCl, 50mM HEPES 7.5, 1mM DTT, protease inhibitors). After ultrasonic treatment, the lysate was precipitated at 30,000×g at 4°C for 30 minutes. The supernatant was applied to a Ni column (GE) with 10mL beads at room temperature, and the beads were pre-washed with lysis buffer. The protein was eluted with elution buffer (25mmol / L Tris pH 8.0, 300mmol / L NaCl, 200mmol / L imidazole). The protein was further treated with 0.1mg / ml ribonuclease A (Thermo Fisher) to remove RNA and then purified by a Superdex 200 16 / 200 column (GE) balanced in SEC buffer (400mM NaCl, 50mM HEPES 7.5, 1mM DTT). The eluted fractions were analyzed by SDS-PAGE, pooled, concentrated, filtered, flash frozen in liquid nitrogen, and stored at -80°C.

[0133] Preparation of recombinant TRIM72 protein and its truncated fragments

[0134] Based on the amino acid sequence of human TRIM72 (SEQ ID NO: 2), codon optimization and gene synthesis were performed, and a 6×His tag was added to the amino terminus. The synthesized TRIM72 gene was used as a template to construct pcDNA3.1-6His-TRIM72, pcDNA3.1-6His-coiled-coil, pcDNA3.1-6His-PRYSPRY and pcDNA3.1-6His-coiled-PRYSPRY plasmids by gene amplification. Figure 1 ). High-quality endotoxin-free plasmids were obtained by plasmid amplification using E. coli. HEK293F cells were transfected, harvested and lysed, and protein lysates were collected. After the lysates were purified by Ni-affinity columns, TRIM72 (full-length) protein and its truncated fragments (coiled-coil (SEQ ID NO: 5), PRYSPRY (SEQ ID NO: 6), and coiled--PRYSPRY (SEQ ID NO: 11)) were prepared.

[0135] Plasmids and lentiviral vectors

[0136] A DNA fragment corresponding to the full length of TRIM72 was amplified from a mouse cDNA library by PCR and inserted between the SalI and XhoI sites of the pCMV-N-3×Flag expression vector using a seamless cloning kit (Beyotime) to generate Flag-tagged TRIM72. A series of Flag-tagged TRIM72 mutants were generated from the wild-type TRIM72 construct by point mutation: C14A (cysteine ​​at position 14 was replaced by alanine); C242A (cysteine ​​at position 242 was replaced by alanine). A series of Flag-tagged TRIM72 domain deletions were generated from the full-length TRIM72 expression vector: ΔRING domain (domain 14-69 deleted, SEQ ID NO: 7); ΔB-box domain (domain 81-122 deleted, SEQ ID NO: 8); ΔCoiled-coil domain (domain 135-232 deleted, SEQ ID NO: 9); ΔPRYSPRY domain (domain 278-470 deleted, SEQ ID NO: 10) constructs. Flag-tagged TRIM72 coiled-coil domain (135-232 aa) or PRYSPRY domain (278-470 aa) or coiled-coil domain plus PRYSPRY domain (135-470 aa) constructs were generated from the full-length TRIM72 expression vector.

[0137] To construct a TRIM72 stable expression cell line, the DNA fragment of the Flag-tagged TRIM72 mutant or domain-deleted or single-domain series was amplified from the corresponding TRIM72 expression vector by PCR and inserted between the BsrGI and EcoRI sites of the pLJM1-EGFP lentiviral vector to generate a fusion of the Flag-tagged TRIM72 mutant or domain-deleted or single-domain series with EGFP in the lentiviral vector.

[0138] Lentivirus expressing Flag-tagged TRIM72 constructs was generated from the corresponding EGFP fused to the Flag-tagged TRIM72 vector.

