WO2018170796A1

WO2018170796A1 - Trim72 as potential therapeutic target for als through ubiquitinating mutant fus protein

Info

Publication number: WO2018170796A1
Application number: PCT/CN2017/077712
Authority: WO
Inventors: Yichang Jia; Xue ZHANG
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2017-03-22
Filing date: 2017-03-22
Publication date: 2018-09-27
Anticipated expiration: 2019-09-22
Also published as: CN110753557A; CN117982625A; CN110753557B

Abstract

Provided is a pharmaceutical composition comprising a scavenger, wherein the scavenger is used for clearing away mutant FUS. A method for treating or preventing or alleviating ALS is also provided.

Description

TRIM72 AS A POTENTIAL THERAPEUTIC TARGET FOR ALS THROUGH UBIQUITINATING THE MUTANT FUS PROTEIN

RELATED APPLICATIONS

TECHNICAL FIELD

Embodiments of the present disclosure generally relate to biological medicine, more particularly, to a pharmaceutical composition, use of the scavenger used for clearing away mutant FUS in the manufacture of a medicament and a method for treating or preventing or alleviating ALS.

BACKGROUND

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 which control voluntary muscles. Some also use the term "motor neuron disease" for a group of conditions of which ALS is the most common. ALS is characterized by stiff muscles, muscle twitching, and gradually worsening weakness due to muscles decreasing in size. This results in difficulty in speaking, swallowing, and eventually breathing. The cause is not known in 90％to 95％of cases. About 5–10％of cases are inherited from a person's parents. About half of these genetic cases are due to four specific genes, SOD1, TDP-43, FUS, and C9orf72. The diagnosis is based on a person's signs and symptoms with testing done to rule out other potential causes. No cure for ALS is known. A medication called riluzole may extend life by about two to three months. Non-invasive ventilation may result in both improved quality and length of life. The disease usually starts around the age of 60 and in inherited cases around the age of 50. The average survival from onset to death is two to four years. About 10％survive longer than 10 years. Most die from respiratory failure. In much of the world, rates of ALS are unknown. In Europe and the United States, the disease affects about two people per 100,000 per year.

Physical therapy plays a large role in rehabilitation for individuals with ALS. Specifically, physical and occupational therapists can set goals and promote benefits for individuals with ALS by delaying loss of strength, maintaining endurance, limiting pain, improving speech and swallowing, preventing complications, and promoting functional independence. Occupational therapy and special equipment such as assistive technology can also enhance people's independence and safety throughout the course of ALS. Gentle, low-impact aerobic exercise such as performing activities of daily living, walking, swimming, and stationary bicycling can strengthen unaffected muscles, improve cardiovascular health, and help people fight fatigue and depression. Range of motion and stretching exercises can help prevent painful spasticity and shortening (contracture) of muscles. Physical and occupational therapists can recommend exercises that provide these benefits without overworking muscles. They can suggest devices such as ramps, braces, walkers, bathroom equipment (shower chairs, toilet risers, etc. ) , and wheelchairs that help people remain mobile. Occupational therapists can provide or recommend equipment and adaptations to enable ALS people to retain as much safety and independence in activities of daily living as possible.

Moreover, scientists have also been working on the study of ALS pathogenic genes to provide more and more effective therapeutic targets with ALS.

Emerging evidence suggests that RNA metabolism abnormalities, including gain-of-function of RBPs, loss-of-function of RNA helicases, and misprocessing of pre-mRNA splicing, lead to neurodegenerative diseases. Among them, mutations in genes encoding two structurally similar RBPs, TDP-43 and FUS, have been associated with ALS, the neurodegenerative disorders sharing genetic and pathological overlaps. More strikingly, the ubiquitin-positive and mislocalized TDP-43 and FUS were found in a large proportion of ALS, even though many of them do not carry these two RBP mutations, underscoring the critical roles of RBP dysfunction in the pathogenesis. However, the disease mechanisms underlying neurodegeneration caused by dysfunction of these RBPs are still largely unknown.

Recently, TDP-43 and FUS, among many other RBPs, which contain low complexity domain (LCD) also known as intrinsically disordered region (IDR) , have been identified in a non-translating cytoplasmic mRNA complex, also known as stress granule (SG) , a structure that often appears under stress conditions to temporarily cease the cytosolic mRNA translation initiation. Like TDP-43 and FUS, mutations in those RBP genes have also been linked to neurodegenerative diseases, including ALS and FTD. In ALS and FTD patient specimen, the mislocalized TDP-43 co-localized with SG markers, and in cultured cells, overexpressed mutant TDP-43 and FUS were found in the SG induced by stresses. The mutations in VCP, a gene encodes protein involved in autophagic clearance of SGs, have been also closely associated with ALS and FTD. Taken together, these data argued that the misprocessing of SG may contribute the disease etiology. However, how the endogenous level of wild type and mutant RBPs behave and function in the SG formation and phase change, especially in the disease target neurons facing the stress challenge, is unclear, partially due to the emergent needs for rational cell and animal models.

Therefore, the method for treating ALS need to be further explored and improved urgently.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent, or to provide a consumer with a useful commercial choice.

Embodiments of a first broad aspect of the present disclosure provide a pharmaceutical composition. According the embodiments, wherein the pharmaceutical composition comprising scavenger, wherein the scavenger is used for clearing away mutant FUS. The inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model. The pharmaceutical composition disclosed here can treat or prevent or alleviate ALS effectively.

According to the embodiment of the invention, the pharmaceutical composition may further include at least one of the following additional technical features.

According the embodiments, the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation. The pharmaceutical composition disclosed here can treat or prevent or alleviate ALS more effectively and safely.

