WO2020140858A1 - Method for treating amyotrophic lateral sclerosis - Google Patents
Method for treating amyotrophic lateral sclerosis Download PDFInfo
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- WO2020140858A1 WO2020140858A1 PCT/CN2019/129704 CN2019129704W WO2020140858A1 WO 2020140858 A1 WO2020140858 A1 WO 2020140858A1 CN 2019129704 W CN2019129704 W CN 2019129704W WO 2020140858 A1 WO2020140858 A1 WO 2020140858A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to a method for treating amyotrophic lateral sclerosis in a subject, comprising administering to the subject suffering from amyotrophic lateral sclerosis a composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
- NRIP nuclear receptor interaction protein
- the NRIP protein comprises 860 amino acids which contains seven WD-40 repeats (40 amino acids terminating with a tryptophan-aspartic acid dipeptide) and one IQ motif.
- the molecular function of NRIP has been studied in prostate cancer cell line.
- NRIP is an androgen receptor (AR) -interaction protein as a transcriptional coactivator to stabilize AR and activates its own expression.
- AR androgen receptor
- the physiological role of NRIP is characterized by NRIP global knockout (gKO) mice and muscle-specific NRIP knockout (cKO) mice.
- gKO NRIP
- NRIP neuromuscular junction
- MNDs The motor neuron diseases
- ALS amyotrophic lateral sclerosis
- SBMA spinal bulbar muscular atrophy
- SMA spinal muscular atrophy
- ALS amyotrophic lateral sclerosis
- SBMA spinal bulbar muscular atrophy
- SMA spinal muscular atrophy
- the disease patients also find that the degeneration of motor neurons is accompanied by abnormalities at the NMJs. Disturbed function of NMJ will disrupt signal transmissions between nerve terminals and motor endplates, leading to muscle weakness in the end. Hence, the NMJ function is characterized to be a therapeutic target for MNDs.
- ALS Amyotrophic lateral sclerosis
- SOD1 G93A transgenic mouse is the most commonly used animal model of ALS, which displays a number of features similar to human ALS.
- the present invention relates to a method for treating amyotrophic lateral sclerosis in a subject, comprising administering to the subject suffering from amyotrophic lateral sclerosis a composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
- NRIP nuclear receptor interaction protein
- Fig. 1 shows that NRIP is one component of NMJ complex.
- Fig. 1A shows that NRIP locates at neuromuscular junction (NMJ) complex.
- Immunofluorescence assay (IFA) with NRIP antibody (green) , ⁇ -bungarotoxin [ ⁇ -BTX; red, for acetylcholine receptors (AChRs) ] and anti-rapsyn (blue) is made in paraffin sections of 16-week-old wild type mice gastrocnemius (GAS) muscle.
- NRIP co-localizes with AChRs and rapsyn at NMJ (arrow head indicates co-localization of NRIP, AChRs and rapsyn) .
- Scale bar 20 ⁇ m.
- FIG. 1B shows IFA with anti-NRIP (green) , ⁇ -BTX (red) and anti- ⁇ -actinin (ACTN2; blue) .
- Fig. 1C shows IFA with anti-ACTN2 (green) , anti-rapsyn (red) and DAPI (blue) .
- Fig. 1D shows that NRIP is a novel protein in AChR cluster complex. The protein lysates from gastrocnemius muscles of wild type are incubated with biotin-labeled BTX (B-BTX) and pulled down with streptavidin-coupled agarose beads; control (c) is streptavidin-coupled agarose beads only.
- the pull-down BTX contains NRIP, rapsyn and ACTN2; indicating that NRIP is in AChR complex.
- Fig. 1E shows the association of NRIP, rapsyn, ACTN2 and AChR in C2C12 myotubes by B-BTX pulldown assay.
- Fig. 2 shows that NRIP acts as a scaffold protein to stabilize protein-protein interaction.
- Fig. 2C shows the binding affinity between rapsyn and ACTN2 ( ⁇ - actinin 2) in NRIP cKO is reduced in comparison with WT; but the expression of rapsyn and ACTN2 is comparable between WT and NRIP cKO.
- GAPDH is the internal control.
- FIG. 3 shows that intramuscular NRIP gene therapy improves muscle oxidative functions of NRIP cKO mice.
- Fig. 3A shows schematic protocol for AAV-NRIP gene therapy.
- AAV-NRIP or AAV-GFP control
- i.m. intramuscular injection
- Fig. 3B shows western blot analysis of NRIP expression in gastrocnemius muscle from cKO mice after 10-weeks treatment.
- FIG. 3C shows frozen sections (10 ⁇ m thickness) stained for slow myosin expression in soleus (SOL) muscle from treated cKO mice. Dark brown staining represents positive-slow myosin (oxidative myofibers) . Scale bar, 100 ⁇ m.
- Fig. 3D shows quantification analysis. The proportion of positive-slow myosin in AAV-NRIP-treated cKO mice is higher than the control (50.84 %vs. 41.55 %, P ⁇ 0.05) .
- Fig. 3E shows CSA (cross section area) .
- CSA was measured by ImageJ software from slow myosin staining (Panel C) .
- Fig. 3F shows rotarod test. Data are mean ⁇ SEM by student’s t-test. *P ⁇ 0.05. ns, not significant.
- Fig. 4 shows that intramuscular NRIP gene therapy improves motor neurons survival of NRIP cKO mice.
- Fig. 4A shows immunofluorescence assay of NeuN (red) , ChAT (green) and DAPI (blue) in frozen sections from the spinal cord L3-L5 regions in AAV-NRIP treated cKO mice 10 weeks after virus injection. Yellow: NeuN and ChAT double-positive cells with cross section area (CSA) larger than 500 ⁇ m 2 are counted as ⁇ -motor neurons. Scale bar, 50 ⁇ m.
- Fig. 4B shows quantification analysis. The ⁇ -motor neuron number per spinal anterior horn demonstrates higher ⁇ -motor neuron number in AAV-NRIP-treated cKO mice than the control after 10 weeks treatment (21.3 vs. 18.2 per section, P ⁇ 0.05) .
- Fig. 5 shows that intramuscular NRIP gene therapy improves neuromuscular junction degeneration of NRIP cKO mice.
- Fig. 5A shows immunofluorescence assay with ⁇ -BTX (red, for acetylcholine receptors (AChRs) ) and anti-synaptophysin (SYN) and anti-neurofilament (NF) antibodies (both green, for axonal terminals) in gastrocnemius (GAS) muscle from treated cKO mice 10 weeks after virus injection. Scale bar, 10 ⁇ m.
- Fig. 5B shows quantification analysis of NMJ area from NRIP cKO mice infected with AAV-GFP and AAV-NRIP.
- Fig. 5C shows axon innervation.
- AAV-NRIP-treated cKO mice Comparison of the proportion of innervated endplates shows that innervation ratio is increased in AAV-NRIP-treated cKO mice compared with the control mice after 10-weeks treatment (97.41 %vs. 94.87 %, P ⁇ 0.01) .
- Fig. 5D shows axon denervation.
- the proportion of denervated endplates is decreased in AAV-NRIP-treated cKO mice compared with the control mice (2.59 %vs. 5.13 %, P ⁇ 0.01) .
- Data are mean ⁇ SEM by two-tailed student’s t-test. *P ⁇ 0.05 and **P ⁇ 0.01.
- Fig. 6 shows that protein expression of NRIP is reduced in SOD1 G93A mice.
- Fig. 6A shows western blot analysis of NRIP expression in spinal cord from 8-week-old WT and SOD1 G93A mice. Total proteins of L3-L5 spinal cord are subjected to western blot (WB) analysis for NRIP expression. GAPDH is the loading control.
- Fig. 6B shows western blot analysis of NRIP expression in gastrocnemius (GAS) muscles from 8-week-old WT and SOD1 G93A mice. Total proteins of GAS are subjected to WB analysis for NRIP expression.
- GAS gastrocnemius
- GAPDH is the loading control.
- Fig. 6C shows western blot analysis of NRIP expression of tibialis anterior (TA) muscles from 8-week-old WT and SOD1 G93A mice. Total proteins of tibialis anterior (TA) muscles are subjected to WB analysis for NRIP expression.
- GAPDH is the loading control.
- Fig. 7 shows the therapeutic effect on muscle weight and rotarod performance in SOD1 G93A mice infected with AAV-NRIP.
- Fig. 7C shows that the rotarod test.
- Data are mean ⁇ SEM by student’s t-test. **P ⁇ 0.01, ***P ⁇ 0.001 and ****P ⁇ 0.0001. ns, not significant.
- Fig. 8 shows the therapeutic effect on MN number of SOD1 G93A mice infected with AAV-NRIP.
- Fig. 8A shows immunofluorescence assay of neuronal DNA binding protein (NeuN) (red) , ChAT (green) , and DAPI (blue) expression and co-expression in lumbar spinal cord by confocal microscopy.
- the spinal cord is dissected from the wild-type and SOD1 G93A treated with AAV-GFP and AAV-NRIP, respectively.
- L3-L5 spinal cord sections are stained with NeuN and ChAT for ⁇ -MNs expression. Size >500 ⁇ m 2 is counted as ⁇ -motor neurons. Scale bar: 100 ⁇ m.Fig.
- Fig. 9 shows the therapeutic effect on NMJ formation in SOD1 G93A mice infected with AAV-NRIP.
- Fig. 9A shows the NMJ area of gastrocnemius muscles stained with ⁇ -BTX from WT, SOD1 G93A infected with AAV-GFP and SOD1 G93A infected with AAV-NRIP at post-infected day 70.
- FIG. 9C shows the innervation percentage of gastrocnemius muscles co-stained with ⁇ -BTX and synaptophysin from WT, SOD1 G93A infected with AAV-GFP and SOD1 G93A infected with AAV-NRIP at post-infected day 70.
- Data are mean ⁇ SEM by student’s t-test. *P ⁇ 0.05 and ***P ⁇ 0.001. ns, not significant.
- Fig. 10 shows locomotor function analysis of AAV-NRIP treated SOD1 G93A mice.
- the statistical data are analyzed by Prism software (GraphPad Software) . Data are presented as mean ⁇ SEM. All P value are determined by student’s t test for comparison between two groups. P ⁇ 0.05 is considered statistically significant.
- NRIP can interact with various proteins to regulate different cellular functions.
- NRIP can interact with androgen receptor (AR) protein to stabilize AR protein and enhance AR transcriptional activity which mediates prostate cancer progression;
- AR androgen receptor
- CaM CaM
- NRIP expression is colocalized with acetylcholine receptors (AChR) and AChR associated proteins, actinin and rapsyn, in WT mice muscles (Fig. 1A) .
