AAV gene therapy for treatment of nervous system diseases

Abstract

Provided is a recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or functional fragment thereof, where the recombinant AAV expression vector comprises a neuron-specific promoter. A pharmaceutical composition comprising the rAAV expression vector and uses thereof are also provided.

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, of which ALS is the most common. ALS is characterized by muscle stiffness, muscle twitching, and a gradually worsening sense of weakness due to muscle loss. This can lead to difficulty speaking, swallowing, and eventually breathing. The cause of 90% to 95% of cases is unknown. About 5-10% of cases are inherited from parents. About half of these inherited cases are due to four specific genes: SOD1, TDP-43, FUS, and C9orf72. There are currently only three drugs clinically used to treat ALS. One is riluzole, which was approved more than 20 years ago. Clinical trial results show that riluzole can only extend patients' survival by a few months. The second, Edaravone, appears to be effective only in the early stages of the disease, slowing the progression of the disease by more than two months, but may actually accelerate the progression of the disease in the middle and late stages. Patients in the AMX0035 treatment group showed only a slight statistically significant and clinically significant improvement, extending the patients' disease-free survival time by 4.9 months. As far as drugs in clinical trials are concerned, there have been few breakthroughs to date, and they can only slow the progression of the disease by less than five months. For decades, drug development for ALS has been difficult. Now, gene therapy is becoming the most promising treatment for neurodegenerative diseases, including ALS. Gene therapy makes more molecules available for drug treatment. However, almost all current ALS gene therapy strategies are limited to ALS patients with familial mutations, and there are no viable solutions for sporadic ALS patients. Oxidative stress is a common pathological process in all ALS patients, so the development of gene therapy drugs targeting oxidative stress is a potentially feasible way to treat familial and sporadic ALS.

[0002] TRIM72 is a triple motif (TRIM) family protein composed of a RING finger, a B-box motif, a coiled-coil region, and a C-terminal PRYSPRY domain. It is involved in the sarcolemmal repair process and is associated with the insulin signaling pathway. It is also involved in protecting the heart from ischemia / reperfusion (IR) injury. It has been reported that full-length TRIM72 is a potential target for ALS by ubiquitinating mutant FUS protein. However, the protective effect of TRIM72 on neurons needs to be further explored, and different types of TRIM72 protein / gene products also need to be further developed. Summary of the invention

[0003] The present disclosure provides a recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or functional fragment thereof. The rAAV expression vector has one or more of the following characteristics: (1) it can highly express the TRIM72 protein in vivo, for example, in neurons; (2) it can effectively protect neurons; (3) it can reduce oxidative stress; (4) it can treat, prevent and / or alleviate neurological diseases.

[0004] On the one hand, the present application provides a recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or a functional fragment thereof, wherein the recombinant AAV expression vector comprises a neuron-specific promoter.

[0005] In some embodiments, the neuron-specific promoter comprises a human promoter.

[0006] In some embodiments, the promoter is one or more selected from the following groups: an excitatory neuron-specific promoter, a cerebral neocortex and hippocampus excitatory neuron-specific promoter, a short neuron-specific promoter, a dopaminergic neuron-specific promoter, a glutamatergic neuron-specific promoter, a GABAergic neuron-specific promoter, a cholinergic neuron-specific promoter, and a serotonergic neuron-specific promoter.

[0007] In some embodiments, the promoter is selected from the group consisting of human synapsin (hSyn), calcium / calmodulin-dependent kinase IIa (CamKⅡa), c-fos, methylated CpG binding protein 2 (Mecp2), neuron-specific enolase (NSE), somatostatin (SST), human vesicular GABA (γ-aminobutyric acid) transporter (hVGAT), choline acetyltransferase (ChAT), serotonin transporter (SERT) and tyrosine hydroxylase (TH).

[0008] In some embodiments, the serotype of the AAV vector is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh, AAVDJ and AAVhull.

[0009] In some embodiments, the AAV is single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

[0010] In some embodiments, the TRIM72 protein is a human TRIM72 protein.

[0011] In some embodiments, the TRIM72 protein comprises a full-length TRIM72 protein.

[0012] In some embodiments, the TRIM72 protein comprises a wild-type TRIM72 protein.

[0013] In some embodiments, the human TRIM72 protein comprises the amino acid sequence as shown in SEQ ID NO:2.

[0014] In some embodiments, the TRIM72 protein comprises a TRIM72 truncated protein.

[0015] In some embodiments, the TRIM72 truncated protein comprises the PRYSPRY domain of the TRIM72 protein or a functional fragment thereof.

[0016] In some embodiments, the PRYSPRY domain comprises amino acid positions 278aa-470aa of the TRIM72 protein.

[0017] In some embodiments, the PRYSPRY domain comprises the amino acid sequence as shown in SEQ ID NO:6.

[0018] In some embodiments, the TRIM72 truncated protein further comprises a coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

[0019] In some embodiments, the TRIM72 truncated protein does not comprise the coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

[0020] In some embodiments, the coiled-coil domain comprises amino acid positions 135aa-232aa of the TRIM72 protein.

[0021] In some embodiments, the coiled-coil domain comprises the amino acid sequence as shown in SEQ ID NO:5.

[0022] In some embodiments, the TRIM72 truncated protein further comprises the B-box domain of the TRIM72 protein or a functional fragment thereof.

[0023] In some embodiments, the TRIM72 truncated protein does not comprise the B-box domain of the TRIM72 protein or a functional fragment thereof.

[0024] In some embodiments, the B-box domain comprises amino acid positions 86aa-117aa of the TRIM72 protein.

[0025] In some embodiments, the B-box domain comprises the amino acid sequence shown in SEQ ID NO:4.

[0026] In some embodiments, the TRIM72 truncated protein further comprises the RING finger domain of the TRIM72 protein or a functional fragment thereof.

[0027] In some embodiments, the TRIM72 truncated protein does not comprise the RING finger domain of the TRIM72 protein or a functional fragment thereof.

[0028] In some embodiments, the RING finger domain comprises amino acid positions 14aa-56aa of the TRIM72 protein.

[0029] In some embodiments, the RING finger domain comprises the amino acid sequence as shown in SEQ ID NO:3.

[0030] 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.

[0031] In some embodiments, the TRIM72 protein or a variant or functional fragment thereof comprises an amino acid mutation at position C14.

[0032] In some embodiments, the TRIM72 protein or a variant or functional fragment thereof comprises the amino acid mutation C14A.

[0033] In some embodiments, the TRIM72 protein or its variant or functional fragment does not contain an amino acid mutation at position C242.

[0034] In some embodiments, rAAV expression vectors are used to protect neurons by reducing oxidative stress.

[0035] In some embodiments, the rAAV expression vector is used to prevent or treat a neurological disease.

[0036] In some embodiments, the rAAV expression vector is used to prevent or treat ALS or stroke.

[0037] On the other hand, the present application provides a host cell comprising the rAAV expression vector.

[0038] On the other hand, the present application provides a pharmaceutical composition comprising the rAAV expression vector or the host cell and a pharmaceutically acceptable adjuvant.

[0039] On the other hand, the present application provides a method for protecting neurons in a subject, comprising administering an effective amount of the rAAV expression vector, the host cell and / or the pharmaceutical composition to a subject in need thereof.

[0040] On the other hand, the present application provides a method for preventing and / or treating a nervous system disease, comprising administering an effective amount of the rAAV expression vector, the host cell and / or the pharmaceutical composition to a subject in need thereof.

[0041] In some embodiments, the neurological disease comprises ALS or stroke.

[0042] On the other hand, the present application provides a use of the rAAV expression vector, the host cell and / or the pharmaceutical composition in the manufacture of a drug for preventing and / or treating a nervous system disease.

[0043] In some embodiments, the neurological disease comprises ALS or stroke.