[0139] Cell culture, lentiviral packaging, and lentiviral infection

[0140] HEK293FT cells were cultured in DMEM (Invitrogen) with 10% fetal bovine serum (Gemini) in a cell culture incubator (37°C, 5% CO2). For lentiviral packaging, HEK293FT cells were seeded in growth medium in three 10-cm culture dishes. When the density reached about 90%, the cells were co-transfected with VSVG (10 μg), pxPAX2 (15 μg) and pLJM1-EGFP lentiviral vectors or pLentiCRISPRv2 (Addgene) or pLenticas9-Blast (Addgene) (20 μg) using PEI (Sigma) according to the manufacturer's instructions, and fresh growth medium was replaced 5-6 h after transfection. The culture medium was harvested 72 hours after transfection and centrifuged at 20,000 rpm, 4°C for 2 hours. After centrifugation, the lentivirus was enriched in the precipitate. The lentivirus was resuspended in 100 μl DPBS and stored at -80°C.

[0141] HEK293FT cells or Hela cells were infected with the indicated lentiviruses. Three days after infection, infected cells were selected with 2 μg / ml puromycin or 10 μg / ml blasticidin, depending on plasmid resistance, for at least one week. Puromycin- or blasticidin-selected cells were used for further analysis.

[0142] Cell viability assay

[0143] Cell viability was assessed using CCK-8. 3 Cells were seeded into 96-well plates at a density of 7 × 10 cells / well for arsenite treatment or 7 × 10 3Cells were inoculated at a density of 10 cells / well for H2O2 treatment. Arsenite (Sigma) was added to each well at a concentration of 0.125 mM, 0.25 mM, or 0.5 mM, and the cells were washed after incubation at 37°C for 2 hours. For H2O2 treatment experiments, scAAV9 infection (estimated multiplicity of infection (MOI): 10,000 vg / cell) was performed 17 hours after cell inoculation for 24 hours. Then, H2O2 was added to each well at a concentration of 300 μM, and the cells were washed after incubation at 37°C for 1 hour. After treating the cells with arsenite or H2O2, a total of 10 μL of CCK-8 solution (Yeasen) was added to each well. After incubation for another 2 hours at 37°C, 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 three times to obtain an average value.

[0144] Exosome purification

[0145] The protocol for purifying exosomes from 100 ml supernatant of 293FT cells includes two steps: ultrafiltration and polyethylene glycol (PEG) precipitation. First, pour the collected supernatant into a centrifuge tube and centrifuge at 3000 × g for 20 minutes to remove cell debris. Then, filter the supernatant once with a 0.45 μm filter. After that, take a new Amicon ULTRA-15 ultrafiltration tube and wash it with PBS or autoclaved water. Then immediately add the supernatant to the ultrafiltration tube and centrifuge at 3000 × g for 5-10 minutes. Discard the filtrate, continue to add 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 separation reagent to the concentrate. Mix the sample thoroughly by vortexing or aspirating. Finally, place the sample at 2-8 ° C overnight. The next day, centrifuge the sample at 10,000 × g for 1 hour at 4 ° C. The supernatant was discarded and the exosomes were in the pellet.

[0146] Western blot analysis

[0147] The total protein content in tissues and cells was extracted using RIPA lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM EDTA, 0.1% SDS, 1% TritonX-100, 0.5% sodium deoxycholate) added with phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (Bimake), and the lysate was incubated on ice for 30 minutes. After centrifugation at 12000rpm for 10 minutes, the supernatant was extracted and incubated at 95°C for 10 minutes after mixing with SDS loading buffer. Then, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Afterwards, the membrane was blocked for 1 hour at room temperature using 5% skim milk. Then incubate with diluted primary antibodies overnight at 4°C, including GAPDH (Ameribio), tubulin (Ameribio), TRIM72 antibody (kind gift from Dr. Jianjie Ma), CCDC22 (Sigma), GFP (Abcam), Myc (Abmart), TSG101 (Abcam), ITGAV (Abcam), H3 (Abcam), and Flag (Abmart). The membrane was then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Images were analyzed using Fiji ImageJ to obtain integrated intensity.

[0148] Arsenite intragastric administration

[0149] Arsenite was prepared at 1 mg / ml in ddH2O and then injected intragastrically into mice at 6 mg / kg body weight as described in the experiment (Zhang et al., 2020).