- AChR acetylcholine receptors
- AChR associated proteins actinin and rapsyn
- WT mice muscles Fig. 1A
- NRIP also can be pull-downed with AChR and AChR associated proteins in muscles and C2C12 cells (Figs. 1D and 1E) in the tissue stain.
- the loss of NRIP disrupts the association of AChR complex (Fig. 2) .
- NRIP acts as a scaffold protein which can be a novel AChR associated protein for AChR complex stabilization.
- the previous study demonstrates that the global NRIP knockout (gKO) mice shows reduction of mitochondrial activity and impaired motor performance.
- the previous report further generates muscle-specific NRIP knockout (cKO) mice and finds that cKO mice shows muscular abnormality as well as abnormal neuromuscular junction (NMJ) integrity, even motor neuron degeneration is detected.
- NMJ neuromuscular junction
- the previous studies indicate that ⁇ -MNs degeneration in NRIP cKO mice is a “dying back” pattern resulted from the NMJ degeneration.
- intramuscular injection of AAV-NRIP can improve the abnormal NMJ (Fig. 5) and retrogradely improve the survival of ⁇ -MNs (Fig.
- NRIP can be a therapeutic agent for abnormal NMJ-caused motor neuron diseases (MNDs) .
- MNDs abnormal NMJ-caused motor neuron diseases
- AAV-NRIP shows no obvious therapeutic effect on motor performance (Fig. 3F) . This may be because AAV-NRIP is only locally injected into hindlimb muscles, which are just partially imoproved but not enough to affect the overall motor performance. In sum, AAV-NRIP gene therapy can improve muscle and motor neuron functions.
- ALS is caused by a “dying back” mechanism, where degeneration starts in muscle, travels up the axons to the spinal cord motor neurons and then to the cortical layer V motor neurons in the brain.
- AAV6 has been shown to efficiently transduce skeletal muscle as well as undergo retrograde transport to motor neurons after intramuscular injection.
- SOD1-specific shRNA SOD1 mice
- An intravenous injection of AAV6-shRNA vector resulted in 50%reduction in SOD1 protein in skeletal muscle of 6-week old SOD mutant mice.
- the amount of tranceduced motor neurons were less than 5%, and there was no apparent reduction in SOD1 levels in the spinal cord.
- NMJ innervation was improved about 29.41%, but there was no change in survival rate.
- the NMJ denervation is improved about 30.13% (Fig. 9) by the muscular injection of AAV-NRIP into 8-week old SOD1 mice.
- the therapeutic effect of NMJ is similar to treatment with SOD1 protein depletion but the treatment time of AAV-NRIP is more close to the time of disease onset than SOD1 RNAi treatment.
- Intravenous treatment with AAV-Dok7 improves muscle innervation and motor function in SOD1 G93A mice, but there are no obvious effects on proximal motor neuron degeneration.
- NRIP is a potential therapeutic agent for SOD1 mice therapy.
- the present invention provides a method for treating amyotrophic lateral sclerosis in a subject, comprising administering to the subject suffering from amyotrophic lateral sclerosis a composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
- NRIP nuclear receptor interaction protein
- ALS Amyotrophic lateral sclerosis
- ALS is a complex and multifactorial disease and multiple mechanisms hypothesized as responsible for ALS pathogenesis include, but are not limited to, dysfunction of protein degradation, glutamate excitotoxicity, mitochondrial dysfunction, apoptosis, oxidative stress, inflammation, protein misfolding and aggregation, aberrant RNA metabolism, and altered gene expression.
- treating refers to therapeutic treatments, wherein the subject is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with ALS.
- treating includes, but is not limited to, a preventive treatment and/or a therapeutic treatment.
- the subject is an animal, preferably a mammal, more preferably a human.
- the nuclear receptor interaction protein (NRIP) is a transcription factor that only expresses in cell nuclei. It is reported that lack of NRIP gene expression in clinical muscular dystrophy and NRIP knockout mice display weaker muscle strength, indicating that NRIP plays a role in muscle function.
- the peptide sequence of the NRIP comprises SEQ ID NO: 2.
- the NRIP is an acetylcholine receptor (AChR) complex protein.
- the NRIP is capable of stabilizing the AChR complex.
- the NRIP improves an abnormal muscle defect, a neuromuscular junction (NMJ) degeneration, a ⁇ -motor neuron degeneration or motor neuron loss for treating the ALS.
- NMJ neuromuscular junction
- the form of the NRIP is a gene, protein or a nucleic acid encoding the protein.
- a “gene” refers to the smallest, independently functional unit of genetic material that can code for and drive the expression of a protein, e.g., NRIP, or whose presence or absence has a phenotypic consequence on a cell or organism.
- the administration of the composition can be carried out via a variety of routes. These routes are designed to provide a local or systemic effect as required. These routes include, but are not limited to, oral, topical, pulmonary, rectal, subcutaneous, intradermal, intranasal, intracranial, intramuscular, intraocular, or intra-articular injection, and the like. The most typical route of administration is intravenous followed by subcutaneous, although other routes can be equally effective. Intramuscular injection can also be performed in the arm or leg muscles. In one embodiment, the administration of the composition is intramuscular injection.
- the NRIP is delivered to the subject systemically.
- systemic delivery means include injection and/or direct injection.
- the systemic injection techniques include intravenous delivery by intravenous injection. Intravenous injection is to a peripheral vein of the subject. For this administration route, the NRIP is adminstered/injected directly into the blood stream of the subject.
- the NRIP can be delivered to the subject by intramuscular injection.
- the form of the composition can be a gene therapy product, so the NRIP of the composition can be prepared into a form of a nucleotide encoding the NRIP.
- the sequence of the nucleotide encoding the NRIP comprises SEQ ID NO: 1.
- the nucleotide encoding the NRIP is administered to the subject by a gene delivery system.
- the term “gene delivery system” as used herein, refers to any forms of carriers that harbor and transport exogenous nucleic acid molecules to a host cell or tissue. The ideal gene delivery system should be harmless to human body, suitable for mass production, and capable of effective transportation of the target gene.
- the gene delivery system is a plasmid, a virus, a polymer, DNA, a bacterium, a plant, a nanomaterial, a liposome, or a niosome.
- the gene delivery system is a gene delivery vector.
- the virus comprises recombinant adenovirus, adeno-associated virus (AAV) , retrovirus, herpes simplex virus, vaccinia virus, measles virus, poxvirus, Semliki Forest virus, and lentiviral vector.
- AAV adeno-associated virus
- retrovirus herpes simplex virus
- vaccinia virus measles virus
- poxvirus poxvirus
- Semliki Forest virus Semliki Forest virus
- lentiviral vector lentiviral vector.
- the viru is AAV. Therefore, the nucleotide encoding NRIP is administered to the subject by viral mediated delivery techniques.
- a recombinant viral gene delivery vector which directs the expression of the NRIP in a form suitable for the treatment of ALS.
- the gene delivery vector is a viral vector.
- the gene delivery vector is a recombinant viral gene delivery vector.
- the recombinant viral gene delivery vector is in a form for administration systemically, for example by intramuscular administration.
- Suitable recombinant viral gene delivery vectors include, for example, retroviral vectors, herpes simplex virus (HSV) -based vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV) -based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors.
- retroviral vectors include, for example, retroviral vectors, herpes simplex virus (HSV) -based vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV) -based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors.
- HSV herpes simplex virus
- the recombinant viral gene delivery vector comprises an adeno-associated virus (AAV) vector, adenovirus vector and lentiviral vector.
- the recombinant viral gene delivery vector is an adeno-associated virus (AAV) vector.
- AAV adeno-associated virus
- the AAV vector comprises a nucleotide encoding NRIP.
- the AAV can be any one of AAV serotype 1 to 11.
- terapéuticaally effective amount used herein is a therapeutic dose which can prevent, decrease, stop or reverse a symptom developed in a subject under specific conditions, or partially, completely alleviates symptoms already exist under specific conditions when the subject begins receiving the treatment.
- the nucleotide encoding the NRIP is capable of being contained in a AAV vector to form an AAV-NRIP.
- the therapeutically effective amount of the AAV-NRIP ranges from 4x10 12 vg per kg of body weight of the subject to 8x10 13 vg per kg of body weight of the subject (vg/kg) .
- the therapeutically effective amount of the AAV-NRIP ranges from 8x10 11 vg per kg of body weight of the subject to 8x10 13 vg per kg of body weight of the subject.
- the therapeutically effective amount of the AAV-NRIP ranges from 8x10 11 vg per kg of body weight of the subject to 8x10 12 vg per kg of body weight of the subject.
- composition of the present invention further comprises a pharmaceutically acceptable carrier which may be administered to a subject through a number of different routes known in the art.
- the composition comprising the NRIP
- a pharmaceutically acceptable carrier are administered externally, intravenously, subcutaneously, topically, orally or by muscle or inhalation.
- the term “pharmaceutically acceptable carrier” is determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
- Carrier can include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, microspheres and emulsions.
- pharmaceutically acceptable refers to compounds and compositions which can be administered to mammals without undue toxicity.
- the present invention also provides an use of a composition for preparing a drug for treating amyotrophic lateral sclerosis in a subject, wherein the composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
- NRIP nuclear receptor interaction protein
- the form of the composition is a gene therapy product.
- the composition further comprises a gene delivery vector.
- the gene delivery vector is a viral vector.
- the viral vector is an adeno-associated virus (AAV) vector. Therefore, the NRIP in the composition can be prepared into a form of a nucleotide encoding the NRIP, and the nucleotide encoding the NRIP is delivered into the subject by the gene delivery vector.
- AAV adeno-associated virus
- the present invention further provides an use of a composition for preparing a drug for treating amyotrophic lateral sclerosis in a subject, wherein the composition comprising a therapeutically effective amount of a gene delivery vector comprising a nucleotide for encoding a nuclear receptor interaction protein (NRIP) .
- NRIP nuclear receptor interaction protein
- the gene delivery vector is a viral vector.
- the viral vector comprises retroviral vector, herpes simplex virus (HSV) -based vector and parvovirus-based vector.
- the parvovirus-based vector comprises adeno-associated virus (AAV) -based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors.
- the viral vector is an adeno-associated virus (AAV) vector.
- the composition is formulated for systemic administration. In a preferred embodiment, the composition is formulated for intramuscular administration or intravenous administration. In a more preferred embodiment, the composition is formulated for injection, infusion or implantation. In another embodiment, the composition is administered by intramuscular injection.