[0044] Other aspects and advantages of the present disclosure will be apparent to those skilled in the art from the detailed description below. Only illustrative embodiments of the present disclosure are shown and described in the detailed description below. As will be appreciated, the present disclosure may have other different embodiments, and several details may be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and descriptions are to be regarded as illustrative in nature, and not restrictive.

[0045] Incorporated by Reference

[0046] 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

[0047] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention may be better understood by referring to the following detailed description of illustrative embodiments and the accompanying drawings (also referred to herein as "figures"), which illustrate the principles of the invention, wherein:

[0048] Figure 1 Different pAAV-ITR vectors containing TRIM72 or control genes are shown. Each construct contains ssAAV 5'ITR and 3'ITR or scAAV 5'ITR and 3'ITR-Δtrs, introns or no introns, neuron (hSyn1) or astrocyte (GfaABC1D, a truncated GFAP promoter) specific promoters, mouse Trim72 (mTrim72) or human TRIM72 (hTRIM72) cDNA, SV40 polyadenylation signal (SV40 polyA) or bovine growth hormone polyadenylation signal (BGH polyA), and woodchuck hepatitis virus post-transcriptional regulatory element (WHV's post-transcriptionalregulatory element, WPRE) or no introns.

[0049] Figure 2The dosing schedule is shown. Evaluation of the neuropathological features of the SOD1-G93A transgenic mouse model showed that the mice had impaired motor ability at 80 days of age, which continued until the mice reached the experimental endpoint. At approximately 120 days of age, body weight began to decrease significantly and continued until the mice reached the experimental endpoint. It is expected that the mice will reach the experimental endpoint at 150 to 180 days of age. In this experiment, we completed AAV injections when the mice were 90 days old. The weight of the mice was recorded from 90 days of age until they reached the experimental endpoint. Behavioral testing of the mice began at 180 days of age until 152 days of age.

[0050] Figure 3 The body weight of SOD1-G93A mice after ssAAV-TRIM72 treatment is shown. All male mice were weighed every two days using a standard electronic scale. The weight changes of mice were recorded after injection of ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72 or ssAAV(PHP.eB)-hSyn1-EGFP until the mice reached the experimental endpoint. Number of group members at the beginning: EGFP: n=15; EGFP-TRIM72: n=15. *p<0.05 (log rank).

[0051] Figure 4 The motor behavior of SOD1-G93A mice after ssAAV-TRIM72 treatment is shown. Starting from 108 days of age, male mice were tested once a week in the open field test, and the total distance traveled by the mice was recorded. In the open field test performed at 152 days of age, there was a significant difference in the total distance traveled by the mice. The values ​​of all data points are shown in the form of box plots. *p<0.05 (t-test), NS=not statistically significant.

[0052] Figure 5 The rotarod behavior of SOD1-G93A mice after ssAAV-TRIM72 treatment is shown. Five experiments were performed on male mice between 112 and 132 days of age. The residence time of the mice during the experiment was recorded. In the last experiment, the mice injected with ssAAV-TRIM72 had a longer residence time, and the difference was significant. The values ​​of all data points are shown in the form of box plots. *p<0.05 (t test), NS=not statistically significant.

[0053] Figure 6 Shown are the survival curves of SOD1-G93A mice after ssAAV-TRIM72 treatment.

[0054] Figure 7EGFP-TRIM72 or EGFP expression in the motor cortex and spinal cord after intravenous AAV delivery is shown. (A) Representative fluorescence images of brain slices from SOD1-G93A mice injected with ssAAV (PHP.eB) -hSyn1-EGFP-TRIM72 or ssAAV (PHP.eB) -hSyn1-EGFP. (B) Representative fluorescence images of lumbar 4-5 spinal cord slices from SOD1-G93A mice treated with different AAVs. All slices were stained for neurons (NeuN), transgene expression (GFP) and nuclei (DAPI), while spinal cord slices were additionally stained for ChAT to obtain motor neuron fluorescence.

[0055] Figure 8 A-8B shows the relationship between the efficiency of ssAAV(PHP.eB)-EGFP or ssAAV(PHP.eB)-TRIM72-EGFP infection in SOD1-G93A mice and the number of days the animals survived.

[0056] Fig. 9 Shown are the survival curves of TDP43-A315T mice treated with ssAAV-TRIM72, and the relationship between the infection efficiency of ssAAV(PHP.eB)-EGFP or ssAAV(PHP.eB)-TRIM72-EGFP and the survival days of TDP43-A315T mice.

[0057] Fig.10 Representative fluorescent images of astrocytes from wild-type mice injected with ssAAV(PHP.eB)-GfaABC1D-EGFP-TRIM72 are shown. Arrows indicate astrocytes.

[0058] Fig.11 No significant exogenous TRIM72 expression is shown in non-neuronal tissues. Expression of Flag-tagged TRIM72 in mouse cerebral cortex, spinal cord (SC), heart, kidney, liver, lung, muscle and spleen. Tissues were from wild-type C57BL / 6 mice injected with Flag-tagged ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72.

[0059] Fig.12 The rotarod behavior of SOD1-G93A mice after scAAV-hSyn1-TRIM72 treatment is shown. The residence time of mice during the experiment was recorded. At day 140, the residence time of mice injected with scAAV-hSyn1-TRIM72 was longer, and the difference was significant. The values ​​of all data points are shown in the form of box plots. **p<0.01 (t test).

[0060] Fig.13 A-13B shows the therapeutic effect of TRIM72 in scAAV vector on ALS animal model. As shown by 15% weight loss (A) and prolonged median survival time of SOD1-G93A mice (B), scAAV9-hSyn1-TRIM72 treatment can significantly delay disease progression. *p<0.05 (log rank).

[0061] Fig.14 Survival curves of TDP43-A315T mice after scAAV-TRIM72 treatment are shown. The scAAV-hSyn1-TRIM72 treatment group exhibited a longer median survival time, while ssAAV-GfaABC1D-TRIM72 treatment was not as effective as the control group.

[0062] Fig.15 A-15B shows that the loss of TRIM72 function accelerates the progression of disease in FUS-R521C knock-in mice. (A) Loss of TRIM72 function significantly shortens the lifespan of FUS-R521C knock-in mice. Mice, male, n=25 (+ / +), 19 (C / C), 17 (C / C; - / -) and 18 (- / -). (B) Rotarod test at 4 months of age. Mice, male, n=10 (+ / +), 10 (C / C), 12 (C / C; - / -) and 9 (- / -). Values ​​are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 (ANOVA, SPSS). NS, not statistically significant.

[0063] Fig.16 A-16B shows that TRIM72 is upregulated in neural tissues but not in non-neural tissues in mice with the indicated genotypes. (A) Total protein was visualized with Commassie blue. Cere., cerebellum; Hippo., hippocampus. Quantification is shown in B, and values ​​are expressed as mean ± SEM. ***p<0.001 (t test), *p<0.05 (t test), NS, not statistically significant.

[0064] Fig.17 A-17B shows the expression of TRIM72 in wild-type (+ / +) and FUS-R521C (C / C) knock-in mutant rats (A). GAPDH was used as a loading control. Quantification is shown in B, and values ​​are expressed as mean ± SEM. ***p<0.001 (t test), **p<0.01 (t test), NS, not statistically significant.

[0065] Fig.18A-18D shows that TRIM72 in ChAT-, Vglut1- and Vgat-positive neurons is upregulated in FUS-R521C knock-in mice. ChAT-positive neurons: cholinergic neurons (lower motor neurons) in the spinal cord, Vglut1-positive neurons: excitatory neurons in the motor cortex. Vgat-positive neurons: inhibitory neurons in the motor cortex. GFAP: marker of astrocytes. IBA1: marker of microglia. ChAT, GFAP and IBA1 were detected using immunostaining. TRIM72, Vglut1 and Vgat mRNA were detected using RNAscope. Data are summarized and shown in B, C and D, and the values ​​are expressed as mean ± SEM. ***p<0.001 (t test), NS, not statistically significant.