[0150] ROS measurement

[0151] ROS levels were measured using dihydroethidium (DHE, Invitrogen) and MitoSOX (Invitrogen). Cortical neurons were plated on 24-well chamber slides and cultured to DIV12-14. Neurons were then incubated with DHE (20 μM in culture medium) or MitoSOX (5 μM in culture medium) at 37 ° C for 20 minutes. After incubation, neurons were fixed with 4% PFA for 10 minutes at room temperature. Images were captured by a Nikon A1 confocal microscope. DHE fluorescence was excited at 535nm and emission was collected at 610nm. MitoSOX fluorescence was excited at 510nm and emission spectra were collected at 580nm. The average intensity of DHE or MitoSOX fluorescence was assessed by FiJi ImageJ software. And the relative average intensity of DHE or MitoSOX fluorescence in different groups was used to express ROS levels. RMI = the average intensity of DHE or MitoSOX fluorescence divided by the background average intensity.

[0152] For lentiviral infection, cortical neurons were plated on 24-well chamber slides and cultured until DIV3-5, and 10 μl of purified lentivirus was added to each well for further culture. When cultured to DIV12-14, neurons were stained with DHE.

[0153] Cortical neuron culture

[0154] Cortices were dissected from postnatal day 0 mice in HBSS (Invitrogen) and incubated with trypsin in Neurobasal™ (Invitrogen) for 15 min at 37°C. During digestion, DNase I (Sigma, 10 μg / ml) was added in the last 10 min. The trypsinization process was stopped by adding Neurobasal medium with 10% FBS. After digestion, the tissue was dissociated by slow titration to obtain a single cell suspension. The suspension was passed through a 70 μm filter. After centrifugation at 800 g for 10 min, the cells were resuspended in 10% FBS, GlutaMAX TM Neurobal (Invitrogen) TM In 2×10 5 The cells were plated at a density of 10 cells / ml in a 24-well plate, with 500 ml per well. After incubation for 5-6 hours (5% CO2, 37°C), the cells were plated with B27 (Invitrogen), GlutaMAX TM Neurobal (Invitrogen) TM The culture medium was replaced. Cultured neurons (12-14 days in vitro) were stained with DHE or MitoSOX.

[0155] Example 1 TRIM72 knockout accelerates disease progression shown in FUS-R521C KI mice

[0156] TRIM72, a protein with undetectable levels in the central nervous system, was significantly upregulated in FUS-R521C KI mice ( Figure 1 A). 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 knockout of TRIM72 (C / C;- / -), and TRIM72 knockout alone (- / -) animals. At one year of age, significantly greater loss of motor neurons in the spinal cord was observed in C / C;- / - animals compared to C / C mutants ( Figure 1 However, TRIM72 knockout mice showed no motor neuron loss compared to + / + controls ( Figure 1 C). The survival curves of the four genotypes showed that C / C;- / - animals had a shorter lifespan (about 40% of C / C;- / - animals died at one year of age) compared with the other three genotypes ( Figure 1 D).

[0157] Example 2 TRIM72 knockout increases oxidative stress in FUS-R522C KI mice

[0158] To investigate the mechanism by which loss of TRIM72 function accelerates disease progression in FUS-R521C KI mice, we cultured cortical neurons from animals of the four genotypes and measured ROS by dihydroethidium (DHE) and MitoSOX staining, both of which are superoxide indicators. As a positive control, wild-type cultures treated with arsenite (AS, an oxidative stress inducer) and H2O2 showed higher relative mean intensity (RMI) of DHE and MitoSOX staining ( Figure 2 A-2C). Although RMI values ​​were similar between + / +, - / -, and C / C cortical neurons, values ​​were significantly higher in cortical neurons from C / C;- / - animals ( Figure 4 G, 4H), indicating that loss of function of TRIM72 actually increases ROS accumulation in FUS-R521C mice.