- the therapeutically effective amount of the gene delivery vector ranges from 4x10 12 vg/kg to 8x10 13 vg/kg. In a preferred embodiment, the therapeutically effective amount of the gene delivery vector ranges from 8x10 11 vg/kg to 8x10 13 vg/kg. In a more preferred embodiment, the therapeutically effective amount of the gene delivery vector ranges from 8x10 11 vg/kg to 8x10 12 vg/kg.
- the nucleotide encoding the NRIP is capable of being contained in the AAV vector to form an AAV-NRIP when the the gene delivery vector is a AAV vector.
- the therapeutically effective amount of the AAV-NRIP ranges from 4x10 12 vg/kg to 8x10 13 vg/kg. In a preferred embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 8x10 11 vg/kg to 8x10 13 vg/kg. In a more preferred embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 8x10 11 vg/kg to 8x10 12 vg/kg.
- the present invention relates to NRIP induced improvement of muscle defects for treating ALS.
- NRIP can improve the muscle defects and motor neuron degeneration, thereby ameliorating symptoms of ALS.
- L5 spinal cord was snap frozen in OCT compound (Tissue- Tek, Torrance, CA) and sectioned into 30 ⁇ m thickness. The sections were then stained with anti-NeuN (1: 500, Millipore, Temecula, CA) and anti-ChAT (1: 200, Millipore, Temecula, CA) antibody. After washing with PBS, the fluorescent signal was produced by the incubation of Cy3-conjugated goat anti-rabbit or 488-conjugated donkey anti-goat secondary antibody (Jackson Lab., Bar Harbor, ME) . The images were acquired with a Leica TSC SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) .
- NMJ neuromuscular junction
- muscles were fixed with 2%paraformaldehyde for 2 hours at room temperature.
- Tissues were washed by PBS and dehydrated with 30%sucrose and incubated overnight at 4 °C.
- the dehydrated tissues were then embedded in OCT and sectioned to 20 ⁇ m thickness.
- the sections were incubated with anti-neurofiliment and anti-synaptophysin for overnight at 4°C.
- slides were incubated with a mixture containing 488-conjugated donkey anti-rabbit secondary antibody and anti-BTX antibody.
- the images were visualized by Leica TSC SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) .
- the size of NMJ were analyzed by the Image J softeware.
- Soleus and gastrocnemius muscles were collected and fixed in 4%paraformaldehyde.
- the paraffin-embedded sections were antigen retrieved by 0.1 M citric acid (pH 6.0) and then incubated with the slow myosin antibody (1: 200, Abcam, Cambridge, MA) at 4°C. for overnight. After washing with PBS and HRP-Polymer (BioGenex, Fremont, CA) incubation for 1 hr in room temperature, the slow myosin positive signal was detected by HRP-Polymer reacted with DAB chromogen (BioGenex, Fremont, CA) .
- Muscle lysates were extracted by RIPA lysis buffer (150 mM NaCl, 1%NP-40, 50 mM Tris) , proteins were separated on SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, USA) . The membranes were blocked in 5%non-fat milk at room temparature for 1 hour. Then the blots were reacted with primary antibodies diluted in blocking buffer at 4 °C for overnight. The primary antibody that the present invention used were listed in Table 1. The blots were washed by TBST for 10 mins, and then reacted with HRP-conjugated secondary antibody at room temperature for 1 hour. After a serial wash by TBST buffer, proteins expression was detected by using an ECL Western blotting detection system (GE Healthcare Life Sciences, USA) .
- ECL Western blotting detection system GE Healthcare Life Sciences, USA
- HEK293T cells were co-transfected with the pAAV-MCS-NRIP or pAAV-GFP, pAAV-DJ/8 and the adenovirus helper plasmid pHelper by using calcium phosphate transfection and cultured for 72 hours.
- the sequence of the nucleotide encoding the NRIP comprises SEQ ID NO: 1.
- the AAV particles were purified by CsCl density-gradient ultracentrifugation and dialyzed by dialysis cassette (Slide-A-Lyzer dialysis cassettes, Thermo; MWCO 10K) in dialysis buffer containing 350 mM NaCl with 5%sorbitol in 1X PBS at 4°C.
- the viral titers were determined by a dot blot assay. The viral particles were divided into small portions and stored at -80°C until further use.
- mice were anaesthetized by intraperitoneal injection of 2.5%avertin (300 ⁇ l) , then intramuscular injected with AAV-GFP (control) or AAV-NRIP (10 ⁇ l, 2 ⁇ 10 12 vg/ml) in a single shot into each bilateral gastrocnemius and tibialis anterior muscles at age 6 weeks. The mice were monitored daily after recovery until 10 weeks after gene therapy. The therapeutic effects were analyzed for motor performance, muscle oxidative function, ⁇ -motor neuron survival, NMJ integrity and axonal innervation at 16 weeks.
- AAV-GFP control
- AAV-NRIP 10 ⁇ l, 2 ⁇ 10 12 vg/ml
- the protein lysates from gastrocnemius muscles, WT C2C12 cells and C2C12 KO19 cells were extracted by RIPA buffer (137 mM NaCl, 20 mM Tris-HCl Ph 8.0) .
- Total protein lysates (5mg) were incubated with biotin-BTX (B-BTX) at 4°C for 4hrs, which was followed by incubation of streptavidin beads in at 4°C for 2hrs. The beads were washed by RIPA buffer for three times.
- the B-BTX conjugated proteins were then eluted by SDS sample buffer and subjected to western blot (WB) analysis for AChR complex detection.
- NRIP was a novel acetylcholine receptor (AChR) complex protein
- NRIP neuromuscular junction
- NMJ neuromuscular junction
- actinin 2 which was reported to interact with rapsyn, a well-known NMJ component.
- the present invention suggested that NRIP could associate with AChR complex to support AChR cluster integrity.
- the present invention firstly determined the localization of NRIP with NMJ components including AChR, rapsyn and actinin through IFA stain of gastrocnemius muscles from 16-week-old WT mice.
- the present invention labeled the AChR cluster by Alexa-594-conjugated ⁇ -bungarotoxin ( ⁇ -BTX) and found that NRIP was colocalized with rapsyn and actinin at NMJ (Figs. 1A and 1B) .
- the present invention also confirmed that rapsyn was colocalized with actinin in gastrocnemius muscle (Fig. 1C) .
- ⁇ -BTX was a competitive antagonist that specifically binds to AChR, hence, biotin-labeled BTX (B-BTX) could be used for AChR pull-down to identify the AChR associated proteins.
- the present invention further used the B-BTX pull-down assay to determine whether NRIP was a AChR complex protein that could be pull-downed with AChR complex in gastrocnemius muscle and C2C12 myotubes.
- NRIP was pull-downed with AChR and was accompanied by AChR associated protein, rapsyn and actinin (Fig. 1D) .
- NRIP, rapsyn and actinin were also pull-downed with AChR in C2C12 myotubes (Fig. 1E) .
- rapsyn was capable of interacting with AChR to stabilize the AChR cluster at NMJ.
- NRIP was capable of colocalizing and associating with rapsyn and actinin (Fig. 1) .
- the present invention suggested that NRIP was required for AChR-rapsyn interaction.
- the protein expression of rapsyn was the same in gastrocnemius muscles from wild-type (WT) and NRIP cKO mice, however, in B-BTX pull-down assay, the AChR-interacted rapsyn was decreased in NRIP cKO mice compared to WT mice (Fig. 2A) .
- the muscle-specific NRIP cKO mice showed the phenotypes similar to MNDs, such as NMJ abnormality, motor neuron degeneration and motor function defects at adult stage (16 wk) .
- AAV-NRIP gene therapy could improve NMJ function and motor neuron deficits in cKO mice
- the present invention injected AAV-NRIP or AAV-GFP (control) (2 ⁇ 10 10 vg in a total volume of 10 ⁇ l for each muscles) into bilateral GAS and TA muscles of cKO mice at age 6 weeks and examined the muscle function (such as slow myosin protein, COX, and SDH) , motor neurons survival and NMJ integrity at age 16 weeks (10 weeks after virus injection) (Fig. 3A) .
- NRIP protein expression in injected muscles of AAV-NRIP-treated cKO mice could be detected 10 weeks after injection (Fig. 3B) .
- the present invention first dissected soleus (SOL) muscles from 16-week-old treated cKO mice to check oxidative fiber expression pattern by immunohistochemistry (IHC) staining. Fresh-frozen SOL sections were cut into 10- ⁇ m-thick slices by cryostat microtome and stained for anti-slow myosin heavy chain antibody. The dark brown stained fibers were slow myosin positive myofibers (Fig. 3C) .
- the spinal cord of the L3-L5 segment was dissected to check the number of ⁇ -motor neurons by immunofluorescence assay (IFA) .
- the present invention labeled the MNs by staining the neuronal nuclear protein (NeuN) and choline acetyltransferase (ChAT) .
- the NeuN and ChAT double-positive (yellow) MNs with size larger than 500 ⁇ m 2 were defined as ⁇ -motor neurons (Fig. 4A) .
- Quantitative data showed that the ⁇ -motor neuron number was significantly higher in AAV-NRIP-treated cKO mice than the control treated cKO at age 16 weeks (21.3 vs. 18.2 per section, P ⁇ 0.05; Fig. 4B)
- NRIP improved the NMJ structure in NRIP cKO mice
- the present invention found that overexpression of NRIP in NRIP-deleted muscle improved the muscle function and MN survival (Fig. 3 and Fig. 4) .
- the present invention would like to examine whether the retrogrdely improvement of MN survival was resulted from NMJ stabilization in NRIP-improved skeletal muscle.
- the sections of gastrocnemius muscles were stained by alpha-bungarotoxin ( ⁇ -BTX) , neurofilament (NF) and synaptophysin (SYN) used to label acetylcholine receptors (AChRs) , motor axons and nerve terminals respectively at the NMJs.
- ⁇ -BTX alpha-bungarotoxin
- NF neurofilament
- SYN synaptophysin
- AChR axonal innervation
- AChR red
- axonal denervation Fig. 5A
- the proportion of denervated endplates were significantly decreased in AAV-NRIP-treated cKO mice than the control at age 16 weeks respectively (2.59 %vs. 5.13 %, respectively, P ⁇ 0.01; Fig. 5C) .
- muscle administering AAV-NRIP in NRIP cKO mice was capable of improving the abnormal muscle defect, NMJ degeneration and motor neuron loss in muscle-specific NRIP cKO mice.