According the embodiments, the scavenger is Trim72 or the nucleic acid encoding Trim72. The inventors of the patent surprisingly discover that Trim72 can target the mutant FUS for ubiquitination and proteasome-mediated degradation. The pharmaceutical composition comprising Trim72 or the nucleic acid encoding Trim72 can treat or prevent or alleviate ALS more effectively and safely.

According the embodiments, wherein the mutant FUS is human ALS mutant FUS. The pharmaceutical composition disclosed here is more suitable for human.

According the embodiments, wherein the mutant FUS has mutation in FUS nuclear localization signal. Optionally, wherein the amino acid site of the nuclear localization signal of FUS is 513-526 (NP_004951.1) . Optionally, wherein the mutant FUS is inclined to localizing in cytosol. The mutant FUS disclosed herein has close relationship with ALS and is key pathogenic mutant. Pharmaceutical composition comprising the scavenger used for clearing away the mutant FUS described above will be used for treating or preventing or alleviating ALS more effectively.

According the embodiments, the pharmaceutical composition disclosed here is used for treating or preventing or alleviating ALS. As described above, the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model effectively. The pharmaceutical composition disclosed here can treat or prevent or alleviate ALS effectively.

According the embodiments, the scavenger is in at least one of the following forms: (a) protein or functional fragment； (b) Nucleic acid molecule encoding (a) ； (c) construct having (b) . As described above, the scavenger can be used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation, such as Trim72. Trim72 or the functional fragment of Trim72 used for ubiquitination and proteasome-mediated degradation of mutant FUS or the Nucleic acid molecule encoding Trim72 or the functional fragment or the construct having the Nucleic acid molecule can be used as scavenger for clearing away mutant FUS effectively.

According the embodiments, wherein the nucleic acid molecule comprising at least one of DNA and RNA. There is no special restriction on the form of the nucleic acid molecule According the embodiments, DNA or RNA has double or single train encoding protein, such as Trim72 or the functional fragment thereof can be used as mutant FUS scavenger effectively.

According the embodiments, wherein the construct comprising at least one of plasmid and virus. There is no special restriction on the form of the construct. As long as the construct can carry the nucleic acid molecule encoding protein, such as Trim72 or the functional fragment thereof and the protein, such as Trim72 or the functional fragment thereof can express in suitable environment. According the embodiments, the construct can be at least one of plasmid and virus.

According the embodiments, the pharmaceutical composition further comprising pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ. And then, the pharmaceutical composition disclosed here can treat ALS more effectively.

According the embodiments, the acceptable carrier is drugs, toxins, cytokines, radioactive elements, carrier proteins, enzymes, lectins, fluorescent quantum dots, or high absorption coefficient of chromophore. The acceptable carrier disclosed above can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ more effectively.

Embodiments of a second broad aspect of the present disclosure provide use of the scavenger used for clearing away mutant FUS in the manufacture of a medicament for treating or preventing or alleviating ALS. As described above, the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model. The scavenger is used in the manufacture of a medicament and the medicament can treat or prevent or alleviate ALS effectively.

According to the embodiment of the invention, the use may further include at least one of the following additional technical features.

According the embodiments, the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation. The medicament disclosed herein can treat or prevent or alleviate ALS more effectively and safely.

According the embodiments, the scavenger is Trim72 or the nucleic acid encoding Trim72. The inventors of the patent surprisingly discover that Trim72 can target the mutant FUS for ubiquitination and proteasome-mediated degradation. The medicament prepared with Trim72 or the nucleic acid encoding Trim72 can treat or prevent or alleviate ALS more effectively and safely.

According the embodiments, wherein the mutant FUS is human ALS mutant FUS. The medicament disclosed here is more suitable for human.

According the embodiments, wherein the mutant FUS has mutation in FUS nuclear localization signal. Optionally, wherein the amino acid site of the nuclear localization signal of FUS is 513-526 (NP_004951.1) . Optionally, wherein the mutant FUS is inclined to localizing in cytosol. The mutant FUS disclosed herein has close relationship with ALS and is key pathogenic mutant. medicament prepared with the scavenger used for clearing away the mutant FUS described above will be used for treating or preventing or alleviating ALS more effectively.

According the embodiments, the medicament further comprising pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ. And then, the medicament disclosed here can treat ALS more effectively.

Embodiments of a third broad aspect of the present disclosure provide a method for treating or preventing or alleviating ALS, wherein the method comprising administration of the scavenger used for clearing away FUS protein mutants to a patient in need. The inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability. Administration of the scavenger used for clearing away FUS protein mutants to a patient in need, such a ALS patient and treat or alleviate the disease or prevent recurrence effectively.

According to the embodiment of the invention, the method may further include at least one of the following additional technical features.

According the embodiments, the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation. The method disclosed herein is more effective and safety.

According the embodiments, the scavenger is Trim72 or the nucleic acid encoding Trim72. The inventors of the patent surprisingly discover that Trim72 can target the mutant FUS for ubiquitination and proteasome-mediated degradation. The method disclosed herein is more effectively and safely.

According the embodiments, wherein the mutant FUS is human ALS mutant FUS. The method disclosed herein is more suitable for human.

According the embodiments, wherein the mutant FUS has mutation in FUS nuclear localization signal. Optionally, wherein the amino acid site of the nuclear localization signal of FUS is 513-526 (NP_004951.1) . Optionally, wherein the mutant FUS is inclined to localizing in cytosol. The mutant FUS disclosed herein has close relationship with ALS and is key pathogenic mutant. Administration of the scavenger used for clearing away FUS protein mutants described above to a patient in need will be more effective in treating or preventing or alleviating ALS.