[0159] Example 3 Overexpression of TRIM72 protects cells from oxidative stress

[0160] 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 protecting against oxidative stress, 293FT cell lines overexpressing full-length TRIM72 or domain-disrupted TRIM72 mutants were constructed by lentiviral infection and puromycin selection ( Figure 3 B). Cell viability after arsenite (AS, an oxidative stress inducer) treatment was measured using CCK-8. Indeed, it was observed that cell viability decreased under AS treatment and overexpression of TRIM72 increased cell viability ( Figure 3 C). It was found that TRIM72 mutants with disrupted coiled-coil domain and PRYSPRY domain abolished the protective effect of TRIM72, while TRIM72 mutants with disrupted RING domain or B-box domain retained the same protective effect as the wild type ( Figure 3 C).

[0161] Previous studies have determined that TRIM72 can sense changes in the oxidative environment and form oligomeric complexes to complete membrane repair. Cytidine residues (C242) play a key role in the formation of TRIM72 oligomers. In addition, cytidine residues (C14) are key to the E3 ligase activity of TRIM72. Mutation of C424 to alanine (C242A) blocks the protective effect of TRIM72, while its E3 ligase-inactive mutant (C14A) retains the same protective effect as the wild type ( Figure 3 D). Therefore, it is inferred that TRIM72 protects cells from oxidative stress and mainly depends on its oligomerization rather than E3 ligase activity.

[0162] 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 neurons expressed TRIM72, and reduced DHE staining could be observed in neurons expressing TRIM72 ( Figure 4 A, 4B).

[0163] Example 4 Recombinant TRIM72 protein protects cortical neurons from oxidative stress

[0164] Next, to investigate whether recombinant TRIM72 protein could induce survival function in culture, TRIM72 protein purified from Escherichia coli was used. It was found that TRIM72 at concentrations exceeding 20 μg / ml significantly reduced ROS levels in cultured cortical neurons and exhibited a dose-dependent effect ( Figure 5 ). In addition, it was shown that the C-terminal, but not the N-terminal, fragment of TRIM protein mediates the ROS-reducing effect ( Figure 5), which means that the PRYSPRY single domain exhibits endogenous antioxidant capacity, significantly reducing ROS levels and increasing cell viability. The loss of the coiled-coil domain in the TRIM72 protein did not abolish the protective effect, a result different from Figure 3 Gene expression results in C.

[0165] Example 5 AAV-TRIM72 packaging and scAAV-TRIM72 protect N2a cells from oxidative stress

[0166] The AAV packaging system is a commonly used three-plasmid system. By transfecting these three plasmids into mammalian cells (e.g., HEK293) at the same time, all components required for AAV packaging can be expressed in this cell and assembled into viral particles. In this article, we use a modified three-plasmid system from PackGene (Guangzhou PackGene Biotech Co., Ltd). This system consists of three plasmids: pAAV-ITR containing target genes, serotype vector pRepCapX and auxiliary vector pADHelper. The target vector pAAV-ITR contains eukaryotic promoters and other components required for high-level gene expression in mammalian cells when foreign sequences are cloned into the multiple cloning site (MCS). The vector also contains AAV reverse terminal repeats (ITR) that guide viral replication and packaging. The vector pRepCapX contains AAV rep and CAP genes encoding replication proteins and viral capsid proteins. The stability of rep and CAP gene expression levels is a key step in obtaining the desired high-titer viral product. The vector pADHelper contains a set of adenoviral genes VA, E2A and E4, which are essential for cells to produce high titers of virus. Figure 6 The construction of the pAAV-ITR vector of scAAV is shown.

[0167] To further investigate which key domains of TRIM72 protein are required for protecting neurons, TRIM72 lacking different domains was constructed and overexpressed in N2a cell line by scAAV9 infection ( Figure 6 A). CCK-8 was used to measure cell viability after H2O2 treatment. In fact, cell viability decreased after H2O2 treatment ( Figure 6 B). Notably, overexpression of either the coiled-PRYSPRY or PRYSPRY single domains was sufficient to protect cells from oxidative stress, but overexpression of the coiled-coil single domain was not, indicating that the coiled-PRYSPRY construct or PRYSPRY alone was able to elicit protection ( Figure 6 B).