- NRIP might also be a therapeutic agent for motor neuron diseases (MNDs) .
- MNDs motor neuron diseases
- ALS Amyotrophic lateral sclerosis
- SOD1 G93A transgenic mice which displays a number of features similar to human ALS.
- the present invention used SOD1 G93A mice to examine whether NRIP could be a novel therapeutic agent for ALS.
- the present invention first extracted protein lysates of spinal cord, GAS and tibialis anterior (TA) muscles from 8-week-old SOD1 G93A and age-matched WT mice and analyzed by the western blot analysis.
- spinal cord the relative NRIP level was approximately 8-fold decreased in SOD1 G93A mice compared with WT (0.12 vs. 0.96 NRIP/GAPDH, P ⁇ 0.0001; Fig. 6A, right panel) .
- the relative NRIP level was approximately 5-and 3-fold decreased in SOD1 G93A mice compared with WT (0.15 vs.
- NRIP might play a role in SOD1 G93A mice and AAV-NRIP could be a therapeutic agent for ALS mice.
- NRIP expression was downregulated in skeletal muscle of SOD1 G93A mice (Fig. 6) ; and SOD1 G93A mice had been reported that it performed muscle dystrophy.
- the present invention would like to know whether the muscle dystrophy in SOD1 G93A mice could be improved by intramuscular (IM) injection of AAV-NRIP in TA and GAS muscles.
- the SOD1 G93A mice received the AAV-NRIP and AAV-GFP injection at P60, the gastrocnemius and soleus muscles were harvested and weighted at P120.
- the muscle weight (MW) of gastrocnemius and soleus were measured and normalized to body weight (BW) .
- Results showed that the MW/BW ratio of gastrocnemius and soleus muscles were decreased in SOD1 G93A injected with AAV-GFP compared to WT mice and the injection of AAV-NRIP did not improve the muscle loss of SOD1 G93A mice (Figs. 7A and 7B) .
- the present invention also measured the therapeutic benefit on rotarod performance of SOD1 G93A mice treated with AAV-NRIP. Results showed that SOD1 G93A mice infected with AAV-NRIP were fell off faster than WT mice, the injection of NRIP did not improve the motor deficits on SOD1 G93A mice (Fig. 7C) . These results indicated that the local IM injection of NRIP had no therapeutic effect on muscle and motor dysfunction.
- the present invention calculated the area of NMJ and found that NMJ area was decreased in SOD1 G93A mice injected with AAV-GFP compared to WT (220.1 vs. 264.2 ⁇ m 2 ; Fig. 9A) .
- the NMJ area was increased to 234.7 ⁇ m 2 compared to AAV-GFP group but not significant (Fig. 9A) .
- the number of NMJ was improved in SOD1 G93A mice injected with AAV-NRIP compared to SOD1 G93A mice injected with AAV-GFP (237.5 ⁇ m 2 vs. 195.2, P ⁇ 0.05; Fig. 9) .
- the innervation is the important parameter for neuron and muscle interaction, thus the present invention also compared the percentage of innervated NMJ to determine the therapeutic effect of retrograde improvement of MN number from muscle injected AAV-NRIP.
- the results showed that AAV-NRIP improved the NMJ innervation in SOD1 G93A mice compared to AAV-GFP group (70.42 %vs. 59.97 %, P ⁇ 0.05; Fig. 9C) .
- These data indicated that the improvement of NMJ number and innervation retrogradely improved the ⁇ -MN survival in ALS mice treated with AAV-NRIP.
- the NRIP could be a novel agent of gene therapy on ALS mice.
- the present invention determined the therapeutic effect of AAV-NRIP in locomotor activity of SOD G93A mutant mice.
- the open field test was used as assessment of locomotor function and voluntary activity for individual mice.
- mice were tracked using EthoVision Video Tracking Software (Noldus) .
- the total distance moving (total distance mice traveled in the box) and the rearing frequency (frequency which the mice stands on its hind limb in the box) were measured during the test.
- the results showed that AAV-NRIP could improve the performance of total distance moving compared to the AAV-GFP (2569.08 cm vs. 1858.26 cm, P ⁇ 0.05; Fig. 10A) .
- the rearing frequency was also improved in AAV-NRIP treatment compared to AAV-GFP treatment (31.60 vs. 13.80, P ⁇ 0.05; Fig. 10B) .
- AAV-NRIP was a potential ALS therapeutic drug for AAV-NRIP intramusle injection of SOD1 G93A mice at day 60; and the present invention found that AAV-NRIP treatment of SOD1 mice could improve motor neuron number growth, increase the size of neuromuscle junction and decrease axon denervation at day 120; along with increased locomotor activities such as total distance travel in the box and the rearing frequency of mice standing on its hind limb in the box at day 126.
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Abstract
A method for treating amyotrophic lateral sclerosis in a subject by administering to the subject a composition which has a therapeutically effective amount of a nuclear receptor interaction protein (NRIP).
Description
The present invention relates to a method for treating amyotrophic lateral sclerosis in a subject, comprising administering to the subject suffering from amyotrophic lateral sclerosis a composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
The NRIP protein comprises 860 amino acids which contains seven WD-40 repeats (40 amino acids terminating with a tryptophan-aspartic acid dipeptide) and one IQ motif. The molecular function of NRIP has been studied in prostate cancer cell line. NRIP is an androgen receptor (AR) -interaction protein as a transcriptional coactivator to stabilize AR and activates its own expression. The physiological role of NRIP is characterized by NRIP global knockout (gKO) mice and muscle-specific NRIP knockout (cKO) mice. NRIP (gKO) mice display a decline in muscle strength and susceptibility to muscle fatigue. And muscle-specific NRIP cKO mice also show muscular abnormality and motor dysfunctions; and intriguingly retrogradely cause the neuromuscular junction (NMJ) abnormality and motor neuron number decreases; these pathologic characters are motor neuron degeneration. Although NRIP is required for NMJ maintenance, the molecular interplay between NRIP and NMJ is still unclear.
The motor neuron diseases (MNDs) are a group of progressive neurodegenerative disorders that characterized by destroy of motor neurons, causing muscle weakness and atrophy. There are several types of MNDs including the amyotrophic lateral sclerosis (ALS) , spinal bulbar muscular atrophy (SBMA, Kenney disease) and spinal muscular atrophy (SMA) . In addition to muscle weakness, the disease patients also find that the degeneration of motor neurons is accompanied by abnormalities at the NMJs. Disturbed function of NMJ will disrupt signal transmissions between nerve terminals and motor endplates, leading to muscle weakness in the end. Hence, the NMJ function is characterized to be a therapeutic target for MNDs.
Amyotrophic lateral sclerosis (ALS) is the most common type of MNDs and usually onset in adult life (approximately 50-60 years old) . No treatment is currently available, but supportive treatment can improve the quality of life. The human superoxide dismutase 1 (SOD1) G93A transgenic mouse is the most commonly used animal model of ALS, which displays a number of features similar to human ALS. Several gene therapies for ALS mice have been developed. Silencing the expression of mutant SOD1 through AAV-delivered SOD1 shRNA or miRNA has therapeutic benefit on survival and disease onset of ALS mice; overexpression of Dok-7, a muscle specific protein, through intravenous injection of AAV-Dok-7 can improve the neuromuscular junction structure and improve the survival of SOD1 G93A mice. Although the therapeutic mechanism is still lack, previous studies have showed that the AAV-delivered gene therapy has therapeutic effect on ALS mice.
Hence, there is still a need of developing new selective, efficacious novel compounds for the treatment and prevention of ALS disease is under active investigation.
SUMMARY OF THE INVENTION
The present invention relates to a method for treating amyotrophic lateral sclerosis in a subject, comprising administering to the subject suffering from amyotrophic lateral sclerosis a composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
Fig. 1 shows that NRIP is one component of NMJ complex. Fig. 1A shows that NRIP locates at neuromuscular junction (NMJ) complex. Immunofluorescence assay (IFA) with NRIP antibody (green) , α-bungarotoxin [α-BTX; red, for acetylcholine receptors (AChRs) ] and anti-rapsyn (blue) is made in paraffin sections of 16-week-old wild type mice gastrocnemius (GAS) muscle. NRIP co-localizes with AChRs and rapsyn at NMJ (arrow head indicates co-localization of NRIP, AChRs and rapsyn) . Scale bar, 20 μm. Fig. 1B shows IFA with anti-NRIP (green) , α-BTX (red) and anti-α-actinin (ACTN2; blue) . Fig. 1C shows IFA with anti-ACTN2 (green) , anti-rapsyn (red) and DAPI (blue) . Fig. 1D shows that NRIP is a novel protein in AChR cluster complex. The protein lysates from gastrocnemius muscles of wild type are incubated with biotin-labeled BTX (B-BTX) and pulled down with streptavidin-coupled agarose beads; control (c) is streptavidin-coupled agarose beads only. The pull-down BTX contains NRIP, rapsyn and ACTN2; indicating that NRIP is in AChR complex. Fig. 1E shows the association of NRIP, rapsyn, ACTN2 and AChR in C2C12 myotubes by B-BTX pulldown assay.
Fig. 2 shows that NRIP acts as a scaffold protein to stabilize protein-protein interaction. Fig. 2A shows that the interaction between rapsyn and AChR in NRIP cKO is lower than WT. Gastrocnemius protein lysates are extracted and subjected to B-BTX pulldown assay and western blot for rapsyn. Right: quantification of relative AChR-rapsyn binding affinity (n=3) . Fig. 2B shows the binding affinity of rapsyn and AChR in C2C12 myotubes. 5 mg proteins extracted from KO (NRIP-null cells) and C2C12 myotubes are incubated with B-BTX for pulled down assay. Right: quantification (n=3) . Fig. 2C shows the binding affinity between rapsyn and ACTN2 (α- actinin 2) in NRIP cKO is reduced in comparison with WT; but the expression of rapsyn and ACTN2 is comparable between WT and NRIP cKO. GAPDH is the internal control. Fig. 2D shows the binding affinity of rapsyn and actinin in C2C12 myotubes. Right: quantification (n=3) . Data are mean ± SEM by student t test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3 shows that intramuscular NRIP gene therapy improves muscle oxidative functions of NRIP cKO mice. Fig. 3A shows schematic protocol for AAV-NRIP gene therapy. At age 6-week-old cKO mice, AAV-NRIP or AAV-GFP (control) is injected into bilateral gastrocnemius and tibialis anterior muscles of cKO mice by intramuscular (i.m. ) injection; 10 weeks after virus injection the mice are analyzed for motor performance, muscle oxidative function, α-motor neuron number and NMJ integrity. Fig. 3B shows western blot analysis of NRIP expression in gastrocnemius muscle from cKO mice after 10-weeks treatment. Fig. 3C shows frozen sections (10 μm thickness) stained for slow myosin expression in soleus (SOL) muscle from treated cKO mice. Dark brown staining represents positive-slow myosin (oxidative myofibers) . Scale bar, 100 μm. Fig. 3D shows quantification analysis. The proportion of positive-slow myosin in AAV-NRIP-treated cKO mice is higher than the control (50.84 %vs. 41.55 %, P < 0.05) . AAV-NRIP treatment, n = 7 mice; AAV-GFP control, n = 5 mice. Fig. 3E shows CSA (cross section area) . CSA was measured by ImageJ software from slow myosin staining (Panel C) . Fig. 3F shows rotarod test. Data are mean ± SEM by student’s t-test. *P < 0.05. ns, not significant.