[0066] Fig.19 The TRIM72 domain annotation is shown, and the key residues for TRIM72 function are labeled.

[0067] Fig. 20 The Flag-tagged domain fused to EGFP is shown to disrupt the expression of TRIM72. GAPDH was used as a protein loading control. FL, full-length TRIM72.

[0068] Fig.21 The effect of domain disruption of TRIM72 on cell viability after stress challenge is shown. Disruption of the coiled-coil domain or PRYSPRY domain, but not the ring domain or B-box domain, abolished the protective effect of TRIM72. Values ​​are expressed as mean ± SEM. ***p < 0.001 (t test), **p < 0.01 (t test), NS, not statistically significant.

[0069] Fig. 22 Different full-length and truncated TRIM72 or TRIM40 constructs in pAAV-ITR vectors are shown.

[0070] Fig.23 The cell viability under H2O2 stress by expressing full-length or different truncated forms of TRIM72 or full-length TRIM40 by scAAV9 infection is shown. The values ​​of all data points are expressed as mean ± SEM, and t-tests were performed to compare the data sets with the control group. **p<0.01, *p<0.05, NS, not statistically significant.

[0071] Fig.24 A schematic diagram showing the timeline of treatment and behavioral testing of the middle cerebral artery occlusion / reperfusion (MCAO / R) model is shown.

[0072] Fig.25AB shows that expression of full-length TRIM72 by scAAV9 infection promoted functional recovery after ischemia in the MCAO model using the nerve test and sticker removal test. Values ​​are expressed as mean ± SEM. **p < 0.01 (t test).

[0073] Fig.26 Cell viability of N2a cells expressing full-length TRIM72 by scAAV9 infection after OGD treatment is shown. Values ​​are expressed as mean ± SEM. **p<0.01 (t-test), *p<0.05 (t-test).

[0074] Fig. 27 Shown is the effect of expression of TRIM72 mutants on cell viability after stress challenge. C242A mutation, but not C14A mutation, abolished the anti-stress effect of TRIM72. Values ​​are from at least three independent experiments (n≥3). *p<0.05, **p<0.01, ***p<0.001 (ANOVA). NS, not statistically significant. DETAILED DESCRIPTION

[0075] 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. Those skilled in the art may make various changes, modifications and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0076] In this application, the term "adeno-associated viral vector" generally refers to a nucleic acid derived from any AAV serotype, such as AAV1, AAV2, AV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 serotypes or any other virus or serotype whose capsid protein sequence is homologous to the capsid protein of an AAV serotype. The term "recombinant adeno-associated virus" or "rAAV" refers to an infectious, replication-defective virus composed of an AAV protein shell encapsulating a nucleic acid molecule of interest, with the nucleic acid molecule flanked on one or both sides by AAV ITRs. As used herein, reference to a particular AAV serotype refers to an AAV having at least one capsid protein of that AAV serotype. For example, the term "AAV9" refers to an AAV having at least one AAV serotype 9 capsid protein.

[0077] In the present application, the term "TRIM72 protein" can be used interchangeably with "MG53" protein, and generally includes 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 an amino acid site of 14aa-56aa of TRIM72 protein or a functional fragment thereof. For example, the B-box domain may include an amino acid site of 86aa-117aa of TRIM72 protein or a functional fragment thereof. For example, the coiled coil domain may include an amino acid site of 135aa-232aa of TRIM72 protein or a functional fragment thereof. For example, the PRYSPRY domain may include an amino acid site of 278aa-470aa of TRIM72 protein. The term may also include TRIM72 protein derived from any known species having TRIM72 protein.

[0078] In the present application, the term "truncated protein" generally refers to a protein that lacks one or more amino acids compared to the full-length protein. For example, a truncated protein may contain a major functional fragment of the protein. For example, a truncated protein also includes (but is not limited to) variants, functional fragments, analogs, and homologs thereof.

[0079] In the present application, "amino acid mutation Xn" refers to an amino acid mutation occurring at 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 at amino acid residue C at position 14 of the amino acid sequence as shown in SEQ ID NO: 2.

[0080] The amino acid mutation of the present application can be a non-conservative mutation. The non-conservative mutation may include changing the amino acid residues in the target protein or polypeptide in a non-conservative manner, such as replacing an amino acid residue with a specific side chain size or specific characteristics (e.g., hydrophilicity) with an amino acid residue with a different side chain size or different characteristics (e.g., hydrophobicity).

[0081] The amino acid substitution may also be a conservative substitution. The conservative substitution may include changing the amino acid residues in the target protein or polypeptide in a conservative manner, such as an amino acid residue with a specific side chain size or a specific characteristic (e.g., hydrophilicity) being replaced with an amino acid residue with the same or similar side chain size or the same or similar characteristic (e.g., still hydrophilicity). 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 as a mutant of a fusion protein, a fragment thereof, or a variant thereof that has undergone one or more amino acid substitutions may include conservative amino acid substitutions that do not significantly change the structure or function of the protein.

[0082] For example, substitutions between the amino acids in each of the following groups may still be considered conservative substitutions in the present application:

[0083] The group of amino acids with nonpolar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0084] The group of uncharged amino acids with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0085] A group of negatively charged amino acids with polar side chains: aspartic acid and glutamic acid.

[0086] Positively charged basic amino acid group: lysine, arginine, and histidine.

[0087] Group of amino acids with a phenyl group: phenylalanine, tryptophan, and tyrosine.

[0088] 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 expression elements may be provided by the host cell or may be provided 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., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0089] In this 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 a biological process, and the polymers and macromolecules have a determined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a determined amino acid sequence and the resulting biological properties. Therefore, if the mRNA corresponding to a gene produces a protein in a cell or other biological system after transcription and translation, the gene encodes the protein. The coding strand whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence table, as well as the non-coding strand used as a template for transcription of a gene or cDNA, can be referred to as a protein or other product encoding the gene or cDNA. In this application, the term "coding element" generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein.

[0090] In the present application, the terms "host cell", "cell" and "host" are used interchangeably and generally refer to a plasmid or vector of a single cell, cell line or cell culture that may or has included a nucleic acid molecule of the present application, or may express a protein, a fragment thereof or a variant thereof of the present application. The host cell may contain offspring of a single host cell. Due to natural, accidental or deliberate mutations, the morphology or genome of the offspring cells and the original parent cell may not be exactly the same, as long as they can express the protein or a fragment thereof of the present application. The host cell can be obtained by transfecting cells in vitro with the vector of the present application.

[0091] As used herein, the term "treating" generally refers to slowing or ameliorating the progression, severity and / or duration of a proliferative disease, or ameliorating one or more symptoms (eg, one or more identifiable symptoms) of a proliferative disease by administering one or more therapies.

[0092] In this application, the term "subject" generally refers to any human or non-human animal. The term "non-human animal" may include all vertebrates, such as mammals and non-mammals, for example, non-human primates, goats, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0093] In addition to the specific proteins and nucleotides mentioned herein, the present application may also include functional variants, derivatives, analogs, homologues and fragments thereof.