[0168] Example 6. TRIM72 can be secreted via exosomes

[0169] Exosomes are small extracellular biological vesicles that are released into the surrounding body fluids through the fusion of multivesicular bodies and plasma membranes, and contain proteins, nucleic acids, lipids and other bioactive substances. Exosomes play an important role in information exchange between cells by releasing bioactive substances that fuse with receptor cell membranes or bind to cell surface receptors. Full-length TRIM72 or TRIM72 mutants with different domains destroyed were constructed and stably overexpressed in the 293FT cell line by lentiviral infection. We found that TRIM72 was enriched in TSG101-labeled exosomes, which means that TRIM72 can affect the biological processes of surrounding cells through the exosome secretion pathway ( Figure 7 ). Further analysis revealed that both the coiled-coil domain and the PRYSPRY domain are essential for its exosome secretion, and the absence of either domain prevents TRIM72 from being secreted via exosomes ( Figure 6 In contrast, removal of the RING domain or the B-box domain had only a limited effect on the secretion efficiency of TRIM72 ( Figure 8 ).

[0170] Although preferred embodiments of the present invention have been shown and described herein, it is apparent to those skilled in the art that these embodiments are provided by way of example only. This does not mean that the present invention is limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not meant to be interpreted in a limiting sense. Without departing from the present invention, those skilled in the art will now expect many variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted to practice the present invention. Therefore, it is expected that the present invention will also cover any such substitutions, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A TRIM72 truncated protein, comprising the PRYSPRY domain of the TRIM72 protein or a functional fragment thereof. 2 . The TRIM72 truncated protein according to claim 1 , wherein the TRIM72 protein is a human TRIM72 protein. 3 . The TRIM72 truncated protein according to any one of claims 1 to 2 , wherein the TRIM72 protein comprises the amino acid sequence of SEQ ID NO:

2. 4 . 4 . The TRIM72 truncated protein according to claim 1 , wherein the PRYSPRY domain comprises amino acid sites 278aa-470aa of the TRIM72 protein. 5 . The TRIM72 truncated protein according to any one of claims 1 to 4 , wherein the PRYSPRY domain comprises the amino acid sequence shown in SEQ ID NO:

6. 6 . The TRIM72 truncated protein according to any one of claims 1 to 5 , further comprising a coiled-coil domain of the TRIM72 protein or a functional fragment thereof. 7 . The TRIM72 truncated protein according to any one of claims 1 to 5 , which does not comprise the coiled-coil domain of the TRIM72 protein or a functional fragment thereof. 8 . The TRIM72 truncated protein according to claim 6 , wherein the coiled-coil domain comprises the amino acid positions 135aa-232aa of the TRIM72 protein. 9 . The TRIM72 truncated protein according to any one of claims 6 to 8 , wherein the coiled-coil domain comprises the amino acid sequence shown in SEQ ID NO:

5. 10 . The TRIM72 truncated protein according to any one of claims 1 to 9 , further comprising a B-box domain of the TRIM72 protein or a functional fragment thereof. The TRIM72 truncated protein according to any one of claims 1 to 9, which does not comprise the B-box domain of the TRIM72 protein or a functional fragment thereof. 12 . The TRIM72 truncated protein according to claim 10 , wherein the B-box domain comprises amino acid positions 86aa-117aa of the TRIM72 protein. 13 . The TRIM72 truncated protein according to any one of claims 10 to 12, wherein the B-box domain comprises the amino acid sequence shown in SEQ ID NO:

4. 14 . The TRIM72 truncated protein according to any one of claims 1 to 13 , further comprising a RING finger domain of the TRIM72 protein or a functional fragment thereof. 15 . The TRIM72 truncated protein according to any one of claims 1 to 13 , which does not comprise the RING finger domain of the TRIM72 protein or a functional fragment thereof. 16 . The TRIM72 truncated protein according to claim 14 , wherein the RING finger domain comprises amino acid positions 14aa-56aa of the TRIM72 protein. 17 . The TRIM72 truncated protein according to any one of claims 1 to 16 , wherein the RING finger domain comprises the amino acid sequence shown in SEQ ID NO:

3. 18 . The TRIM72 truncated protein according to any one of claims 1 to 17 , comprising an amino acid sequence as shown in any one of SEQ ID NOs: 6, 7, 8, 9 and 11. 19 . The TRIM72 truncated protein according to any one of claims 1 to 18 , comprising a variant thereof. 20 . The TRIM72 truncated protein according to any one of claims 1 to 19 , which does not comprise an amino acid mutation at position C242. 21 . The TRIM72 truncated protein according to any one of claims 1 to 20, further comprising an amino acid mutation at position C14. 22 . The TRIM72 truncated protein according to any one of claims 1 to 21 , further comprising an amino acid mutation C14A.