Fig. 4 shows that intramuscular NRIP gene therapy improves motor neurons survival of NRIP cKO mice. Fig. 4A shows immunofluorescence assay of NeuN (red) , ChAT (green) and DAPI (blue) in frozen sections from the spinal cord L3-L5 regions in AAV-NRIP treated cKO mice 10 weeks after virus injection. Yellow: NeuN and ChAT double-positive cells with cross section area (CSA) larger than 500 μm
2 are counted as α-motor neurons. Scale bar, 50 μm. Fig. 4B shows quantification analysis. The α-motor neuron number per spinal anterior horn demonstrates higher α-motor neuron number in AAV-NRIP-treated cKO mice than the control after 10 weeks treatment (21.3 vs. 18.2 per section, P < 0.05) . AAV-NRIP treatment, n = 8 mice; control, n = 6 mice. Data are mean ± SEM by student’s t-test. *P < 0.05.
Fig. 5 shows that intramuscular NRIP gene therapy improves neuromuscular junction degeneration of NRIP cKO mice. Fig. 5A shows immunofluorescence assay with α-BTX (red, for acetylcholine receptors (AChRs) ) and anti-synaptophysin (SYN) and anti-neurofilament (NF) antibodies (both green, for axonal terminals) in gastrocnemius (GAS) muscle from treated cKO mice 10 weeks after virus injection. Scale bar, 10 μm. Fig. 5B shows quantification analysis of NMJ area from NRIP cKO mice infected with AAV-GFP and AAV-NRIP. Fig. 5C shows axon innervation. Comparison of the proportion of innervated endplates shows that innervation ratio is increased in AAV-NRIP-treated cKO mice compared with the control mice after 10-weeks treatment (97.41 %vs. 94.87 %, P < 0.01) . AAV-NRIP treatment, n = 9 mice; control, n = 6 mice. Fig. 5D shows axon denervation. The proportion of denervated endplates is decreased in AAV-NRIP-treated cKO mice compared with the control mice (2.59 %vs. 5.13 %, P < 0.01) . AAV-NRIP treatment, n = 9 mice; control, n = 6 mice. Data are mean ± SEM by two-tailed student’s t-test. *P < 0.05 and **P < 0.01.
Fig. 6 shows that protein expression of NRIP is reduced in SOD1 G93A mice. Fig. 6A shows western blot analysis of NRIP expression in spinal cord from 8-week-old WT and SOD1 G93A mice. Total proteins of L3-L5 spinal cord are subjected to western blot (WB) analysis for NRIP expression. GAPDH is the loading control. Right panel: quantification of NRIP expression. WT, n = 3 mice; SOD1 G93A mice, n = 5 mice. Fig. 6B shows western blot analysis of NRIP expression in gastrocnemius (GAS) muscles from 8-week-old WT and SOD1 G93A mice. Total proteins of GAS are subjected to WB analysis for NRIP expression. GAPDH is the loading control. Right panel: quantification of NRIP expression. WT, n = 3 mice; SOD1 G93A, n = 5 mice. Fig. 6C shows western blot analysis of NRIP expression of tibialis anterior (TA) muscles from 8-week-old WT and SOD1 G93A mice. Total proteins of tibialis anterior (TA) muscles are subjected to WB analysis for NRIP expression. GAPDH is the loading control. Right panel: quantification of NRIP expression. WT, n = 3 mice; SOD1 G93A, n = 5 mice. Data are mean ± SEM by student’s t-test. *P < 0.05, ***P < 0.001 and ****P < 0.0001.
Fig. 7 shows the therapeutic effect on muscle weight and rotarod performance in SOD1 G93A mice infected with AAV-NRIP. Fig. 7A shows the average GAS muscle weight to body weight ratio of WT and SOD1 G93A mice infected with AAV-GFP and AAV-NRIP (WT, n=4; SOD1 G93A infected AAV-GFP, n=7; SOD1 G93A infected AAV-NRIP, n=7) . Fig. 7B shows the average soleus muscle weight to body weight ratio of WT and SOD1 G93A mice infected with AAV-GFP and AAV-NRIP (WT, n=4; SOD1 G93A infected AAV-GFP, n=7; SOD1 G93A infected AAV-NRIP, n=7) . Fig. 7C shows that the rotarod test. WT and SOD1 G93A mice infected with AAV-GFP and AAV-NRIP are placed on a rod rotating at 20 rpm, and riding time is measured (WT, n=12; SOD1 G93A infected AAV-GFP, n=10; SOD1 G93A infected AAV-NRIP, n=12) . Data are mean ± SEM by student’s t-test. **P < 0.01, ***P < 0.001 and ****P < 0.0001. ns, not significant.
Fig. 8 shows the therapeutic effect on MN number of SOD1 G93A mice infected with AAV-NRIP. Fig. 8A shows immunofluorescence assay of neuronal DNA binding protein (NeuN) (red) , ChAT (green) , and DAPI (blue) expression and co-expression in lumbar spinal cord by confocal microscopy. The spinal cord is dissected from the wild-type and SOD1 G93A treated with AAV-GFP and AAV-NRIP, respectively. L3-L5 spinal cord sections are stained with NeuN and ChAT for α-MNs expression. Size >500 μm
2 is counted as α-motor neurons. Scale bar: 100 μm.Fig. 8B shows that quantification of motor neuron number per spinal anterior horn shows lower number in SOD1 G93A mice than WT mice. The MN number is increased in SOD1 G93A mice infected with AAV-NRIP compared to SOD1 G93A mice infected with AAV-GFP (WT, n=10; SOD1 G93A infected AAV-GFP, n=10; SOD1 G93A infected AAV-NRIP, n=10) . Data are mean ± SEM by student’s t-test. *P < 0.05 and ***P < 0.001.
Fig. 9 shows the therapeutic effect on NMJ formation in SOD1 G93A mice infected with AAV-NRIP. Fig. 9A shows the NMJ area of gastrocnemius muscles stained with α-BTX from WT, SOD1 G93A infected with AAV-GFP and SOD1 G93A infected with AAV-NRIP at post-infected day 70. Theα-BTX-positive area is defined as NMJ area and is measured by ImageJ software. (WT, n=11; SOD1 G93A infected AAV-GFP, n=9; SOD1 G93A infected AAV-NRIP, n=11) . Fig. 9B shows the NMJ number of gastrocnemius muscles stained with α-BTX from WT, SOD1 G93A infected with AAV-GFP and SOD1 G93A infected with AAV-NRIP at post-infected day 70. Quantification analysis of NMJ number are counted from the sum of three sections (30μm thickness) of gastrocnemius muscles. (WT, n=9; SOD1 G93A infected AAV-GFP, n=9; SOD1 G93A infected AAV-NRIP, n=11) . Fig. 9C shows the innervation percentage of gastrocnemius muscles co-stained with α-BTX and synaptophysin from WT, SOD1 G93A infected with AAV-GFP and SOD1 G93A infected with AAV-NRIP at post-infected day 70. Quantification analysis of NMJ innervation is defined by the percentage of innervated NMJ in total NMJ. (WT, n=11; SOD1 G93A infected AAV-GFP, n=9; SOD1 G93A infected AAV-NRIP, n=11) . Data are mean ± SEM by student’s t-test. *P < 0.05 and ***P < 0.001. ns, not significant.
Fig. 10 shows locomotor function analysis of AAV-NRIP treated SOD1 G93A mice. Fig. 10A shows that the total distance which AAV-NRIP and AAV-GFP treated SOD1 G93A travelled are measured at the post-infected day 70. N=5 for each group. Fig. 10B shows that the times of rearing performed by AAV-NRIP and AAV-GFP treated SOD1 G93A are measured at the post-infected day 70. N=5 for each group. The statistical data are analyzed by Prism software (GraphPad Software) . Data are presented as mean ± SEM. All P value are determined by student’s t test for comparison between two groups. P < 0.05 is considered statistically significant.
In previous studies, NRIP can interact with various proteins to regulate different cellular functions. For example, NRIP can interact with androgen receptor (AR) protein to stabilize AR protein and enhance AR transcriptional activity which mediates prostate cancer progression; NRIP also can interact with CaM to activate CaN and CaMK to regulate contraction of skeletal muscle. In the present invention, NRIP expression is colocalized with acetylcholine receptors (AChR) and AChR associated proteins, actinin and rapsyn, in WT mice muscles (Fig. 1A) . NRIP also can be pull-downed with AChR and AChR associated proteins in muscles and C2C12 cells (Figs. 1D and 1E) in the tissue stain. The loss of NRIP disrupts the association of AChR complex (Fig. 2) . Hence, NRIP acts as a scaffold protein which can be a novel AChR associated protein for AChR complex stabilization.
The previous study demonstrates that the global NRIP knockout (gKO) mice shows reduction of mitochondrial activity and impaired motor performance. The previous report further generates muscle-specific NRIP knockout (cKO) mice and finds that cKO mice shows muscular abnormality as well as abnormal neuromuscular junction (NMJ) integrity, even motor neuron degeneration is detected. The previous studies indicate that α-MNs degeneration in NRIP cKO mice is a “dying back” pattern resulted from the NMJ degeneration. In the present invention, intramuscular injection of AAV-NRIP can improve the abnormal NMJ (Fig. 5) and retrogradely improve the survival of α-MNs (Fig. 4) ; which further supports the dying back theory of MN degeneration and further demonstrates that NRIP can be a therapeutic agent for abnormal NMJ-caused motor neuron diseases (MNDs) . However, AAV-NRIP shows no obvious therapeutic effect on motor performance (Fig. 3F) . This may be because AAV-NRIP is only locally injected into hindlimb muscles, which are just partially imoproved but not enough to affect the overall motor performance. In sum, AAV-NRIP gene therapy can improve muscle and motor neuron functions.