[0094] The term "functional variant" refers to a polypeptide having substantially the same amino acid sequence as a naturally occurring sequence or being encoded by a substantially identical nucleotide 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 sequence of residues (amino acid residues or nucleotide residues) is modified so that the polypeptide or polynucleotide substantially retains at least one endogenous function. The variant sequence can be obtained by adding, deleting, replacing, modifying, substituting 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 retained. In the present application, the term "derivative" generally refers to any substitution, change, modification, replacement, deletion and / or addition of one (or more) amino acid residues in the sequence of the polypeptide or polynucleotide of the present application, provided that the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

[0095] In the present application, with respect to a polypeptide or polynucleotide, the term "analog" generally includes any mimetic of the polypeptide or polynucleotide, i.e., a compound having at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics. In general, amino acids may be substituted, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 or more) amino acids may be substituted, provided that the modified sequence substantially retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs. The proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues, wherein the amino acid residues undergo silent changes and produce functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic 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; and amino acids containing uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0096] 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" can be equivalent to the "identity" of a sequence. A homologous sequence can 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 subject sequence. In general, a homologue will contain an active site that is identical to the subject amino acid sequence, etc. Homology can be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions), or homology can be expressed according to sequence identity. In the present application, a sequence with a percentage identity in any SEQ ID NO of the amino acid sequence or nucleotide sequence refers to a sequence with a percentage identity over the entire length of the SEQ ID NO. To determine sequence identity, sequence alignment can be performed in a variety of ways 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.

[0097] In this application, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 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.

[0098] In this application, the term "comprising" generally means including, containing, having or covering. In some cases, it also means "being" or "consisting of...".

[0099] 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.

[0100] Recombinant adeno-associated virus (rAAV) expression vector

[0101] On the one hand, the present application provides a recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or a functional fragment thereof, wherein the recombinant AAV expression vector comprises a neuron-specific promoter.

[0102] In the present application, rAAV may include an AAV genome or a derivative thereof, and / or an AAV capsid protein or a derivative thereof. In the present application, rAAV may be a chimeric AAV, a reorganized AAV or a capsid modified AAV. In the present application, the AAV genome or AAV capsid protein may be from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh, AAVDJ and AAVhull. In the present application, rAAV may be a hybrid AAV (e.g., AAV-DJ, AAV-DJ / 8 or AAV-DJ / 9). In the present application, rAAV (e.g., AAV7m8 or AAV-PHP.B) may be developed by directed evolution and / or rational design. In the present application, rAAV may comprise one or more capsid mutations (e.g., YF, KR, TA, SA and / or TV mutations, (e.g., AAV2 having one or more capsid mutations in Y444F, Y500F, Y730F, Y252F, Y272F, Y700F, Y704F and T491V or corresponding mutations of different AAV serotypes (e.g., AAV2 / 8 (Y733F), AAV2 (Y444F+Y500F+Y730F) and AAV2 (quadY-F+TV))). In the present application, the serotype of rAAV may be selected from the following group: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 1, AAV12, Anc80, rh10 and ShH10. In the present application, the rAAV vector can be selected from the following group: AAV2 / 5, AAV2 / 8, AAV2 / 8 (Y733F), AAV2 (Y444F + Y500F + Y730F), AAV2 / 1, AAV2 / 4, AAV2 / 9, AAV2 / 6, AAV2 / 7, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, Anc80, AAV7m8, AAV-DJ, ShH10, AAV-PHP.B or its hybrid, derivative or variant. In the present application, the rAAV vector can be a single-stranded AAV vector or a self-complementary AAV (scAAV) vector.

[0103] In some embodiments, the serotype of the AAV vector can be AAV9.

[0104] In the present application, the AAV packaging system can be used to deliver genes encoding TRIM72 protein or its variants or functional fragments. For example, the AAV packaging system can be a three-plasmid system. For example, the AAV packaging system can include pAAV-ITR containing genes encoding TRIM72 protein or its variants or functional fragments, serotype vector pRepCapX and auxiliary vector pADHelper.

[0105] In the present application, the neuron-specific promoter may include a human promoter.

[0106] In the present application, the promoter may include a universal neuron-specific promoter. In the present application, the promoter may include an excitatory neuron-specific promoter. In the present application, the promoter may include a cerebral neocortex and hippocampus excitatory neuron-specific promoter. In the present application, the promoter may include an inhibitory neuron-specific promoter. In the present application, the promoter may include a short neuron-specific promoter. In the present application, the promoter may include a dopaminergic neuron-specific promoter. In the present application, the promoter may include a glutamatergic neuron-specific promoter. In the present application, the promoter may include a GABAergic neuron-specific promoter. In the present application, the promoter may include a cholinergic neuron-specific promoter. In the present application, the promoter may include a serotonergic neuron-specific promoter.

[0107] In the present application, the neuron-specific promoter can be selected from the following group: human synapsin (hSyn), calcium / calmodulin-dependent kinase IIa (CamKⅡa), c-fos, methylated CpG binding protein 2 (Mecp2), neuron-specific enolase (NSE), somatostatin (SST), human vesicular GABA (γ-aminobutyric acid) transporter (hVGAT), choline acetyltransferase (ChAT), serotonin transporter (SERT) and tyrosine hydroxylase (TH). For example, the promoter can be a Syn1 promoter. For example, the Syn1 promoter can include a nucleotide sequence as shown in SEQ ID NO: 12 or a variant thereof.

[0108] In the present application, the rAAV expression vector comprises a gene encoding the TRIM72 protein or a variant or functional fragment thereof.

[0109] In the present application, the TRIM72 protein may include a full-length TRIM72 protein. In the present application, the TRIM72 protein may include a wild-type TRIM72 protein. In the present application, the human TRIM72 protein includes the amino acid sequence shown in SEQ ID NO: 2.

[0110] In the present application, the TRIM72 protein may include a TRIM72 truncated protein.

[0111] In the present application, the TRIM72 truncated protein comprises a PRYSPRY domain or a functional fragment thereof. For example, the TRIM72 truncated protein may comprise an amino acid site of 278aa-470aa of the TRIM72 protein. For example, the TRIM72 truncated protein may comprise an amino acid sequence as shown in SEQ ID NO:6.

[0112] In the present application, the TRIM72 truncated protein may also contain other domains of the TRIM72 protein.

[0113] For example, the TRIM72 truncated protein may include a PRYSPRY domain and a coiled-coil domain. For example, the TRIM72 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.

[0114] 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 a TRIM72 protein coiled-coil domain and a ring finger domain.

[0115] 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.

[0116] For example, the TRIM72 truncated protein may comprise a PRYSPRY domain, a coiled-coil domain and a RING finger domain. For example, the TRIM72 truncated protein may comprise a deletion of the B-box domain of the TRIM72 protein.

[0117] 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 TRIM72 protein RING finger domain.

[0118] 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 TRIM72 protein coiled-coil domain.

[0119] In the present application, the TRIM72 protein or its fragment may include a variant thereof. For example, the TRIM72 protein may include one or more amino acid mutations compared to the corresponding wild-type sequence.

[0120] In the present application, the TRIM72 protein may include an amino acid mutation at position C14. For example, the amino acid mutation may be C14A.

[0121] According to the applicant's research, the amino acid cysteine ​​at position 242 is particularly important for the oligomer formation of TRIM72 protein. Substitution of amino acid C242 may block the protective function of neurons. Therefore, amino acid substitution at position C242 (e.g., C242A) may not be included in the TRIM72 protein of the present application.

[0122] In the present application, rAAV expression vectors can be used to protect neurons by reducing oxidative stress.

[0123] In the present application, the rAAV expression vector can be used to prevent or treat nervous system diseases. For example, the present application can be used to prevent and / or treat ALS.

[0124] Host cells, pharmaceutical compositions

[0125] In another aspect, the present application provides a cell (eg, a host cell) that may contain the rAAV expression vector of the present application.

[0126] In some embodiments, the host cell is a bacterial cell, an Escherichia coli cell, a plant cell, an insect cell, or a mammalian cell. In some embodiments, the cell is a somatic cell or a stem cell. In some embodiments, the rAAV expression vector is delivered to the host cell so that the Cas encoded by the nucleic acid molecule is expressed in the cell.

[0127] On the other hand, the present application provides a pharmaceutical composition comprising the rAAV expression vector or the host cell and a pharmaceutically acceptable adjuvant.

[0128] In some embodiments, the pharmaceutically acceptable adjuvant may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counter ion, a metal complex and / or a nonionic surfactant, etc.