23. A TRIM72 truncated protein according to any one of claims 1 to 22 for use in protecting neurons by reducing oxidative stress.

24. The TRIM72 truncated protein according to any one of claims 1 to 23, which is secreted via exosomes.

25. The TRIM72 truncated protein according to any one of claims 1 to 24, for use in preventing or treating a nervous system disease. The TRIM72 truncated protein according to claim 25 , wherein the nervous system disease is a nerve damage disease induced by oxidative stress.

27. The TRIM72 truncated protein according to any one of claims 1 to 26, for use in preventing and / or treating ALS (amyotrophic lateral sclerosis), PD (Parkinson's disease) and / or stroke.

28. A recombinant protein comprising the TRIM72 truncated protein according to any one of claims 1 to 27.

29. One or more isolated nucleic acid molecules encoding a TRIM72 truncated protein according to any one of claims 1 to 27.

30. A vector comprising the nucleic acid molecule according to claim 29.

31. The vector of claim 30, comprising a viral vector.

32. The vector of any one of claims 30 to 31, comprising an AAV vector.

33. The vector of any one of claims 31 to 32, wherein the viral vector is an AAV9 vector.

34. A cell comprising the nucleic acid molecule of claim 29 or the vector of any one of claims 30 to 33.

35. A fusion protein comprising the TRIM72 truncated protein according to any one of claims 1 to 27.

36. A pharmaceutical composition comprising a TRIM72 truncated protein according to any one of claims 1 to 27, a recombinant protein according to claim 28, a nucleic acid molecule according to claim 29, a vector according to any one of claims 30 to 33, a cell according to claim 34 and / or a fusion protein according to claim 35, and a pharmaceutically acceptable adjuvant.

37. The pharmaceutical composition of claim 36, wherein 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.

38. A method for protecting neurons in a subject, comprising administering to a subject in need thereof an effective amount of a TRIM72 truncated protein according to any one of claims 1 to 27, a recombinant protein according to claim 28, a nucleic acid molecule according to claim 29, a vector according to any one of claims 30 to 33, a cell according to claim 34, a fusion protein according to claim 35 and / or a pharmaceutical composition according to any one of claims 36 to 37.

39. A method for preventing and / or treating a nervous system disease, comprising administering to a subject in need thereof an effective amount of a TRIM72 truncated protein according to any one of claims 1 to 27, a recombinant protein according to claim 28, a nucleic acid molecule according to claim 29, a vector according to any one of claims 30 to 33, a cell according to claim 34, a fusion protein according to claim 35 and / or a pharmaceutical composition according to any one of claims 36 to 37.

40. The method according to claim 39, wherein the nervous system disease is a nerve damage disease induced by oxidative stress.

41. The method of any one of claims 39 to 40, wherein the nervous system disease is a neurodegenerative disease.

42. The method of any one of claims 39 to 41, wherein the neurological disease comprises ALS, PD and / or ALS.

43. Use of a TRIM72 truncated protein according to any one of claims 1 to 27, a recombinant protein according to claim 28, a nucleic acid molecule according to claim 29, a vector according to any one of claims 30 to 33, a cell according to claim 34, a fusion protein according to claim 35 and / or a pharmaceutical composition according to any one of claims 36 to 37 in the manufacture of a medicament for preventing and / or treating a nervous system disease.

44. The use according to claim 43, wherein the nervous system disease is a nerve damage disease induced by oxidative stress.

45. The use according to any one of claims 43 to 44, wherein the nervous system disease is a neurodegenerative disease.

46. ​​The use according to any one of claims 43 to 45, wherein the neurological disease comprises ALS, PD and / or stroke.

Images