According the embodiments, wherein the scavenger is supplied in the form of a pharmaceutical composition, and the pharmaceutical composition further comprising pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ. And then, the pharmaceutical composition disclosed here can treat ALS more effectively.

The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The Figures and the detailed description which follow more particularly exemplify illustrative embodiments.

Additional aspects and advantages of embodiments of present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference to the accompanying drawings, in which:

Fig. 1 shows generation of mFUS-R513C KI mutant mouse for ALS by CRISPR/Cas9,

wherein,

A shows alignment of last 12 amino acids of FUS in different mammal species. The amino acids are highly conserved from rodent to human. The NCBI accession number for H. sapiens (NP_004951.1) , B. Taurus (XP_005224884.1) , R. norvegicus (NP_001012137) , and M. musculus (NP_631888.1) . The human FUS sequences are used as a reference for amino acid positioning. The R521 is labeled with asterisk；

B shows the mouse genome structure of FUS gene. The mouse FUS R513 corresponds to human R521. Two-nucleotide mutations labeled in red were introduced into mouse FUS locus 2bp upstream of PAM site (3 capital letters in dark black) . The underlined genomic DNA sequences correspond to gRNA sequences. A novel PstI cutting site was built into the locus for future genotyping；

C shows DNA chromatograms；

D shows gel electrophoresis. The PCR products including the insertion were treated with PstI. +/+, wild type C57BL/6J mouse, C/+ and C/C, hetero-and homozygous for mFUS-R513C KI mice ； and

E shows the expression of FUS in mouse various tissues. The tissues from wild type (+/+) and mFUS-R513C KI mutant (C/C) mice at 8 months of age were blotted with a homemade FUS antibody. The GAPDH was used as loading control. Hippo. , hippocampus； S. C. spinal cord.

Fig. 2 shows the aged mFUS-R513C mutant KI mice showed motor decline and the reduced number of motor nerve fibers,

wherein,

A shows the travel distance measured by Open Field (TopScan behavioral analysis system, CleverSys. , USA) and the significant decline of travel distance was documented in aged hetero-(C/+) and homozygous (C/C) KI mutant (6.5 months) , but not in the younger (4 months) mutant animals. 4 months, n＝9 (+/+ and C/+, respectively) , male. 6.5 months, n＝25 (+/+) , n＝16 (C/+) , n＝12 (C/C) , male. In Open field test, the standing time (seconds in 10-minute interval) on the hind legs in aged (6.5 months) mice was calculated. The value are presented as mean ±SEM. *p <0.05, **p <0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance；

B shows the standing time was significantly reduced in the heterozygous (C/+) and homozygous (C/C) KI mutant groups, compared to the wild type (+/+) group. The value are presented as mean ± SEM. *p <0.05, **p <0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance；

C and D show rotarod performance (4-day interval, Med Associates Inc. , USA) was carried out in wild type (+/+) , the hetero- (C/+) and/or homozygous (C/C) KI mutant mice at 4 (C) and 6.5 (D) months of age. The stay time on the rotarod was significantly decreased in the aged KI mutant (C/+ and C/C, 6.5 months) mice, but not in the younger (4 months) mutant animals. 4 months, n＝8 (+/+ and C/+, respectively) , male. 6.5 months, n＝25 (+/+) , n＝14 (C/+) , n＝15 (C/C) , male. The value are presented as mean ± SEM. *p <0.05, **p <0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance；

E shows toluidine blue-stained cross sections of femoral nerve motor branches from wild type (+/+) and KI mutant (C/C) animals at 8 months of age. Axon degeneration (arrow) was shown in high magnification image. Scale bar, 50 μm in low and 100 μm in high magnification images. The diameter of a red dot in low magnification image is 5 μm, which is used for the fiber size measurement. The value are presented as mean ± SEM (n＝3) . **p <0.01 (t-test, SPSS) ； and

F shows the big size (diameter ≥ 5 μm) , but not the small size (< 5 μm) , nerve fiber numbers of femoral motor branches were significantly reduced in the KI mutant (C/C, 8 months) animals, compared to their wild type (+/+) littermate controls. In (A, B, C and D) , the value are presented as mean ± SEM. *p <0.05, **p <0.01 (one-way ANOVA, SPSS) . NS, no statistical significance； and

Fig. 3 shows Trim72, encoding an E3 ligase, was upregulated in the mFUS-R513C KI spinal cords and the E3 ligase targeted mutant FUS for degradation,

wherein,

A shows the differentially expressed genes in the spinal cords at 1.5 and 7 month of age between the wild type and KI mutant (C/C) genotypes were identified by RNA-seq. Among the limited numbers of differentially expressed gene (7 genes for 1.5 month, and 13 genes for 7 months) , Trim72 was the only differentially expressed gene at both time points；

B shows the RPKM values (mean ± SEM) of indicated genes at two age points between the genotypes were plotted. N＝3, male；

C shows experimental strategy for detecting the interaction of Trim72 with the mutant FUS (hFUS-R521C) . Upper, the inducible expression system for Myc-BirA*-Trim72. BirA*, a previously reported biotin ligase (doi/10.1073/pnas. 1406459111) . Lower, the experimental procedure；

D shows the HEK293 cells were infected with Myc-BirA*-Trim72 expression lentivirus. After the doxycycline (Dox) administration, the expression of Myc-BirA*-Trim72 was induced, and the biotinylation of hFUS-R521C was detected by streptavidin M-280 beads. However, the interaction was largely attenuated when the MG132 was removed from the culture medium, suggesting that Trim72 ubiquitinate the ALS-associated hFUS-R521C and result in its degradation；

E and F show compared to hFUS-R521C, wild type hFUS and hFUS truncation (Δ513-526) had much less interactions with Trim72. The interactions were quantitated by relative Flag intensity (the band density ratio between the biotinylated and total input) from at least three independent experiments. The value are presented as mean ± SEM. **p <0.01 (t-test, SPSS) .