[0129] 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 according to the procedures in Remington: The Science and Practice of Pharmacy, 19th edition, edited by Gennaro, Mack Publishing Company, Easton, Pennsylvania, 1995.

[0130] 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 administration of the drug via a subcutaneous depot.

[0131] 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 (e.g., ALS or stroke), and / or can alleviate and / or stabilize the disease state.

[0132] The pharmaceutical composition of the present application may comprise a therapeutically effective amount of the rAAV expression vector. The therapeutically effective amount is the dose required to prevent and / or treat (at least partially treat) a disease (e.g., ALS or stroke) and / or any complications thereof in a subject who is ill or at risk of the disease.

[0133] Preparation, method and use

[0134] On the other hand, the present application provides a method for protecting neurons in a subject, comprising administering an effective amount of a rAAV expression vector, a host cell and / or a pharmaceutical composition to a subject in need thereof.

[0135] On the other hand, the present application provides a method for preventing and / or treating a nervous system disease, comprising administering an effective amount of a rAAV expression vector, a host cell and / or a pharmaceutical composition to a subject in need thereof.

[0136] On the other hand, the present application provides a use of a rAAV expression vector, a host cell and / or a pharmaceutical composition in the manufacture of a drug for preventing and / or treating a nervous system disease.

[0137] On the other hand, the present application provides rAAV expression vectors, host cells and / or pharmaceutical compositions for preventing and / or treating nervous system diseases.

[0138] In the present application, nervous system diseases may include neurodegenerative diseases or other diseases.

[0139] In the present application, the neurological disease comprises ALS or stroke. For example, the stroke can be an ischemic stroke.

[0140] Example

[0141] The following examples are listed to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, but 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 values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should also be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase pairs; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; rpm, revolutions per minute, etc.

[0142] Materials and methods

[0143] animal

[0144] In this project, overexpression of ALS-causing mutant SOD1 was used G93A The ALS mouse model SOD1-G93A or the ALS mouse model hTDP43-A315T generated by using cDNA encoding human TDP43 with an N-terminal Flag tag with the A315T mutation, while age-matched wild-type (WT) mice were used as controls in each experiment. The animal facility of Tsinghua University has been fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International since 2014. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, NHR). All mice were housed in an isolated ventilated cage (maximum 6 mice per cage) barrier facility at Tsinghua University. Mice were maintained under conditions of 12 / 12 h light / dark cycle, 22-26°C, humidity 40-70%, free access to sterile pelleted food and water. Cages were checked daily to ensure animal welfare. Body weight was assessed regularly to ensure that there was no weight loss. When using animals for research, we follow the 3Rs (Replacement, Refinement, or Reduction) rule.

[0145] Behavioral testing

[0146] In the open field behavior test, a single animal was placed in the center of an open field area (60×60 cm), and multiple parameters including total distance, average speed, and distance traveled in the center area were tracked at 10-minute intervals using the TopScan behavior analysis system (CleverSys, USA). Rotarod behavior was tested by an automated system (Med Associates Inc.). In brief, the animal was placed on an accelerating spindle (5-40 rpm) for 5 minutes per test, three times a day in a row. There was a 20-minute rest between each test. When a mouse fell off the spindle within a 5-minute interval, the system automatically calculated the time it fell from the spindle. The residence time was calculated by subtracting the fall time from 5 minutes, and statistical analysis was performed as the average of the residence time for three consecutive tests per day. Mice were assigned to different experimental groups according to their age, genotype, and treatment. Behavioral assays were performed randomly in different groups. The sticker removal experimental test was used to assess neurological dysfunction after stroke, which is a sensitive method for assessing sensorimotor deficits in mice with focal cerebral ischemia. Briefly, mice were trained before surgery until they were able to remove a sticky dot on their paw within 12 seconds. Animals were tested 3, 5, or 7 days after ischemia by a researcher blinded to the experimental groups. The time required to perceive the stimulus and remove the sticker strip was measured.

[0147] Experimental endpoint assessment

[0148] Mice were weighed three times per week and assessed for weakness as described previously. The endpoint was reached when mice were unable to right themselves within 30 seconds after being placed supine.

[0149] Immunoblot analysis

[0150] The total protein content in cells or tissues was homogenized in RIPA buffer (50Mm Tris-HCl, pH 8.0, 150mMNaCl, 0.25% sodium deoxycholate, 0.1% SDS, 1% NP-40, supplemented with a complete protease inhibitor cocktail; BimakeB15001), and the lysate was incubated on ice for 30 minutes. After centrifugation at 12000rpm for 10 minutes, the supernatant was extracted, mixed with SDS loading buffer and incubated at 95°C for 10 minutes. Then, the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% skim milk at room temperature for 1 hour. Then, the membranes were incubated with diluted primary antibodies, including GAPDH (Ameribio), TUBULIN (Ameribio), TRIM72 antibody (gift from Dr. Jianjie Ma), TSG101 (Abcam), ITGAV (Abcam), and H3 (Abcam) at 4°C overnight. The membranes were then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Images were analyzed using FijiImageJ to obtain integrated intensity.

[0151] Exosome purification

[0152] The protocol for purifying exosomes from 100 ml of 293FT cell supernatant includes two steps: ultrafiltration and polyethylene glycol (PEG) precipitation. First, the collected supernatant is poured into a centrifuge tube and centrifuged at 3000xg for 20 minutes to remove cell debris. The supernatant is then filtered once with a 0.45 μm filter. Then, a new Amicon ULTRA-15 ultrafiltration tube that has been washed with PBS or autoclaved water is taken. The supernatant is then immediately added to the ultrafiltration tube and centrifuged at 3000xg for 5-10 minutes. The filtrate is discarded, and the supernatant is continued to be added and centrifuged at 3000xg for 5-10 minutes until all the supernatant is introduced into the ultrafiltration tube. The concentrate is then transferred to a 50 mL centrifuge tube. The separation reagent is added to the concentrate, and the sample is thoroughly mixed by vortexing or pipetting. Finally, the sample is placed at 2-8°C overnight. The next day, the sample is centrifuged at 10,000xg for 1 hour at 4°C. The supernatant was discarded, and the exosomes were in the pellet.

[0153] Middle cerebral artery occlusion / reperfusion (MCAO / R) model

[0154] Male C57BL / 6J mice weighing 25-28 g were used in this study. Mice were anesthetized with 1.2% tribromoethanol. After the neck hair of the mice was shaved and disinfected with povidone-iodine solution, the carotid artery was exposed by incision in the midline, and then the common carotid artery (CCA) and the external carotid artery (ECA) were sutured with 6-0 silk sutures to permanently block the blood flow. A silicone-coated nylon suture (MSMC23B100PK50, RWD) was introduced into the internal carotid artery (ICA) through the arteriotomy site of the CCA stump until a slight resistance was felt, indicating that the suture had reached the skull base. The remaining loose monofilament suture was gently tightened around the CCA to prevent backflow leakage, and then the wound was covered with cotton balls and the mouse was transferred to a heating pad to maintain body temperature. After one hour, the silicone-coated nylon suture was gently withdrawn and the monofilament suture was firmly tied to avoid postoperative bleeding. Finally, the wound was closed with absorbable sutures.

[0155] Oxygen Glucose Deprivation (OGD)

[0156] During OGD, the medium was replaced with HEPES-buffered glucose-free medium containing (in mmol / L) 154 NaCl, 5.6 KCl, 2.3 CaCl2, 1.0 MgCl2, 3.6 NaHCO3, 5 HEPES, pH 7.4. After scAAV9 infection (estimated multiplicity of infection (MOI) of 10,000 vg / cell), N2a cells were cultured in this medium for 24 hours, and then OGD was induced by placing the cells in an anaerobic chamber containing 95% N2 and 5% CO2 at 37°C. After 2, 4, or 6 hours, OGD was terminated by replacing the glucose-free medium back to Neurobasal medium and incubating the cultures in a normal cell culture incubator for an additional 22 hours or the length of reoxygenation (reperfusion, R) specified in individual experiments. Control cells were treated in the same manner, except that they were not exposed to OGD conditions.