Gene therapy for ALS mice has developed in recent years. The number of animal studies using AAV vectors or RNAi have increased rapidly. Multiple studies have approached SOD1-ALS treatment through the use of AAV delivered RNAi in both mouse and rat models of ALS. The AAV9 based study used an H1 promoter driven shRNA, and reached a 39%increase in survival rate when delivered to SOD1 mice at postnatal day 1 (P1) , and 30%when delivered at postnatal day 21 (P21) . Treatment with AAVrh10-amiRNA reduced SOD1 mRNA in transduced cells and increased life span by 21%. The above studies are focused on the treatment of neuron to improve ALS phenotype. It has been hypothesized that ALS is caused by a “dying back” mechanism, where degeneration starts in muscle, travels up the axons to the spinal cord motor neurons and then to the cortical layer V motor neurons in the brain. AAV6 has been shown to efficiently transduce skeletal muscle as well as undergo retrograde transport to motor neurons after intramuscular injection. Thus, several studies used AAV6 to deliver an SOD1-specific shRNA in SOD1 mice, to reduce the mutant protein in muscle. An intravenous injection of AAV6-shRNA vector resulted in 50%reduction in SOD1 protein in skeletal muscle of 6-week old SOD mutant mice. However, the amount of tranceduced motor neurons were less than 5%, and there was no apparent reduction in SOD1 levels in the spinal cord. In a subsequent study, they performed multiple muscle injection of AAV6-shRNA in neonate SOD1 mice, the NMJ innervation was improved about 29.41%, but there was no change in survival rate. In the present invention, the NMJ denervation is improved about 30.13% (Fig. 9) by the muscular injection of AAV-NRIP into 8-week old SOD1 mice. The therapeutic effect of NMJ is similar to treatment with SOD1 protein depletion but the treatment time of AAV-NRIP is more close to the time of disease onset than SOD1 RNAi treatment. Intravenous treatment with AAV-Dok7 improves muscle innervation and motor function in SOD1 G93A mice, but there are no obvious effects on proximal motor neuron degeneration. Hence, NRIP is a potential therapeutic agent for SOD1 mice therapy.
The term “a” or “an” as used herein is to describe elements and ingredients of the present invention. The term is used only for convenience and providing the basic concepts of the present invention. Furthermore, the description should be understood as comprising one or at least one, and unless otherwise explicitly indicated by the context, singular terms include pluralities and plural terms include the singular. When used in conjunction with the word “comprising” in a claim, the term “a” or “an” may mean one or more than one.
The term “or” as used herein may mean “and/or. ”
The present invention provides a method for treating amyotrophic lateral sclerosis in a subject, comprising administering to the subject suffering from amyotrophic lateral sclerosis a composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
Amyotrophic lateral sclerosis (ALS) , an adult-onset neurodegenerative disorder, is a progressive and fatal disease characterized by selective death of motor neurons in the motor cortex, brainstem and spinal cord. The incidence of ALS is about 1.9 per 100,000. Patients diagnosed with ALS develop a progressive muscle phenotype characterized by spasticity, hyperreflexia or hyporeflexia, fasciculations, muscle atrophy and paralysis. These motor impairments are caused by the denervation of muscles due to the loss of motor neurons. The major pathological features of ALS include degeneration of the corticospinal tracts and extensive loss of lower motor neurons (LMNs) or anterior horn cells, degeneration and loss of Betz cells and other pyramidal cells in the primary motor cortex. Therefore, ALS is a complex and multifactorial disease and multiple mechanisms hypothesized as responsible for ALS pathogenesis include, but are not limited to, dysfunction of protein degradation, glutamate excitotoxicity, mitochondrial dysfunction, apoptosis, oxidative stress, inflammation, protein misfolding and aggregation, aberrant RNA metabolism, and altered gene expression.
As used herein, the term “treating” refers to therapeutic treatments, wherein the subject is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with ALS. The term “treating” includes, but is not limited to, a preventive treatment and/or a therapeutic treatment.
In one embodiment, the subject is an animal, preferably a mammal, more preferably a human.
The nuclear receptor interaction protein (NRIP) is a transcription factor that only expresses in cell nuclei. It is reported that lack of NRIP gene expression in clinical muscular dystrophy and NRIP knockout mice display weaker muscle strength, indicating that NRIP plays a role in muscle function. In one embodiment, the peptide sequence of the NRIP comprises SEQ ID NO: 2.
In one embodiment, the NRIP is an acetylcholine receptor (AChR) complex protein. In a preferred embodiment, the NRIP is capable of stabilizing the AChR complex.
In one embodiment, the NRIP improves an abnormal muscle defect, a neuromuscular junction (NMJ) degeneration, a α-motor neuron degeneration or motor neuron loss for treating the ALS.
As used herein, the form of the NRIP is a gene, protein or a nucleic acid encoding the protein. A “gene” refers to the smallest, independently functional unit of genetic material that can code for and drive the expression of a protein, e.g., NRIP, or whose presence or absence has a phenotypic consequence on a cell or organism.
The administration of the composition can be carried out via a variety of routes. These routes are designed to provide a local or systemic effect as required. These routes include, but are not limited to, oral, topical, pulmonary, rectal, subcutaneous, intradermal, intranasal, intracranial, intramuscular, intraocular, or intra-articular injection, and the like. The most typical route of administration is intravenous followed by subcutaneous, although other routes can be equally effective. Intramuscular injection can also be performed in the arm or leg muscles. In one embodiment, the administration of the composition is intramuscular injection.
In one embodiment, the NRIP is delivered to the subject systemically. Ideally, systemic delivery means include injection and/or direct injection. In a preferred embodiment, the systemic injection techniques include intravenous delivery by intravenous injection. Intravenous injection is to a peripheral vein of the subject. For this administration route, the NRIP is adminstered/injected directly into the blood stream of the subject. In addition, the NRIP can be delivered to the subject by intramuscular injection.
In addition, the form of the composition can be a gene therapy product, so the NRIP of the composition can be prepared into a form of a nucleotide encoding the NRIP. In one embodiment, the sequence of the nucleotide encoding the NRIP comprises SEQ ID NO: 1. In a preferred embodiment, the nucleotide encoding the NRIP is administered to the subject by a gene delivery system. The term “gene delivery system” as used herein, refers to any forms of carriers that harbor and transport exogenous nucleic acid molecules to a host cell or tissue. The ideal gene delivery system should be harmless to human body, suitable for mass production, and capable of effective transportation of the target gene. In a preferred embodiment, the gene delivery system is a plasmid, a virus, a polymer, DNA, a bacterium, a plant, a nanomaterial, a liposome, or a niosome. In one embodiment, the gene delivery system is a gene delivery vector.
In another embodiment, the virus comprises recombinant adenovirus, adeno-associated virus (AAV) , retrovirus, herpes simplex virus, vaccinia virus, measles virus, poxvirus, Semliki Forest virus, and lentiviral vector. In a preferred embodiment, the viru is AAV. Therefore, the nucleotide encoding NRIP is administered to the subject by viral mediated delivery techniques. There is provided a recombinant viral gene delivery vector which directs the expression of the NRIP in a form suitable for the treatment of ALS.
In one embodiment, the gene delivery vector is a viral vector. Preferably, the gene delivery vector is a recombinant viral gene delivery vector. The recombinant viral gene delivery vector is in a form for administration systemically, for example by intramuscular administration. Suitable recombinant viral gene delivery vectors include, for example, retroviral vectors, herpes simplex virus (HSV) -based vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV) -based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors. In one embodiment, the recombinant viral gene delivery vector comprises an adeno-associated virus (AAV) vector, adenovirus vector and lentiviral vector. In a preferred embodiment, the recombinant viral gene delivery vector is an adeno-associated virus (AAV) vector. In a more preferred embodiment, there is provided a recombinant adeno-associated virus (AAV) gene delivery vector which directs the expression of NRIP suitable for administration to the subject suffering from ALS.
In one embodiment, the AAV vector comprises a nucleotide encoding NRIP. In another embodiment, the AAV can be any one of AAV serotype 1 to 11.
The term “therapeutically effective amount” used herein is a therapeutic dose which can prevent, decrease, stop or reverse a symptom developed in a subject under specific conditions, or partially, completely alleviates symptoms already exist under specific conditions when the subject begins receiving the treatment.
If the NRIP is prepared into a form of a nucleotide encoding the NRIP, the nucleotide encoding the NRIP is capable of being contained in a AAV vector to form an AAV-NRIP. In one embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 4x10
12 vg per kg of body weight of the subject to 8x10
13 vg per kg of body weight of the subject (vg/kg) . In a preferred embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 8x10
11 vg per kg of body weight of the subject to 8x10
13 vg per kg of body weight of the subject. In a more preferred embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 8x10
11 vg per kg of body weight of the subject to 8x10
12 vg per kg of body weight of the subject.
The composition of the present invention further comprises a pharmaceutically acceptable carrier which may be administered to a subject through a number of different routes known in the art. In one embodiment, the composition (comprising the NRIP) and a pharmaceutically acceptable carrier are administered externally, intravenously, subcutaneously, topically, orally or by muscle or inhalation.
As used herein, the term “pharmaceutically acceptable carrier” is determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Carrier can include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, microspheres and emulsions. The term “pharmaceutically acceptable” refers to compounds and compositions which can be administered to mammals without undue toxicity.
The present invention also provides an use of a composition for preparing a drug for treating amyotrophic lateral sclerosis in a subject, wherein the composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
In one embodiment, the form of the composition is a gene therapy product. In another embodiment, the composition further comprises a gene delivery vector. In a preferred embodiment, the gene delivery vector is a viral vector. In a more preferred embodiment, the viral vector is an adeno-associated virus (AAV) vector. Therefore, the NRIP in the composition can be prepared into a form of a nucleotide encoding the NRIP, and the nucleotide encoding the NRIP is delivered into the subject by the gene delivery vector.
The present invention further provides an use of a composition for preparing a drug for treating amyotrophic lateral sclerosis in a subject, wherein the composition comprising a therapeutically effective amount of a gene delivery vector comprising a nucleotide for encoding a nuclear receptor interaction protein (NRIP) .
In one embodiment, the gene delivery vector is a viral vector. In a preferred embodiment, the viral vector comprises retroviral vector, herpes simplex virus (HSV) -based vector and parvovirus-based vector. In a preferred embodiment, the parvovirus-based vector comprises adeno-associated virus (AAV) -based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors. In another embodiment, the viral vector is an adeno-associated virus (AAV) vector.