DETAILED DESCRIPTION

Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

In addition, terms such as “first” and “second” are used herein for purposes of description and are not intended to indicate or imply relative importance or significance.

pharmaceutical composition

The present invention provides a pharmaceutical composition comprising scavenger, wherein the scavenger is used for clearing away mutant FUS. According to the specific examples of the present invention, the pharmaceutical composition can further comprise pharmaceutically acceptable excipient, carrier, adjuvant, solvent and a combination thereof.

The present invention provides a method of treating, preventing or ameliorating a disease or disorder, comprising administrating a safe and effective amount of a combination of drugs containing the scavenger and one or more therapeutic active agents. Among them, the combination of drugs comprises one or more additional drugs for treatment ALS. Other drugs for treatment of ALS are not limited to: Riluzole or antisense oligoes against mutant FUS RNA.

According the embodiments, the scavenger is in at least one of the following forms: (a) protein or functional fragment； (b) Nucleic acid molecule encoding (a) ； (c) construct having (b) . AAV-mediated gene therapy is also included herein. Recently, AAV-mediated gene therapy has been used for delivering the interested genes for therapy. Following reasons make the AAV-mediated gene therapy become promising for delivering the scavenger gene into the patients: 1) the AAV apparent lack of pathogenicity and also present very low immunogenicity； 2) not like retroviruses, random integration of AAV DNA into the host genome occurs at very low frequency； 3) for non-dividing cell, like neuron, AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus, and these concatemers remain intact for the life of the host cell； 4) one limitation for AAV delivery is the cloning capacity, which is limited to about 4.8kb for the virial vector. However, the human Trim72 recombinant protein is about 477 a. a. (ENST00000322122.7) , and the DNA size for expressing the scavenger is suitable for AAV-mediated gene delivery. Therefore, due to these advantages, AAV-mediated gene therapy will be feasible means to delivery target human Trim72 into the patients. The single stranded (ss) and self-complementary (sc) AAV9 were used to deliver DNA encoding the SMN protein for SMA patients. Therefore, the similar means will be used for the scavenger. Moreover， the amount of the scavenger in the pharmaceutical composition disclosed herein refers to an amount which can be effectively clearing away mutant FUS. The dosage of active ingredient in the compositions of this invention may be varied, however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The active ingredient may be administered to patients (animals or human) in need of such treatment in dosage that will provide optimal pharmaceutical efficacy. The selected dosage upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. The dosage will vary from patient to patient depending upon the nature and severity of disease, the patient's weight, special diet then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize. The dosage range will generally be about 0.5 mg to 1.0 g per patient per day which may be administered in single or multiple doses. In one embodiment, the dosage range will be about 0.5 mg to 500 mg per patient per day； in anther embodiment about 0.5 mg to 200 mg per patient per day； and in yet another embodiment about 5 mg to 50 mg per patient per day.

The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein a safe and effective amount of a compound of Formula (I) disclosed herein can be extracted and then given to the patient, such as with powders or syrups. Generally, dosage levels of between 0.0001 to 10 mg/kg of body weight daily are administered to the patient to obtain effective clearing away mutant FUS. The pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a safe and effective amount of the scavenger disclosed herein. When prepared in unit dosage form, the pharmaceutical compositions of the invention commonly contain from about 0.5 mg to 1 g, or 1 mg to 700 mg, or 5 mg to 100 mg, of the compound.

When the pharmaceutical compositions of the present invention also contain one or more other active ingredients, in addition to a compound of the present invention, the weight ratio of the compound of the present invention to the second active ingredient may be varied and depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the present invention is combined with another agent, the weight ratio of the compound of the present invention to the other agent will generally range from about 1000: 1 to about 1: 1000, such as about 200: 1 to 1: 200. Combinations of a compound of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.

"Pharmaceutically acceptable excipient" as used herein means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled, such that interactions which would substantially reduce the efficacy of the compound of the invention when administered to a patient and would result in pharmaceutically unacceptable compositions are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.

Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting the compound of the present invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.

Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweetners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention. In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company) , The Handbook of Pharmaceutical Additives (Gower Publishing Limited) , and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press) .

In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams &Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, the contents of each of which is incorporated by reference herein, are disclosed various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component (s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention.

The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company) .

Therefore, another aspect of the present invention is related to a method for preparing a pharmaceutical composition. The pharmaceutical composition contains the compound disclosed herein and pharmaceutically acceptable excipient, carrier, adjuvant, vehicle or a combination thereof, the method comprises mixing various ingredients. The pharmaceutical composition containing the compound disclosed herein can be prepared at for example environment temperature and under barometric pressure.

The compound of the invention will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration. For example, dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixers, suspensions, solutions, emulsions, sachets, and cachets； (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution； (3) transdermal administration such as transdermal patches； (4) rectal administration such as suppositories； (5) inhalation such as aerosols, solutions, and dry powders； and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

In one embodiment, the compounds disclosed herein can be prepared to oral. In the other embodiment, the compounds disclosed herein can be prepared to inhalation. In the still other embodiment, the compounds disclosed herein can be prepared to nasal administration. In the yet other embodiment, the compounds disclosed herein can be prepared to transdermal administration. In the still yet other embodiments, the compounds disclosed herein can be prepared to topical administration.

The pharmaceutical compositions provided herein may be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets. Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach. Enteric-coatings include, but are not limited to, fatty acids, fats, phenylsalicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation. Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Film coating imparts the same general characteristics as sugar coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press-coated or dry-coated tablets.