[0157] Plasmids and lentiviral vectors

[0158] A DNA fragment corresponding to the full length of TRIM72 was amplified from a mouse cDNA library by PCR and then inserted into the pCMV-N-3×Flag expression vector between the SalI and XhoI sites using a seamless cloning kit (Beyotime) to generate TRIM72 with a Flag tag. A series of TRIM72 domain deletions with a Flag tag were generated from the full-length TRIM72 expression vector: ΔRing domain (domains 14-69 deleted); ΔB-box domain (domains 81-122 deleted); Δcoiled-coil domain (domains 135-232 deleted); ΔPRYSPRY domain (domains 278-470 deleted) constructs.

[0159] To construct TRIM72 stable expression cell lines, a series of Flag-tagged TRIM72 mutants or domain deletions or single domain DNA fragments were amplified from the corresponding TRIM72 expression vectors by PCR and inserted into the pLJM1-EGFP lentiviral vector between the BsrG I and EcoR I sites to produce a series of Flag-tagged TRIM72 mutants or domain deletions or single domains fused with EGFP in the lentiviral vector.

[0160] Lentivirus expressing Flag-tagged TRIM72 constructs was generated by fusing the corresponding EGFP to the Flag-tagged TRIM72 vector.

[0161] Cell culture, lentiviral packaging, and lentiviral infection

[0162] HEK293FT cells were cultured in DMEM (Invitrogen) containing 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 cells reached approximately 90% confluence, 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 the medium was replaced with fresh growth medium 5-6 hours after transfection. The culture medium was harvested 72 hours after transfection and centrifuged at 20,000 rpm for 2 hours at 4°C. After centrifugation, the lentivirus was enriched in the precipitate. The lentivirus was resuspended with 100 μl DPBS and stored at -80°C.

[0163] 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 the plasmid containing drug resistance, for at least one week. Cells selected with puromycin or blasticidin were used for further analysis.

[0164] Cell viability assay

[0165] Cell viability was assessed using CCK-8. 1.5 × 10 cells / well were plated in 96-well plates. 3 Cells were seeded at a density of 10 cells / well for arsenite treatment or 7 × 10 3Cells were seeded at a density of 100 μM 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) was 10,000 vg / cell) was performed for 24 hours 17 hours after cell inoculation. H2O2 was then 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 the average value.

[0166] Example 1 AAV packaging and construction of AAV vectors

[0167] The AAV packaging system is a commonly used three-plasmid system. By transfecting three plasmids into mammalian cells (such as HEK293) at the same time, all components required for AAV packaging can be expressed in these cells and assembled into viral particles. Here we used a modified three-plasmid system from PackGene (Guangzhou Paizhen Biotechnology Co., Ltd.). The system consists of three plasmids: pAAV-ITR containing the target gene, the serotype vector pRepCapX, and the auxiliary vector pADHelper. The target vector pAAV-ITR contains a eukaryotic promoter and other components required for high-level gene expression in mammalian germ cells when the exogenous sequence is cloned into the multiple cloning site (MCS). The vector also contains AAV inverted terminal repeats (ITR) that direct viral replication and packaging. PRepCapX contains AAV rep and CAP genes encoding replication proteins and viral capsid proteins. Stabilizing the expression levels of rep and CAP genes is a key step in obtaining the desired high-titer viral products. pADHelper contains a series of adenovirus genes VA, E2A, and E4, which are necessary for cells to produce high-titer viruses.

[0168] To construct the pAAV-ITR plasmid for TRIM72 expression, we used self-complementary AAV (scAAV) and single-stranded (ss) AAV vectors. Compared with ssAAV9 vectors, scAAV9 vectors have 10- to 100-fold higher transfection and expression efficiencies, but can only package 2.2 kb of foreign DNA. In addition, in non-dividing cells, scAAV exists as a stable episome, and studies have reported that it can maintain stable transgene expression for many years. Figure 1Different pAAV-ITR vectors containing TRIM72 (SEQ ID NO: 2) or a control gene are shown. The human synapsin 1 (hSyn1) promoter is a neuron-specific promoter. The GfaABC1D promoter, a truncated GFAP promoter, is an astrocyte-specific promoter. Each construct contains the 5'ITR and 3'ITR of ssAAV or the 5'ITR and 3'ITR of scAAV-△trs, a neuron (hSyn1) or astrocyte (GfaABC1D) specific promoter, mouse TRIM72 (mTRIM72) or human TRIM72 (hTRIM72) cDNA, SV40 polyadenylation signal (SV40polyA) or bovine growth hormone polyadenylation signal (BGHpolyA), and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or not.

[0169] In order to deliver sufficient amounts of AAV vectors to the nervous system and provide sufficient gene expression levels, the following administration routes (but not limited thereto) can be used: intracerebral administration, intrathecal administration, intravenous administration, aerosol administration, and intranasal, intramuscular, subcutaneous, intradermal, rectal and other parenteral administration routes. If necessary, administration routes can also be used in combination.

[0170] Example 2 Therapeutic effect of TRIM72 in ssAAV vector on ALS animal model

[0171] To test the therapeutic effect of TRIM72 in AAV vector on ALS, the classic ALS animal models SOD1-G93A transgenic mice (Jackson Laboratory: Stock No. 002726) and hTDP43-A315T transgenic mice (Jackson Laboratory: Stock No. 010700) were used in this paper. Mutated SOD1 is an important genetic factor in the pathogenesis of ALS. SOD1-G93A transgenic mice express high copy numbers of mutant SOD1 and develop adult-onset spinal motor neuron degeneration and progressive motor deficits, leading to paralysis and death. TDP43-A315T transgenic mice express mutant human TAR DNA binding protein cDNA containing an amino acid substitution associated with familial ALS. Hemizygous mice develop a progressive and fatal neurodegenerative disease reminiscent of ALS and frontotemporal lobar degeneration with ubiquitin accumulation.

[0172] In the first experiment, ssAAV(PHP.eB)-EGFP-TRIM72 was used as the experimental group, and AAV(PHP.eB)-EGFP was used as the control group. Figure 2 The AAV (PHP.eB) administration schedule shown was performed by retro-orbital intravenous injection at 10 11SOD1-G93A mice were injected with AAV vectors at a dose of 3 × 10 vg per mouse. 11 vg of AAV vectors were injected into TDP43-A315T mice. The SOD1-G93A model mice showed a significant downward trend in body weight at 130 days of age, which continued until the end of the experiment. However, during the progression of the disease, the EGFP-TRIM72 group showed relatively less weight loss than the AAV(PHP.eB)-EGFP group. This suggests that AAV-TRIM72 has a moderate but significant protective effect against weight loss caused by disease progression in male mice ( Figure 3 ). In addition, to determine the effect of AAV-TRIM72 on the motor ability of SOD1-G93A, the motor open field test and rotarod test were performed during AAV treatment. As the disease progresses, the total movement distance gradually decreases due to the gradual death of motor neurons. This suggests that AAV-TRIM72 has a partial protective effect on the decreased motor ability of SOD1-G93A mice ( Figure 4 In addition, AAV-TRIM72 treatment significantly preserved the motor skills of mice, as assessed by the rotarod behavioral test, with significant differences in the latency to fall in mice 120 days of age and older ( Figure 5 Finally, the survival data showed that AAV-TRIM72 only slightly affected the survival rate of mice ( Figure 6 ). In addition, after TRIM72 was administered via ssAAV, the survival time of TDP43-A315T mice was significantly prolonged. The median survival of the ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72 treatment group was approximately 133 days, while the median survival of the ssAAV(PHP.eB)-EGFP treatment group was approximately 117 days. This suggests that AAV-TRIM72 has a protective effect on the lifespan extension of TDP43-A315T mice ( Fig. 9 A).