In another embodiment, the composition is formulated for systemic administration. In a preferred embodiment, the composition is formulated for intramuscular administration or intravenous administration. In a more preferred embodiment, the composition is formulated for injection, infusion or implantation. In another embodiment, the composition is administered by intramuscular injection.
In one embodiment, the therapeutically effective amount of the gene delivery vector ranges from 4x10
12 vg/kg to 8x10
13 vg/kg. In a preferred embodiment, the therapeutically effective amount of the gene delivery vector ranges from 8x10
11 vg/kg to 8x10
13 vg/kg. In a more preferred embodiment, the therapeutically effective amount of the gene delivery vector ranges from 8x10
11 vg/kg to 8x10
12 vg/kg.
The nucleotide encoding the NRIP is capable of being contained in the AAV vector to form an AAV-NRIP when the the gene delivery vector is a AAV vector. In one embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 4x10
12 vg/kg to 8x10
13 vg/kg. In a preferred embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 8x10
11 vg/kg to 8x10
13 vg/kg. In a more preferred embodiment, the therapeutically effective amount of the AAV-NRIP ranges from 8x10
11 vg/kg to 8x10
12 vg/kg.
Therefore, the present invention relates to NRIP induced improvement of muscle defects for treating ALS. Such NRIP can improve the muscle defects and motor neuron degeneration, thereby ameliorating symptoms of ALS.
EXAMPLES
The embodiment of the present invention could be implemented with different content and is not limited to the examples described in the following text. The following examples are merely representative of various aspects and features of the present invention.
Materials and Methods
Immunofluorescence assay (IFA)
For alpha-MNs staining, L5 spinal cord was snap frozen in OCT compound (Tissue- Tek, Torrance, CA) and sectioned into 30μm thickness. The sections were then stained with anti-NeuN (1: 500, Millipore, Temecula, CA) and anti-ChAT (1: 200, Millipore, Temecula, CA) antibody. After washing with PBS, the fluorescent signal was produced by the incubation of Cy3-conjugated goat anti-rabbit or 488-conjugated donkey anti-goat secondary antibody (Jackson Lab., Bar Harbor, ME) . The images were acquired with a Leica TSC SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) .
For neuromuscular junction (NMJ) staining, muscles were fixed with 2%paraformaldehyde for 2 hours at room temperature. Tissues were washed by PBS and dehydrated with 30%sucrose and incubated overnight at 4 ℃. The dehydrated tissues were then embedded in OCT and sectioned to 20μm thickness. The sections were incubated with anti-neurofiliment and anti-synaptophysin for overnight at 4℃. After PBS washing, slides were incubated with a mixture containing 488-conjugated donkey anti-rabbit secondary antibody and anti-BTX antibody. The images were visualized by Leica TSC SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) . The size of NMJ were analyzed by the Image J softeware.
Immunohistochemistry assay (IHC)
Soleus and gastrocnemius muscles were collected and fixed in 4%paraformaldehyde. The paraffin-embedded sections were antigen retrieved by 0.1 M citric acid (pH 6.0) and then incubated with the slow myosin antibody (1: 200, Abcam, Cambridge, MA) at 4℃. for overnight. After washing with PBS and HRP-Polymer (BioGenex, Fremont, CA) incubation for 1 hr in room temperature, the slow myosin positive signal was detected by HRP-Polymer reacted with DAB chromogen (BioGenex, Fremont, CA) .
Western blotting
Muscle lysates were extracted by RIPA lysis buffer (150 mM NaCl, 1%NP-40, 50 mM Tris) , proteins were separated on SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, USA) . The membranes were blocked in 5%non-fat milk at room temparature for 1 hour. Then the blots were reacted with primary antibodies diluted in blocking buffer at 4 ℃ for overnight. The primary antibody that the present invention used were listed in Table 1. The blots were washed by TBST for 10 mins, and then reacted with HRP-conjugated secondary antibody at room temperature for 1 hour. After a serial wash by TBST buffer, proteins expression was detected by using an ECL Western blotting detection system (GE Healthcare Life Sciences, USA) .
Table 1. Antibody lists
AAV production
For production of AAV-NRIP or AAV-GFP, HEK293T cells were co-transfected with the pAAV-MCS-NRIP or pAAV-GFP, pAAV-DJ/8 and the adenovirus helper plasmid pHelper by using calcium phosphate transfection and cultured for 72 hours. In AAV-NRIP, the sequence of the nucleotide encoding the NRIP comprises SEQ ID NO: 1. The AAV particles were purified by CsCl density-gradient ultracentrifugation and dialyzed by dialysis cassette (Slide-A-Lyzer dialysis cassettes, Thermo; MWCO 10K) in dialysis buffer containing 350 mM NaCl with 5%sorbitol in 1X PBS at 4℃. The viral titers were determined by a dot blot assay. The viral particles were divided into small portions and stored at -80℃ until further use.
AAV injection
Mice were anaesthetized by intraperitoneal injection of 2.5%avertin (300 μl) , then intramuscular injected with AAV-GFP (control) or AAV-NRIP (10 μl, 2 × 10
12 vg/ml) in a single shot into each bilateral gastrocnemius and tibialis anterior muscles at age 6 weeks. The mice were monitored daily after recovery until 10 weeks after gene therapy. The therapeutic effects were analyzed for motor performance, muscle oxidative function, α-motor neuron survival, NMJ integrity and axonal innervation at 16 weeks.
Biotin-BTX pulldown assay
The protein lysates from gastrocnemius muscles, WT C2C12 cells and C2C12 KO19 cells were extracted by RIPA buffer (137 mM NaCl, 20 mM Tris-HCl Ph 8.0) . Total protein lysates (5mg) were incubated with biotin-BTX (B-BTX) at 4℃ for 4hrs, which was followed by incubation of streptavidin beads in at 4℃ for 2hrs. The beads were washed by RIPA buffer for three times. The B-BTX conjugated proteins were then eluted by SDS sample buffer and subjected to western blot (WB) analysis for AChR complex detection.
Results
NRIP was a novel acetylcholine receptor (AChR) complex protein
In a previous study, it found that NRIP was expressed in skeletal muscle especially in neuromuscular junction (NMJ) and was required for NMJ structure maintenance. NRIP also could directly bind with actinin 2 which was reported to interact with rapsyn, a well-known NMJ component. Hence, the present invention suggested that NRIP could associate with AChR complex to support AChR cluster integrity. The present invention firstly determined the localization of NRIP with NMJ components including AChR, rapsyn and actinin through IFA stain of gastrocnemius muscles from 16-week-old WT mice. The present invention labeled the AChR cluster by Alexa-594-conjugated α-bungarotoxin (α-BTX) and found that NRIP was colocalized with rapsyn and actinin at NMJ (Figs. 1A and 1B) . The present invention also confirmed that rapsyn was colocalized with actinin in gastrocnemius muscle (Fig. 1C) . These results indicated that NRIP was colocalized with AChR associated protein, rapsyn and actinin, at NMJ. The α-BTX was a competitive antagonist that specifically binds to AChR, hence, biotin-labeled BTX (B-BTX) could be used for AChR pull-down to identify the AChR associated proteins. The present invention further used the B-BTX pull-down assay to determine whether NRIP was a AChR complex protein that could be pull-downed with AChR complex in gastrocnemius muscle and C2C12 myotubes. In gastrocnemius muscle, NRIP was pull-downed with AChR and was accompanied by AChR associated protein, rapsyn and actinin (Fig. 1D) . NRIP, rapsyn and actinin were also pull-downed with AChR in C2C12 myotubes (Fig. 1E) . These results indicated that NRIP was an AChR complex protein to interact with AChR associated protein, rapsyn and actinin.
NRIP was required for stabilization of AchR complex
It had been revealed that rapsyn was capable of interacting with AChR to stabilize the AChR cluster at NMJ. In IFA and B-BTX pull-down experiments, the present invention demonstrated that NRIP was capable of colocalizing and associating with rapsyn and actinin (Fig. 1) . Thus, the present invention suggested that NRIP was required for AChR-rapsyn interaction. The protein expression of rapsyn was the same in gastrocnemius muscles from wild-type (WT) and NRIP cKO mice, however, in B-BTX pull-down assay, the AChR-interacted rapsyn was decreased in NRIP cKO mice compared to WT mice (Fig. 2A) . The decrease of AChR-rapsyn binding affinity was also observed in NRIP deficient C2C12 myotubes (KO19) compared to WT C2C12 myotubes (Fig. 2B) . Studies also showed that binding of actinin and rapsyn was crucial for AChR cluster formation. Thus, the present invention suggested that NRIP was required for rapsyn-actinin interaction to stabilize AChR cluster. The protein expression of actinin and rapsyn was the same in gastrocnemius from WT and NRIP cKO mice (Fig. 2C, input) . After immunoprecipitation of actinin, the immunoprecipitated rapsyn was decreased in NRIP cKO mice compared to WT mice (Fig. 2C) . Immunoprecipitation of actinin was also performed in KO19 and WT C2C12 myotubes, results showed that rapsyn-actinin binding affinity was decreased in NRIP-deleted C2C12 myotubes (KO19) compared to WT C2C12 myotubes (Fig. 2D) . These results indicated that NRIP was required for AChR-rapsyn-actinin complex stabilization.
Restore the expression of NRIP improved the muscle dysfunction in NRIP cKO mice
It was previously reported that the muscle-specific NRIP cKO mice showed the phenotypes similar to MNDs, such as NMJ abnormality, motor neuron degeneration and motor function defects at adult stage (16 wk) . To know whether AAV-NRIP gene therapy could improve NMJ function and motor neuron deficits in cKO mice, the present invention injected AAV-NRIP or AAV-GFP (control) (2 × 10
10 vg in a total volume of 10 μl for each muscles) into bilateral GAS and TA muscles of cKO mice at age 6 weeks and examined the muscle function (such as slow myosin protein, COX, and SDH) , motor neurons survival and NMJ integrity at age 16 weeks (10 weeks after virus injection) (Fig. 3A) . NRIP protein expression in injected muscles of AAV-NRIP-treated cKO mice could be detected 10 weeks after injection (Fig. 3B) . In order to know whether AAV-NRIP delivery could improve muscle function in cKO mice, the present invention first dissected soleus (SOL) muscles from 16-week-old treated cKO mice to check oxidative fiber expression pattern by immunohistochemistry (IHC) staining. Fresh-frozen SOL sections were cut into 10-μm-thick slices by cryostat microtome and stained for anti-slow myosin heavy chain antibody. The dark brown stained fibers were slow myosin positive myofibers (Fig. 3C) . Quantitative data showed that the abnormal oxidative function was significantly improved in AAV-NRIP-treated cKO mice than control at age 16 weeks by counting the proportion of oxidative myofibers (50.84 %vs. 41.55 %, P < 0.05; Fig. 3D) and oxidative fiber cross-sectional area (CSA; 50.99 %vs. 39.74 %, P < 0.05; Fig. 3E) , but the rotarod data indicated that there was no significant extending tendency of the retention time in AAV-NRIP-treated cKO mice than the control (at 20 rpm, 159.1 s vs. 103.5 s, P = 0.3605; Fig. 3F) .