The tablet dosage forms may be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled-release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.

The pharmaceutical compositions provided herein may be provided as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, or calcium alginate. The hard gelatin capsule, also known as the dry-filled capsule (DFC) , consists of two sections, one slipping over the other, thus completely enclosing the active ingredient. The soft elastic capsule (SEC) is a soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol. The soft gelatin shells may contain a preservative to prevent the growth of microorganisms. Suitable preservatives are those as described herein, including methyl-and propyl-parabens, and sorbic acid. The liquid, semisolid, and solid dosage forms provided herein may be encapsulated in a capsule. Suitable liquid and semisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Pat. Nos. 4,328,245； 4,409,239； and 4,410,545. The capsules may also be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.

The pharmaceutical compositions provided herein may be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in-water or water-in-oil. Emulsions may include a pharmaceutically acceptable non-aqueous liquids or solvent, emulsifying agent, and preservative. Suspensions may include a pharmaceutically acceptable suspending agent and preservative. Aqueous alcoholic solutions may include a pharmaceutically acceptable acetal, such as a di (lower alkyl) acetal of a lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal； and a water-miscible solvent having one or more hydroxy groups, such as propylene glycol and ethanol. Elixirs are clear, sweetened, and hydroalcoholic solutions. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative. For a liquid dosage form, for example, a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.

Other useful liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient (s) provided herein, and a dialkylated mono-or poly-alkylene glycol, including, 1, 2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol. These formulations may further comprise one or more antioxidants, such as butylated hydroxytoluene (BHT) , butylated hydroxyanisole (BHA) , propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.

The pharmaceutical compositions provided herein for oral administration may be also provided in the forms of liposomes, micelles, microspheres, or nanosystems. Miccellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.

The pharmaceutical compositions provided herein may be provided as non-effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form. Pharmaceutically acceptable carriers and excipients used in the non-effervescent granules or powders may include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.

Coloring and flavoring agents can be used in all of the above dosage forms.

The scavenger disclosed herein can also be coupled to soluble polymers as targeted medicament carriers. Such polymers may encompass polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidophenol, polyhydroxyethylaspartamidophenol or polyethylene oxide polylysine, substituted by palmitoyl radicals. The compounds may furthermore be coupled to a class of biodegradable polymers which are suitable for achieving controlled release of a medicament, for example polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

The pharmaceutical compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.

The pharmaceutical compositions provided herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.

The pharmaceutical compositions provided herein may be administered parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

The pharmaceutical compositions provided herein may be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, Remington: The Science and Practice of Pharmacy, supra) .

The pharmaceutical compositions intended for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

Suitable aqueous vehicles include, but are not limited to, water, saline, physiological saline or phosphate buffered saline (PBS) , sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection. Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, and palm seed oil. Water-miscible vehicles include, but are not limited to, ethanol, 1, 3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400) , propylene glycol, glycerin, N-methyl-2-pyrrolidone, N, N-dimethylacetamide, and dimethyl sulfoxide.

Suitable antimicrobial agents or preservatives include, but are not limited to, phenols, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride) , methyl-and propyl-parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffering agents include, but are not limited to, phosphate and citrate. Suitable antioxidants are those as described herein, including bisulfite and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents are those as described herein, including sodium carboxymethylcelluose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80 and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, including α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, and sulfobutylether 7-β-cyclodextrin (

CyDex, Lenexa, Kans. ) .

The pharmaceutical compositions provided herein may be formulated for single or multiple dosage administration. The single dosage formulations are packaged in an ampoule, a vial, or a syringe. The multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.

In one embodiment, the pharmaceutical compositions are provided as ready-to-use sterile solutions. In another embodiment, the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle prior to use. In yet another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile suspensions. In yet another embodiment, the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use. In still another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile emulsions.

The pharmaceutical compositions may be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot. In one embodiment, the pharmaceutical compositions provided herein are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.

Suitable inner matrixes include polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.

Suitable outer polymeric membranes include polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.

In other aspect, the pharmaceutical composition of the invention is prepared to a dosage form adapted for administration to a patient by inhalation, for example as a dry powder, an aerosol, a suspension, or a solution composition. In one embodiment, the invention is directed to a dosage form adapted for administration to a patient by inhalation as a dry powder. In one embodiment, the invention is directed to a dosage form adapted for administration to a patient by inhalation as a dry powder. Dry powder compositions for delivery to the lung by inhalation typically comprise a compound disclosed herein or a pharmaceutically acceptable salt thereof as a finely divided powder together with one or more pharmaceutically-acceptable excipients as finely divided powders. Pharmaceutically-acceptable excipients particularly suited for use in dry powders are known to those skilled in the art and include lactose, starch, mannitol, and mono-, di-, and polysaccharides. The finely divided powder may be prepared by, for example, micronisation and milling. Generally, the size-reduced (e. g. micronised) compound can be defined by a D50 value of about 1 to about 10 microns (for example as measured using laser diffraction) .

Aerosols may be formed by suspending or dissolving a compound disclosed herein or a pharmaceutically acceptable salt thereof in a liquified propellant. Suitable propellants include halocarbons, hydrocarbons, and other liquified gases. Representative propellants include: trichlorofluoromethane (propellant 11) , dichlorofluoromethane (propellant 12) , dichlorotetrafluoroethane (propellant 114) , tetrafluoroethane (HFA-134a) , 1, 1-difluoroethane (HFA-152a) , difluoromethane (HFA-32) , pentafluoroethane (HFA-12) , heptafluoropropane (HFA-227a) , perfluoropropane, perfluorobutane, perfluoropentane, butane, isobutane, and pentane. Aerosols comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof will typically be administered to a patient via a metered dose inhaler (MDI) . Such devices are known to those skilled in the art.