[0173] To examine the expression of EGFP-TRIM72 in the target areas including the motor cortex and spinal cord, mouse tissues were sectioned and stained for GFP labeling, neurons, and motor neurons ( Figure 7). Infected neurons and motor neurons were observed in the motor cortex and spinal cord of SOD1-G93A transgenic mice. The infection efficiency of ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72 was calculated based on the proportion of infected neurons to total neurons. The efficiency in the cerebral cortex was approximately 20.95%, and the efficiency in the spinal cord was approximately 29.8%. Further analysis also showed that in SOD1-G93A and TDP43-A315T mice, there was a positive correlation between the infection efficiency and the number of days the animals survived in the AAV(PHP.eB)-TRIM72-GFP-treated groups, but not in the AAV(PHP.eB)-GFP group ( Figure 8 and Fig. 9 B).

[0174] To characterize the expression of EGFP-TRIM72 in other non-neuronal tissues, the ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72 construct was transfected into each mouse at 10 11 Vg was injected into wild-type C57BL / 6 mice at different doses, and tissues were collected for immunoblot analysis 10 days after expression. No significant expression was detected in non-neuronal tissues such as heart, kidney, liver, lung, muscle, and spleen ( Fig.11 When the hSyn1 promoter was used, the expression of EGFP-Trim or GFP was mainly distributed in the cerebral cortex and spinal cord; when the GfaABC1D promoter was used, the expression of EGFP-Trim or GFP was mainly distributed in astrocytes ( Fig.10 ).

[0175] Example 3 Therapeutic effect of TRIM72 in scAAV vector on ALS animal model

[0176] In order to achieve better therapeutic effects, a higher dose (2×10 14 vg / kg body weight) of scAAV9-hSyn1-TRIM72 (the sequence of Syn1 promoter is shown in SEQ ID NO: 12). Vehicle was used as a control. An enhanced therapeutic effect was observed in the scAAV9-hSyn1-Trim72 treatment group. Rotarod behavior showed improved motor function ( Fig.12 In addition, scAAV9-hSyn1-TRIM2 treatment significantly delayed disease progression in SOD1-G93A mice, as evidenced by a 15% decrease in body weight ( Fig.13 A). Survival data also showed that a dose of 2×10 14 vg / kg body weight of scAAV9-hSyn1-TRIM72 can significantly prolong the median survival time of SOD1-G93A mice ( Fig.13B). GfaABC1D (SEQ ID NO: 13) is an astrocyte-specific promoter. Another efficacy analysis was performed in hTDP43-A315T mice. 12 Significant therapeutic effects were also observed in the scAAV(PHP.eB)-hSyn1-TRIM72 treatment group at a dose of 10 vg / kg body weight, but 13 This effect was not observed in the ssAAV(PHP.eB)-GfaABC1D-TRIM72-treated group (vg / kg body weight) Fig.14 ). The median survival time of the scAAV(PHP.eB)-hSyn1-TRIM72 treatment group was about 124 days, while the median survival time of the control group was about 115 days. However, the ssAAV(PHP.eB)-GfaABC1D-TRIM72 treatment group was not as effective as the control group, with a median survival time of only 103 days. Using riluzole as a reference, its median survival time was the same as the control group.

[0177] These results are also consistent with our previous findings. In particular, FUS-R521C knock-in mice (C / C) did not show a severe ALS phenotype compared with controls, and there were no significant differences in behavior as shown by the rotarod test or survival curves ( Fig.15 Western analysis showed that TRIM72 was overexpressed in the cerebral cortex and spinal cord of mice (C / C) and rats (C / C). Figure 16-17 ). Further analysis showed that TRIM72 was specifically upregulated in neurons of FUS-R521C knock-in mice (C / C), but remained at low levels in wild-type (+ / +) ( Fig.18 ). In addition, loss of Trim72(- / -) function significantly shortened the residence time and lifespan of FUS-R521C knock-in mice in the rotarod test ( Fig.15 ). Therefore, upregulation of TRIM72 in neurons is a key factor in the FUS-R521C knock-in mice not showing severe ALS phenotypes. Therefore, the specific expression of TRIM72 in neurons can effectively treat ALS and other neuronal damage diseases.

[0178] Example 4 Protective effect of truncated TRIM72 in scAAV vector on cultured cells

[0179] TRIM72 protein contains a RING finger motif, a B-box domain, a coiled-coil domain, and a PRYSPRY domain ( Fig.19To investigate which domain of TRIM72 plays a role in protecting cells from oxidative stress, full-length TRIM72 or domain-impaired TRIM72 mutants were constructed and stably overexpressed in the 293FT cell line by lentiviral infection and puromycin selection ( Fig. 20 ). Cell viability after arsenite treatment was measured using CCK-8. We found that TRIM72 mutants with impaired coiled-coil domains and PRYSPRY domains abolished the protective effect of TRIM72, while TRIM72 mutants with impaired RING domains or B-box domains retained the same protective effect as the wild type ( Fig.21 ).

[0180] To further investigate which key domains of TRIM72 protein are required for protecting neurons, we constructed TRIM72 with damaged different domains and simultaneously overexpressed them in N2a cell lines by scAAV9 infection ( Fig. 22 ). CCK-8 was used to measure the cell viability after H2O2 treatment. In fact, the cell viability decreased after H2O2 treatment ( Fig.23 Notably, overexpression of either the coiled-coil PRYSPRY or PRYSPRY single domains, but not the coiled-coil single domain, was sufficient to protect cells from oxidative stress, suggesting that either the coiled-coil PRYSPRY truncation or the PRYSPRY single domains can elicit protection ( Fig.23 ).

[0181] Example 5 scAAV-TRIM72 prevents and alleviates brain damage and behavioral dysfunction in MACO / R mice

[0182] Here, the intraluminal middle cerebral artery occlusion / reperfusion (MCAO / R) model was used as a disease model of ischemic stroke. Fig.24 The dosing schedule shown was as follows: scAAV9-hSyn1-TRIM72 vector was administered at 4×10 14 vg / kg body weight was injected into mice via retro-orbital intravenous injection. TTC staining quantification showed that the infarct size of mice in the scAAV-TRIM72-treated group was reduced compared with the control group (data not shown). It was also shown that scAAV-TRIM72 pretreatment could prevent and alleviate the behavioral deficits in motor and sensory activity tasks in the neurological test and gel removal test 5 or 7 days after MCAO / R. The behavioral scores of the scAAV-TRIM72 pretreatment group were better ( Fig.25 A), and the presence of the tape could be sensed even sooner after MCAO / R. ( Fig.25 B).

[0183] Example 6 scAAV-TRIM72 prevents cell death after OGD

[0184] N2a cells experience increased oxidative stress and cell death when subjected to OGD. To investigate and confirm whether TRIM72 plays a key protective role after OGD treatment, full-length TRIM72 was overexpressed in the N2a cell line by scAAV9 infection (estimated multiplicity of infection (MOI): 10,000 vg / cell). Fig.26 ), and cell viability was measured using CCK-8. Indeed, cell viability decreased after OGD treatment, whereas overexpression of TRIM72 increased cell viability ( Fig.26 ).

[0185] Example 7. TRIM72, but not other TRIM proteins, protects cells from oxidative stress

[0186] To investigate whether other TRIM proteins could also elicit protective effects against oxidative stress, TRIM40 was overexpressed in the N2a cell line by scAAV9 infection (estimated multiplicity of infection (MOI): 10,000 vg / cell). Fig. 22 ). Cell viability after H2O2 treatment was measured using CCK-8. Notably, treatment with TRIM72, but not TRIM40, induced protection against oxidative stress, suggesting that only TRIM72, but not other TRIMs, protects cells from oxidative stress ( Fig.23 ).