Muscle NRIP retrogradely improved the α-MN degeneration
To know whether NRIP gene therapy could retrogrdely improve motor neuron degeneration and NMJ abnormality in cKO mice, the spinal cord of the L3-L5 segment was dissected to check the number of α-motor neurons by immunofluorescence assay (IFA) . The present invention labeled the MNs by staining the neuronal nuclear protein (NeuN) and choline acetyltransferase (ChAT) . The NeuN and ChAT double-positive (yellow) MNs with size larger than 500 μm
2 were defined as α-motor neurons (Fig. 4A) . Quantitative data showed that the α-motor neuron number was significantly higher in AAV-NRIP-treated cKO mice than the control treated cKO at age 16 weeks (21.3 vs. 18.2 per section, P < 0.05; Fig. 4B)
NRIP improved the NMJ structure in NRIP cKO mice
In previous results, the present invention found that overexpression of NRIP in NRIP-deleted muscle improved the muscle function and MN survival (Fig. 3 and Fig. 4) . Next, the present invention would like to examine whether the retrogrdely improvement of MN survival was resulted from NMJ stabilization in NRIP-improved skeletal muscle. The sections of gastrocnemius muscles were stained by alpha-bungarotoxin (α-BTX) , neurofilament (NF) and synaptophysin (SYN) used to label acetylcholine receptors (AChRs) , motor axons and nerve terminals respectively at the NMJs. The overlap of AChR (red) and axonal terminals (green) was defined as axonal innervation, conversely, AChR (red) only considered as axonal denervation (Fig. 5A) . Images were captured by confocal microscopy and the NMJ sizes were evaluated by the ImageJ software. The NMJ size of GAS muscles was significantly larger in AAV-NRIP-treated cKO mice than the control-treated cKO at age 16 weeks (10 weeks after gene therapy) (249.5 μm
2 vs 219.0 μm
2, P < 0.05; Fig. 5B) . The proportion of denervated endplates were significantly decreased in AAV-NRIP-treated cKO mice than the control at age 16 weeks respectively (2.59 %vs. 5.13 %, respectively, P < 0.01; Fig. 5C) . Taken together, muscle administering AAV-NRIP in NRIP cKO mice was capable of improving the abnormal muscle defect, NMJ degeneration and motor neuron loss in muscle-specific NRIP cKO mice.
The expression of NRIP was decreased in SOD1 G93A mice
Because of the present invention found that AAV-NRIP delivering in skeletal muscle improved abnormal phenotypes in muscle-specific NRIP cKO mice (Figs. 3-5) , such as motor neuron loss and NMJ degeneration. Thus, the present invention suggested that NRIP might also be a therapeutic agent for motor neuron diseases (MNDs) . Amyotrophic lateral sclerosis (ALS) is one of the MNDs and usually onset in adult life. The most commonly used model of ALS is the human SOD1 G93A transgenic mice, which displays a number of features similar to human ALS. In this experiment, the present invention used SOD1 G93A mice to examine whether NRIP could be a novel therapeutic agent for ALS. To further investigate the role of NRIP in SOD1 G93A mice, the present invention first extracted protein lysates of spinal cord, GAS and tibialis anterior (TA) muscles from 8-week-old SOD1 G93A and age-matched WT mice and analyzed by the western blot analysis. In spinal cord, the relative NRIP level was approximately 8-fold decreased in SOD1 G93A mice compared with WT (0.12 vs. 0.96 NRIP/GAPDH, P < 0.0001; Fig. 6A, right panel) . In GAS and TA muscles, additionally, the relative NRIP level was approximately 5-and 3-fold decreased in SOD1 G93A mice compared with WT (0.15 vs. 0.81 NRIP/GAPDH, P < 0.001; 0.23 vs. 0.68 NRIP/GAPDH, P < 0.05, respectively; Figs. 6B and 6C, right panel) . To sum up, these results suggested that NRIP might play a role in SOD1 G93A mice and AAV-NRIP could be a therapeutic agent for ALS mice.
The therapeutic effect of NRIP on SOD1 G93A mice
NRIP expression was downregulated in skeletal muscle of SOD1 G93A mice (Fig. 6) ; and SOD1 G93A mice had been reported that it performed muscle dystrophy. Hence, the present invention would like to know whether the muscle dystrophy in SOD1 G93A mice could be improved by intramuscular (IM) injection of AAV-NRIP in TA and GAS muscles. The SOD1 G93A mice received the AAV-NRIP and AAV-GFP injection at P60, the gastrocnemius and soleus muscles were harvested and weighted at P120. The muscle weight (MW) of gastrocnemius and soleus were measured and normalized to body weight (BW) . Results showed that the MW/BW ratio of gastrocnemius and soleus muscles were decreased in SOD1 G93A injected with AAV-GFP compared to WT mice and the injection of AAV-NRIP did not improve the muscle loss of SOD1 G93A mice (Figs. 7A and 7B) . The present invention also measured the therapeutic benefit on rotarod performance of SOD1 G93A mice treated with AAV-NRIP. Results showed that SOD1 G93A mice infected with AAV-NRIP were fell off faster than WT mice, the injection of NRIP did not improve the motor deficits on SOD1 G93A mice (Fig. 7C) . These results indicated that the local IM injection of NRIP had no therapeutic effect on muscle and motor dysfunction. However, when the present invention looked into the therapeutic effect of AAV-NRIP on α-MN survival in L3-L5 spinal segment through NeuN and ChAT costaining (Fig. 8A) , the present invention found that AAV-NRIP which was injected in muscle successfully improved the survival of α-MNs in SOD1 G93A compared to AAV-GFP injected SOD1 G93A mice (18.26 vs. 14.41, P < 0.05; Fig. 8B) . The present invention suggested that NRIP could retrogrdely improve the MN through stabilizing the NMJ structure. Next, the present invention would like to examine whether the NMJ integrity was improved in AAV-NRIP treated skeletal muscle. The present invention calculated the area of NMJ and found that NMJ area was decreased in SOD1 G93A mice injected with AAV-GFP compared to WT (220.1 vs. 264.2 μm
2; Fig. 9A) . In the AAV-NRIP injected SOD1 G93A mice, the NMJ area was increased to 234.7 μm
2 compared to AAV-GFP group but not significant (Fig. 9A) . However, the number of NMJ was improved in SOD1 G93A mice injected with AAV-NRIP compared to SOD1 G93A mice injected with AAV-GFP (237.5 μm
2 vs. 195.2, P < 0.05; Fig. 9) . The innervation is the important parameter for neuron and muscle interaction, thus the present invention also compared the percentage of innervated NMJ to determine the therapeutic effect of retrograde improvement of MN number from muscle injected AAV-NRIP. The results showed that AAV-NRIP improved the NMJ innervation in SOD1 G93A mice compared to AAV-GFP group (70.42 %vs. 59.97 %, P < 0.05; Fig. 9C) . These data indicated that the improvement of NMJ number and innervation retrogradely improved the α-MN survival in ALS mice treated with AAV-NRIP. Thus, the NRIP could be a novel agent of gene therapy on ALS mice.
It had been reported that the locomotor activity of SOD1 G93A mice (ALS model mice) was significantly reduced from disease onset to the end-stage in comparison to WT. Hence, the present invention determined the therapeutic effect of AAV-NRIP in locomotor activity of SOD G93A mutant mice. The open field test was used as assessment of locomotor function and voluntary activity for individual mice. The SOD1 G93A mice were infected with AAV-GFP (N=5) and AAV-NRIP (N=5) at the age of 60 days and subjected to the open field test at the age of 126 days. The mice were allowed to move freely within a clean, opaque acrylic box (40 × 40 × 40 cm
3) for 10 minutes. The movement of the mice were tracked using EthoVision Video Tracking Software (Noldus) . The total distance moving (total distance mice traveled in the box) and the rearing frequency (frequency which the mice stands on its hind limb in the box) were measured during the test. The results showed that AAV-NRIP could improve the performance of total distance moving compared to the AAV-GFP (2569.08 cm vs. 1858.26 cm, P < 0.05; Fig. 10A) . The rearing frequency was also improved in AAV-NRIP treatment compared to AAV-GFP treatment (31.60 vs. 13.80, P < 0.05; Fig. 10B) . These results indicate that AAV-NRIP could improve the locomotor activities of ALS mice. Therefore, AAV-NRIP could be a therapeutic agent for ALS.
AAV-NRIP was a potential ALS therapeutic drug for AAV-NRIP intramusle injection of SOD1 G93A mice at day 60; and the present invention found that AAV-NRIP treatment of SOD1 mice could improve motor neuron number growth, increase the size of neuromuscle junction and decrease axon denervation at day 120; along with increased locomotor activities such as total distance travel in the box and the rearing frequency of mice standing on its hind limb in the box at day 126.
Those skilled in the art recognize the foregoing outline as a description of the method for communicating hosted application information. The skilled artisan will recognize that these are illustrative only and that many equivalents are possible.
Claims (6)
- A method for treating amyotrophic lateral sclerosis in a subject, comprising administering to the subject suffering from amyotrophic lateral sclerosis a composition comprising a therapeutically effective amount of a nuclear receptor interaction protein (NRIP) .
- The method of claim 1, wherein the NRIP is administered to the subject by a gene delivery system.
- The method of claim 2, wherein the gene delivery system is a plasmid, a virus, a polymer, DNA, a bacterium, a plant, a nanomaterial, a liposome, or a niosome.
- The method of claim 3, wherein the virus comprises recombinant adenovirus, adeno-associated virus (AAV) , retrovirus, herpes simplex virus, vaccinia virus, measles virus, poxvirus, Semliki Forest virus and lentiviral vector.
- The method of claim 4, wherein the virus is AAV.
- The method of claim 1, wherein the administration of the composition is intramuscular injection.
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