The aerosol may contain additional pharmaceutically-acceptable excipients typically used with MDIs such as surfactants, lubricants, cosolvents and other excipients to improve the physical stability of the formulation, to improve valve performance, to improve solubility, or to improve taste.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the patient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3 (6) , 318 (1986) .

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Ointments, creams and gels, may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agent and/or solvents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil, or a solvent such as polyethylene glycol. Thickening agents and gelling agents which may be used according to the nature of the base include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethylene and cellulose derivatives, and/or glyceryl monostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents, suspending agents or preservatives.

Topical preparations may be administered by one or more applications per day to the affected area； over skin areas occlusive dressings may advantageously be used. Continuous or prolonged delivery may be achieved by an adhesive reservoir system.

In one embodiment, the therapies disclosed herein comprise administrating a safe and effective amount of the compound or the pharmaceutical composition containing the scavenger used for clearing away mutant FUS to patients in need. Each example disclosed herein comprises the method of treating the diseases above comprising administrating a safe and effective amount of the compound of the inhibitor or the pharmaceutical composition containing the compound of the inhibitor to patients in need.

In one embodiment, the pharmaceutical composition thereof may be administered by any suitable route of administration, including both systemic administration and topical administration. Systemic administration includes oral administration, parenteral administration, transdermal administration and rectal administration. Parenteral administration refers to routes of administration other than enteral or transdermal, and is typically by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion. Topical administration includes application to the skin as well as intraocular, otic, intravaginal, inhaled and intranasal administration. In one embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered orally. In another embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered by inhalation. In a further embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered intranasally.

In one embodiment, the pharmaceutical composition thereof may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. In one embodiment, a dose is administered once per day. In a further embodiment, a dose is administered twice per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for the compound of the invention or the pharmaceutical composition thereof depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the skilled artisan. In addition, suitable dosing regimens, including the duration such regimens are administered, for the compound of the invention or the pharmaceutical composition thereof depend on the disorder being treated, the severity of the disorder being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual patient's response to the dosing regimen or over time as individual patient needs change.

The pharmaceutical composition of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agents. The compounds of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents.

The pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredients for a subject of about 50-70 kg, preferably about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg or about 1-50 mg of active ingredients. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

The above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof. The compounds of the present invention can be applied in vitro in the form of solutions, e.g., preferably aqueous solutions, and in vivo either enterally or parenterally, advantageously intravenously, e.g., as a suspension or in aqueous solution.

In one embodiment, a therapeutically effective dosage of the pharmaceutical composition disclosed herein from about 0.1 mg to about 2,000 mg per day. The pharmaceutical compositions should provide a dosage of from about 0.1 mg to about 2000 mg of the compound. In a special embodiment, pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 2,000 mg, about 10 mg to about 1,000 mg, about 20 mg to about 500 mg, or about 25 mg to about 250 mg of the active ingredient or a combination of essential ingredients per dosage unit form. In a special embodiment, pharmaceutical dosage unit forms are prepared to provide about 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg or 2000 mg of the active ingredient.

Use of the scavenger used for clearing away mutant FUS

Embodiments of a second broad aspect of the present disclosure provide use of the scavenger used for clearing away mutant FUS in the manufacture of a medicament for treating or preventing or alleviating ALS. The inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model. The scavenger is used in the manufacture of a medicament and the medicament can treat or prevent or alleviate ALS effectively.

Method for treating or preventing or alleviating ALS

As used herein, the terms "administration of" and "administering a" the scavenger should be understood to mean providing the scavenger to an individual in need thereof. It is recognized that one skilled in the art can treat a patient presently afflicted with ALS disease with an effective amount of scavenger used for clearing away mutant FUS.

The following examples are provided so that the invention might be more fully understood. However, it should be understood that these embodiments merely provide a method of practicing the present invention, and the present invention is not limited to these embodiments.

The related methods are described as follows:

Materials and Methods:

Mice and mouse behavior tests.

For the generation of the FUS-R513C KI mouse line, we PCR amplified the target sequences from the C57BL/6J (JAX, Stock No. 000664) mouse genomic DNA. The donor DNA fragment contains the R513C mutation (tcg to ctg) , and the flanking left and right homology arms (～1kb) , respectively. The donor DNA, gRNA (gcgagcacagacaggatcgcAGG, PAM site capitalized) , and Cas9 mRNA were injected into C57BL/6J embryos. The injected embryos were transferred into the oviduct ampulla of the pseudo-pregnant ICR (JAX, Stock No. 009122) female recipients. The right genotype offsprings were backcrossed to C57BL/6J for at least five generations to establish the line.

For Open Field behavior test, the single animal was placed in the center of an open field area (60 × 60 cm) and tracked with multiple parameters, including total distance, average speed, and distance traveled in the center region, by TopScan behavioral analysis system (CleverSys., USA) in a 10-minute interval. The rotarod performance was measured by an automated system (Med Associates, Inc) . In brief, the animal was placed on an accelerating spindle (4-40rpm) in 5 minutes per trail and 3 consecutive trials per day. A 20-minute break was set in between each trial. The test was repeated for four days. The fall time from the spindle was auto-calculated by the system when the mouse fell off the spindle within the 5-minute interval. The stay time was calculated by subtraction of the fall time from the 5 minutes, and the mean value of the stay time from 3 consecutive trials per day was used for statistical analysis.

The animal facility at Tsinghua university has been fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) since 2014. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Tsinghua university based on Guide for the Care and Use of Laboratory Animals (Eighth Edition, NHR) . The C57BL/6J and ICR mice were purchased from Charles River Laboratories, Beijing, China.