[0187] Example 8. Key sites in TRIM72 protein that protect cells from oxidative stress

[0188] A series of Flag-tagged TRIM72 mutants were generated from the wild-type TRIM72 construct by point mutation: C14A (cysteine ​​at position 14 was substituted by alanine); C242A (cysteine ​​at position 242 was substituted by alanine).

[0189] Previous studies have determined that TRIM72 senses changes in the oxidative environment and forms oligomeric complexes to complete membrane repair. The cysteine ​​residue (C242) plays a key role in the formation of TRIM72 oligomers. In addition, the cysteine ​​residue (C14) is also essential for TRIM72 E3 ligase activity. Mutating C242 to alanine (C242A) blocked the protective effect of TRIM72, while its E3 ligase-inactive mutant (C14A) retained the same protective effect as the wild type ( Fig. 27 ). It can be concluded that TRIM72 protects cells from oxidative stress mainly by its oligomerization rather than its E3 ligase activity.

[0190] Although preferred 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. The present invention is not intended to be limited to the specific embodiments provided in the specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not meant to be interpreted in a restrictive manner. Without departing from the present invention, many variations, changes and substitutions will occur to those skilled in the art. 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 may be adopted when implementing the present invention. Therefore, it is contemplated that the present invention should 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 recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or a functional fragment thereof, wherein the recombinant AAV expression vector comprises a neuron-specific promoter.

2. The rAAV expression vector of claim 1, wherein the neuron-specific promoter comprises a human promoter.

3. The rAAV expression vector according to any one of claims 1-2, wherein the promoter is one or more selected from the following groups: an excitatory neuron-specific promoter, a cerebral neocortex and hippocampus excitatory neuron-specific promoter, a short neuron-specific promoter, a dopaminergic neuron-specific promoter, a glutamatergic neuron-specific promoter, a GABAergic neuron-specific promoter, a cholinergic neuron-specific promoter, and a serotonergic neuron-specific promoter.

4. The rAAV expression vector according to any one of claims 1-3, wherein the promoter is selected from the group consisting of human synapsin (hSyn), calcium / calmodulin-dependent kinase IIa (CamKⅡa), c-fos, methylated CpG binding protein 2 (Mecp2), neuron-specific enolase (NSE), somatostatin (SST), human vesicular GABA (γ-aminobutyric acid) transporter (hVGAT), choline acetyltransferase (ChAT), serotonin transporter (SERT) and tyrosine hydroxylase (TH).

5. The rAAV expression vector according to any one of claims 1-4, wherein the serotype of the AAV vector is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh, AAVDJ and AAVhull.

6. The rAAV expression vector according to any one of claims 1-5, wherein the rAAV is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

7. The rAAV expression vector according to any one of claims 1-6, wherein the TRIM72 protein is a human TRIM72 protein.

8. The rAAV expression vector according to any one of claims 1-7, wherein the TRIM72 protein comprises a full-length TRIM72 protein.

9. The rAAV expression vector according to any one of claims 1-8, wherein the TRIM72 protein comprises a wild-type TRIM72 protein.

10. The rAAV expression vector according to any one of claims 7-9, wherein the human TRIM72 protein comprises the amino acid sequence shown in SEQ ID NO:

2.

11. The rAAV expression vector according to any one of claims 1-10, wherein the TRIM72 protein comprises a TRIM72 truncated protein. 12 . The rAAV expression vector according to claim 11 , wherein the TRIM72 truncated protein comprises the PRYSPRY domain of the TRIM72 protein or a functional fragment thereof.

13. The rAAV expression vector of claim 11, wherein the PRYSPRY domain comprises amino acid sites 278aa-470aa of the TRIM72 protein.

14. The rAAV expression vector according to any one of claims 12-13, wherein the PRYSPRY domain comprises the amino acid sequence as shown in SEQ ID NO:

6.

15. The rAAV expression vector according to any one of claims 12-14, wherein the TRIM72 truncated protein further comprises a coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

16. The rAAV expression vector according to any one of claims 12-14, wherein the TRIM72 truncated protein does not comprise the coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

17. The rAAV expression vector according to any one of claims 15-16, wherein the coiled-coil domain comprises amino acid sites 135aa-232aa of the TRIM72 protein.

18. The rAAV expression vector of any one of claims 15-17, wherein the coiled-coil domain comprises the amino acid sequence shown in SEQ ID NO:

5.

19. The rAAV expression vector according to any one of claims 12-18, wherein the TRIM72 truncated protein further comprises the B-box domain of the TRIM72 protein or a functional fragment thereof.

20. The rAAV expression vector according to any one of claims 12-18, wherein the TRIM72 truncated protein does not comprise the B-box domain of the TRIM72 protein or a functional fragment thereof.

21. The rAAV expression vector according to any one of claims 19-20, wherein the B-box domain comprises amino acid sites 86aa-117aa of the TRIM72 protein.

22. The rAAV expression vector of any one of claims 19-21, wherein the B-box domain comprises the amino acid sequence shown in SEQ ID NO:

4.

23. The rAAV expression vector according to any one of claims 12-22, wherein the TRIM72 truncated protein further comprises the RING finger domain of the TRIM72 protein or a functional fragment thereof.

24. The rAAV expression vector according to any one of claims 12-22, wherein the TRIM72 truncated protein does not comprise the RING finger domain of the TRIM72 protein or a functional fragment thereof.

25. The rAAV expression vector according to any one of claims 23-24, wherein the RING finger domain comprises amino acid sites 14aa-56aa of the TRIM72 protein.

26. The rAAV expression vector of any one of claims 23-25, wherein the RING finger domain comprises the amino acid sequence shown in SEQ ID NO:

3.

27. The rAAV expression vector according to any one of claims 11-26, wherein the TRIM72 truncated protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 6, 7, 8, 9 and 11.

28. The rAAV expression vector according to any one of claims 1-27, wherein the TRIM72 protein or a variant or functional fragment thereof comprises an amino acid mutation at position C14.

29. The rAAV expression vector according to any one of claims 1-28, wherein the TRIM72 protein or a variant or functional fragment thereof comprises the amino acid mutation C14A.

30. The rAAV expression vector according to any one of claims 1-29, wherein the TRIM72 protein or its variant or functional fragment does not contain an amino acid mutation at position C242.

31. The rAAV expression vector of any one of claims 1-30, for use in protecting neurons by reducing oxidative stress.

32. The rAAV expression vector according to any one of claims 1-31, for use in preventing or treating a nervous system disease.

33. The rAAV expression vector according to any one of claims 1-32, for use in preventing or treating amyotrophic lateral sclerosis (ALS) or stroke.

34. A host cell comprising the rAAV expression vector according to any one of claims 1-33.

35. A pharmaceutical composition comprising the rAAV expression vector according to any one of claims 1-33 or the host cell according to claim 34, and a pharmaceutically acceptable adjuvant.

36. A method for protecting neurons in a subject, the method comprising administering to a subject in need thereof an effective amount of the rAAV expression vector according to any one of claims 1-33, the host cell according to claim 34, and / or the pharmaceutical composition according to claim 35.

37. A method for preventing and / or treating a nervous system disease, the method comprising administering to a subject in need thereof an effective amount of the rAAV expression vector according to any one of claims 1-33, the host cell according to claim 34 and / or the pharmaceutical composition according to claim 35.

38. The method of claim 37, wherein the neurological disease comprises amyotrophic lateral sclerosis (ALS) or stroke.

39. Use of the rAAV expression vector according to any one of claims 1-33, the host cell according to claim 34 and / or the pharmaceutical composition according to claim 35 in the manufacture of a medicament for preventing and / or treating a nervous system disease.

40. The use according to claim 39, wherein the neurological disease comprises amyotrophic lateral sclerosis (ALS) or stroke.

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