Femoral nerve dissection and axon count.

Dissection of femoral nerves was performed as described previously. The femoral nerve was exposed in the sacrificed mice and briefly fixed (2％glutaraldehyde/2％paraformaldehyde in 0.1 M cacodylate buffer) before dissection. The isolated nerves were postfixed overnight in the same fixative. Dissected nerves were processed for plastic embedding and transmission electron microscopy by standard procedures. Nerve cross sections were stained with toluidine blue and examined by light microscopy.

RNA-seq and deferentially expressed gene identification.

The spinal cord total RNAs were extracted by TRIzol (Invitrogen) , and the cDNA library preparation for high-throughput sequencing (Illumina HiSeq 2500) was prepared as described in the manual (NEBNext Ultra) . Reads were aligned to mouse reference genome mm10 by HISAT2 with known splice sites from Ensembl database as a parameter. Differentially expressed genes were called by DESeq2 .

Protein-protein interaction.

For the protein-protein interaction, a previous lentivirus-based inducible system (Addgene, #50661) was modified for doxycycline-induced expression of Myc-BirA*-mTrim72 in HEK293 cells. After blasticidin (Sigma) selection, the infected cells were transiently transfected with plasmids (pCMV-3Tag, Stratagene) expressing human wild type FUS, FUS-R521C, and FUS 513-526 truncation. Doxycycline (1μg/ml, Sigma) were added in the cells to induce the expression of Myc-BirA*-mTrim72 8 hours after transfection. To block the protein degradation, we added the MG132 (20μM, Selleck) in the cells 24 hours after transfection, and harvested the cells 8 hours later for biotinylated-protein purification (streptavidin Dynabead M-280, Invitrogen) . After wash, the IP or immobilized products were probed with anti-Flag (mouse abmart, China) or anti-Myc (mouse abmart, China) antibodies.

EXAMPLES

In the past, significant efforts have been made to generate animal models for ALS by overexpressing recombinant DNA carrying mutations found in ALS families. Although those transgenic animal models significantly gain our insights into the disease mechanisms, people may argue the potential artifacts driven by ectopic overexpression of mutant proteins in the disease models, like 40-time overexpression of human SOD1-G93A in mouse, one of the most popular ALS mouse models. Similar transgenic strategy was applied for generation of TDP-43 and FUS ALS models. However, overexpression of wild type TDP-43 and FUS also generate similar motor phenotypes as that of the mutant transgenes in many animal species, including fly, mouse, and rat, suggesting the potential artifacts in pathogenesis in these models. Besides, the transgenic strategy has other caveats, including uncertain genome insertion sites, unstable copy numbers, potential disruption of genome integrity, ectopic expression pattern driven by exogenous promoter, and lack of endogenous splicing regulation.

In order to avoid the caveats, inventors employed the CRISPR-Cas9-based knock-in (KI) approach. There are three reasons for us to choose the FUS-R521C to generate KI ALS mouse model. First, the R521C mutation locates in FUS C-terminal, a non-classical PY nuclear localization signal (NLS) , in which clusters more than half of ALS-associated FUS mutations. Among these mutations, R521C is the most frequent one and accounts for 30％of the total. Second, the R521C mutation has been found in both familiar and sporadic ALS patients with high disease penetration. Third, the FUS R521C transgenic mouse and rat models present strong ALS-like pathological features.

To generate the KI mouse line, inventors first analyzed the FUS protein conservation across species (Fig. 1A) . The C-terminal NLS encoded by the last exon of mouse FUS is identical to that of human (515GEHRQDRRERPY526) . The mouse corresponding position for human R521 is R513. Downstream of mouse R513 triplet codon, a PAM site for Cas9 recognition appears (Fig. 1B) . The correct genome insertion and germ line transmission were confirmed by breeding the KI founders with C57BL/6J wild type mice (Fig. 1C and 1D) . Meanwhile, inventors backcrossed the KI mutant mouse to C57BL/6J at least 5 generations, in order to reduce the potential off-target effect introduced by CRISPR/Cas9. Using FUS antibody, inventors observed that the expression level of mutant FUS was slightly upregulated in spinal cord (Fig. 1E) , although the mRNA level of mutant FUS was comparable to that of wild type (data not shown) , suggesting that the R513C mutation may increase the protein stability and/or decrease the turnover rate in the target tissue.

Unlike early-onset motor disability and death seen in previously reported wild type and mutant FUS transgenic mice, inventors failed to observe progressive paralysis and shorter life span in our aged KI animals (data not shown) . However, a slight decline of spontaneous locomotor activity detected by open field assay was seen in FUS KI heterozygous group at 4 months of age, and significant motor decline was documented in the both hetero-and homozygous mutant mice at 6.5 months of age (Fig. 2A) . The motor ability decline was further confirmed by the performance on rotarod in the mutant groups. Although the stay time on the rotarod was slightly declined in the 4-month old FUS KI heterozygous group (Fig. 2B) , by 6.5 months of age, both hetero-and homozygous mutant mice had shown further drop of stay time on rotarod, significantly at

day

2 and 3 in comparison with wild type littermates (Fig. 2C) .

The motor ability decline in the aged KI mice was further echoed by the motor axon reduction. At 8 months of age, the degenerating motor axons of femoral nerve were observed in the homozygous KI mutant animals (Fig. 2D) . By counting the axon number, inventors found that the numbers of large-diameter (≥5μm) fibers, but not the small ones (<5μm) , were significant reduced in the homozygous KI mutant mice (Fig. 2D and 2E) , suggesting that the fibers with fast conduction velocity are majorly affected by the expression of mutant FUS.

To further tackle the disease mechanisms, inventors isolated mRNAs from spinal cords of wild type and FUS-R513C KI mice at two age points-1.5 and 7 months of age, and applied for RNA-seq. To inventors’ great surprise, among a few differentially expressed genes between the two genotypes, only Trim72, encoding an E3 ligase, was greatly upregulated in the mutant spinal cords at both age points (Fig. 3A and 3B) . To study the function of Trim72 upregulation in the mutant FUS spinal cord, invnetors expressed the Trim72 together with human wild type and mutant FUS in a heterologous system (Fig. 3C) . Surprisingly, the Trim72 preferentially interacted with FUS-R521C, compared to wild type FUS, but had little interaction with the FUS truncation (Δ513-526) , which lacks the nuclear localization signal (NLS) , suggesting that the NLS of FUS is required for the interaction (Fig. 3C and 3D) . Given the localization of the expressed Trim72 in the cytosol (Fig. 3E) , it is plausible that Trim72 may target the mislocalized FUS for ubiquitination and proteasome-mediated degradation. Indeed, the interaction between Trim72 and FUS-R521C was sensitive to MG132, a proteasome inhibitor (Fig. 3F) .

Reference throughout this specification to “an embodiment, ” “some embodiments, ” “one embodiment” , “another example, ” “an example, ” “aspecific example, ” or “some examples, ” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments, ” “in one embodiment” , “in an embodiment” , “in another example, ” “in an example, ” “in a specific example, ” or “in some examples, ” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments can not be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

A pharmaceutical composition comprising scavenger, wherein the scavenger is used for clearing away mutant FUS.
The pharmaceutical composition of claim 1, wherein the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation.
The pharmaceutical composition of claim 1, wherein the scavenger is Trim72 or the nucleic acid encoding Trim72.
The pharmaceutical composition of claim 1, wherein the mutation FUS is human ALS mutation FUS.
The pharmaceutical composition of claim 4, wherein the mutation FUS has mutation in FUS nuclear localization signal.
The pharmaceutical composition of claim 5, wherein the amino acid site of the nuclear localization signal of FUS is 513-526.
The pharmaceutical composition of claim 4, wherein the mutant FUS is prone to be mislocalized in cytosol.
The pharmaceutical composition of claim 1 is used for treating or preventing or alleviating ALS.
The pharmaceutical composition of claim 1, wherein the scavenger is in at least one of the following forms:

(a) protein or functional fragment；

(b) Nucleic acid molecule encoding (a) ；

(c) construct having (b) .
The pharmaceutical composition of claim 9, wherein the nucleic acid molecule comprising at least one of DNA and RNA.
The pharmaceutical composition of claim 9, wherein the construct comprising at least one of plasmid and virus.
The pharmaceutical composition of claim 1 further comprising pharmaceutically acceptable carrier.
The pharmaceutical composition of claim 12, wherein the acceptable carrier is drugs, toxins, cytokines, radioactive elements, carrier proteins, enzymes, lectins, fluorescent quantum dots, or high absorption coefficient of chromophore.
Use of the scavenger used for clearing away mutant FUS in the manufacture of a medicament for treating or preventing or alleviating ALS.
The use of claim 14, wherein the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation.
The use of claim 14, wherein the scavenger is Trim72 or the nucleic acid encoding Trim72.
The use of claim 14, wherein the mutation FUS is human ALS mutation FUS.
The use of claim 17, the mutation FUS has mutation in FUS nuclear localization signal.
The use of claim 18, the amino acid site of the nuclear localization signal of FUS is 513-526.
The use of claim 18, wherein the mutant FUS is prone to be mislocalized in cytosol.
The use of claim 1, wherein the scavenger is in at least one of the following forms:

(a) protein or functional fragment；

(b) Nucleic acid molecule encoding (a) ；

(c) construct having (b) .
The use of claim 21, wherein the nucleic acid molecule comprising at least one of DNA and RNA.
The use of claim 21, wherein the construct comprising at least one of plasmid and virus.
The use of claim 14, wherein the medicament further comprising pharmaceutically acceptable carrier.
The use of claim 24, wherein the acceptable carrier is drugs, toxins, cytokines, radioactive elements, carrier proteins, enzymes, lectins, fluorescent quantum dots, or high absorption coefficient of chromophore.
A method for treating or preventing or alleviating ALS comprising:

administration of the scavenger used for clearing away mutant FUS to a patient in need.
The method of claim 26, wherein the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation.
The method of claim 26, wherein the scavenger is Trim72 or the nucleic acid encoding Trim72.
The method of claim 26, wherein the mutation FUS is human ALS mutation FUS.
The method of claim 29, wherein the mutation FUS has mutation in FUS nuclear localization signal
The method of claim 30, wherein the amino acid site of the nuclear localization signal of FUS is 513-526.
The method of claim 30, wherein the mutant FUS is prone to be mislocalized in cytosol.
The method of claim 26, wherein the scavenger is in at least one of the following forms:

(a) protein or functional fragment；

(b) Nucleic acid molecule encoding (a) ；

(c) construct having (b) .
The method of claim 33, wherein the nucleic acid molecule comprising at least one of DNA and RNA.
The method of claim 33, wherein the construct comprising at least one of plasmid and virus.
The method of claim 26, wherein the scavenger is supplied in the form of a pharmaceutical composition, wherein the scavenger further comprising pharmaceutically acceptable carrier.
The method of claim 36, wherein the acceptable carrier is drugs, toxins, cytokines, radioactive elements, carrier proteins, enzymes, lectins, fluorescent quantum dots, or high absorption coefficient of chromophore.