Methods for treating polyq disease and novel adipose tissue derived mesenchymal stem cells

Abstract

This invention provides the use of adipose-derived stem cells expressing activin A, or a combination of activin A and modified adipose-derived stem cells, for treating polyQ disorders, such as spinocerebellar ataxia 3 (SCA 3), by reducing misfolded and aggregated polyQ proteins, such as ataxia-3 protein. This invention also provides compositions for reducing misfolded and aggregated polyQ proteins, wherein the composition comprises a modified adipose-derived stem cell population and activin A.

Description

Methods for treating POLYQ diseases and novel adipose tissue-derived mesenchymal stem cells Cross-reference to related applications This application claims the benefit of priority of Australian Provisional Application No. 2023901470, filed on May 15, 2023, the entire content of which is incorporated herein by reference. The present invention relates to a method for treating POLYQ diseases and novel adipose tissue-derived mesenchymal stem cells. There are nine hereditary neurodegenerative disorders, collectively known as polyglutamine (polyQ) diseases, including Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA), and spinocerebellar ataxias (SCA) 1, 2, 3, 6, 7, and 17. PolyQ diseases are caused by polyglutamine (polyQ) expansions in their corresponding disease proteins and share a common pathological feature: nuclear accumulation and inclusion body formation of polyQ disease proteins. The most common pathogenic cascade in polyQ diseases is the misfolding and aggregation of the expanded polyQ protein (T. Takeuchi et al., "Disease protein misfolding and aggregations a therapeutic target for polyQ diseases", Brain Science 2017:128; M. Arrasate et al., "Protein aggregates in Huntington's disease", Exp Neuro 20-12;1-11; A. Klement et al., "Ataxin-1 Nuclear Localization and Aggregation: Role in Polyglutamine-Induced Disease in "SCA1 Transgenic Mice", Cell 1998:41; M Mark et al., "Spinocerebellar Ataxia Type 6 Protein Aggregates Cause Deficits in Motor Learning and Cerebellar Plasticity", The Journal of Neuroscience, 2015:8882 - 8895). To date, there is no definitive treatment to cure or slow the progression of polyQ diseases. There is a need to reduce mutant polyQ proteins and effectively treat polyQ diseases. The present invention addresses this need and others. In one embodiment, the present invention provides a method for treating polyQ diseases, which comprises the step of administering a therapeutically effective amount of each of the following to an individual in need: (a) activin A; and (b) a population of modified adipose tissue-derived stem cells (modified ADMSCs), the population having the phenotypic characteristics of CD273, CD46, CD55, CXCR4, CD90, CD105, and CD73 (but not expressing CD45, CD34, CD11b, CD19, and HLA-DR), and maintaining osteogenic, chondrogenic, and adipogenic differentiation capabilities. The present invention also provides a method for treating polyQ diseases, which comprises the step of administering a therapeutically effective amount of the ADMSCs expressing activin A described herein to an individual in need, the ADMSCs expressing activin A having the phenotypic characteristics of CD273, CD46, CD55, CXCR4, CD90, CD105, and CD73 (but not expressing CD45, CD34, CD11b, CD19, and HLA-DR). Also provided is a method for reducing mutant polyQ proteins (such as ATXN-3) in an individual, which comprises the step of administering a therapeutically effective amount of (a) activin A and (b) the modified ADMSCs described herein to an individual in need. Also provided is a method for reducing mutant polyQ proteins (such as ATXN-3) in an individual suffering from a polyQ disease, which comprises the step of administering a therapeutically effective amount of the ADMSCs expressing activin A described herein to an individual in need. Some embodiments provide isolated populations of modified ADMSCs (ADMSCs expressing activin A) that express adipose tissue-derived stem cell markers including CD90, CD105, and CD73, but not CD45, CD34, CD11b, CD19, and HLA-DR, and maintain osteogenic, chondrogenic, and adipogenic differentiation capabilities. Other embodiments provide pharmaceutical compositions comprising at least one activin A ADMSC described herein and a pharmaceutically acceptable carrier or excipient. As used in this patent, the terms "invention", "the invention", "this invention", and "the present invention" are intended to generally refer to all subject matter of this patent and the following patent application claims. Statements containing these terms should be understood not to limit the subject matter described herein or the meaning or scope of the following patent application claims. The embodiments of the invention covered by this patent are defined by the following claims rather than by this description of the invention. This description of the invention is a high-level overview of various aspects of the invention and introduces some concepts further described in the following detailed description section. This description of the invention is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the appropriate portions of the entire specification, any or all of the drawings, and each claim. The invention will become more apparent when the following drawings and detailed description are read. As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "modified ADMSC" means one modified ADMSC or more than one modified ADMSC. As used herein, "therapeutically effective amount" means an amount of the modified ADMSCs or pharmaceutical compositions disclosed herein sufficient to cause a reduction in mutant polyQ protein (such as ATXN-3) or to ameliorate one or more symptoms and signs of a polyQ disease, including (but not limited to) ataxia, involuntary movements, cognitive impairment, which can be clinically or radiologically detected by various imaging modalities. As used herein, the terms "treating", "treated", or "treatment" refer to palliative use or outcome, and / or slowing or inhibiting the progression of a polyQ disease, the formation of mutant polyQ protein, and neuronal intracellular inclusions. The term "subject" can refer to a vertebrate that has or is suspected of having a polyQ disease or a vertebrate considered in need of treatment for a polyQ disease. Subjects include warm-blooded animals such as mammals, primates and more preferably humans. Non-human primates are also subjects. The term subject includes domesticated animals (such as cats, dogs, etc.), livestock (such as cows, horses, pigs, sheep, goats, etc.) and laboratory animals (such as mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses and pharmaceutical formulations are contemplated herein. The expression level or surface density of a surface marker (such as CD273) on the surface of a modified ADMSC is " + " as long as the expression of the surface marker can be detected by any conventional means. In one exemplary embodiment, " + " means that the cell surface marker can be detected in fluorescence-activated cell sorting / flow cytometry or magnetic beads at a level higher than that of an isotype control; or can be detected at a level higher than background in quantitative or semi-quantitative RT-PCR. The expression level or surface density of a surface marker (such as CD45) is " - " as long as the expression of the surface marker cannot be detected by any conventional means. In one exemplary embodiment, " - " means that the cell surface marker cannot be detected in fluorescence-activated cell sorting / flow cytometry or magnetic beads at a level higher than that of an isotype control; or cannot be detected at a level higher than background in quantitative or semi-quantitative RT-PCR. As used herein, "substantially free of" means less than 2%, 1% or 0.5%. All numbers herein are understood to be modified by "about". As used herein, the term "about" is intended to cover variations of ± 10%. The terms "adipose tissue-derived mesenchymal stem cells" and "adipose tissue-derived stem cells" are used interchangeably. The abbreviation "ADMSC" applies to both "adipose tissue-derived mesenchymal stem cells" and "adipose tissue-derived stem cells". Modified ADMSC The modified ADMSCs of the present invention are derived from native mesenchymal stem cells isolated from adipose tissue. Native mesenchymal stem cells isolated from adipose tissue (native ADMSC) are characterized by CD105 + , CD73 + , CD90 + , CD45 - , CD34 - , CD11b - , CD19 - , HLA-DR - and CD273 - and maintain osteogenic, chondrogenic and adipogenic differentiation capabilities. In one embodiment, the native ADMSC is CD46 - and CD55 - and CXCR4 - . If less than 2% of the ADMSCs express CD45, CD34, CD11b, CD19 and HLA-DR, the population of modified ADMSCs described herein is considered a purified population of modified ADMSCs. Example 1 of this application illustrates an embodiment of a culture method for generating the modified ADMSCs of the present invention, wherein the native ADMSCs are cultured in MSC medium for about 12 hours to about 7 days, and the MSC medium contains α-MEM, UltraGRO and heparin. In another embodiment, the MSC medium contains epidermal growth factor (EGF), fibroblast growth factor 2 (FGF-2) and insulin-transferrin-selenium (ITS) supplement. In one embodiment, the modified ADMSCs are characterized by expressing CD273, CD46, CD55, CXCR4, which are not present in naturally occurring ADMSCs or native ADMSCs. The phenotypic characteristics of the modified ADMSCs are CD273 + and CD46 + and CD55 + and CXCR4 + and CD105 + and CD73 + and CD90 + , but not CD45 - and CD34 - and CD11b - and CD19 - and HLA-DR - . The modified ADMSCs maintain osteogenic, chondrogenic and adipogenic differentiation capabilities. ADMSCs expressing activin A In one embodiment, one or more activin A plasmids are inserted into the modified ADMSCs described herein. The modified ADMSCs can express activin A and are referred to as ADMSCs expressing activin A. The modified ADMSCs and the ADMSCs expressing activin A of the present invention are non-naturally occurring because native ADMSCs do not express CD273. The modified ADMSCs can be from a single individual (i.e., autologous), or pooled from multiple individuals (allogeneic non-autologous). Pharmaceutical compositions In one embodiment, a pharmaceutical composition is provided that comprises at least one of the modified ADMSCs described herein, activin A, and a pharmaceutically acceptable medium, carrier, or excipient. Advantageously, this combination has a synergistic effect on reducing mutant polyQ proteins (such as ATXN-3) or improving one or more symptoms and signs of polyQ diseases. Suitable media are, for example, water, saline, dextrose, glycerol, ethanol, dimethyl sulfoxide (DMSO), trehalose, and combinations thereof. Suitable excipients are, for example, wetting agents, emulsifying agents, or pH buffering agents. The pharmaceutically acceptable carrier can contain physiologically acceptable compounds that are used, for example, to stabilize or increase or decrease the absorption or clearance rate of the pharmaceutical compositions of the present invention. Physiologically acceptable compounds can include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposome carriers; or other stabilizers and / or buffering agents. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives. Excipients can be nonionic surfactants, polyvinylpyrrolidone, human serum albumin, aluminum hydroxide, agents with anesthetic effects, and various unmodified and derivatized cyclodextrins. In one embodiment, the nonionic surfactant can include polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. The polyvinylpyrrolidone can preferably be Plasdone C15, a pharmaceutical grade polyvinylpyrrolidone. The pharmaceutical composition containing the excipient or carrier is formulated by well-known conventional methods. In an exemplary embodiment, the pharmaceutical composition is substantially free of bone marrow-derived mesenchymal stromal cells. The present invention also provides a method for treating polyQ diseases by administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition described herein or ADMSCs expressing activin A described herein. PolyQ diseases include Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and six types of spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17). In some embodiments, a method for reducing mutant polyQ proteins (such as ATXN-3) is provided by administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition described herein or ADMSCs expressing activin A described herein. In one embodiment, the polyQ protein of SCA3 is ATXN-3. In another embodiment of the present invention, the use of the modified ADMSCs and activin A combination or ADMSCs expressing activin A described herein for the manufacture of a medicament for treating polyQ diseases or reducing disease polyQ proteins is provided. The administration routes of the pharmaceutical composition, modified ADMSCs and activin A combination, and ADMSCs expressing activin A of the present invention include (but are not limited to): intravenous, intramuscular, subcutaneous, oral, topical, subcutaneous, intradermal, transdermal, subcutaneous, parenteral, rectal, spinal, intrathecal, intraventricular, intracranial, or intervertebrate administration. In one embodiment, the pharmaceutical composition, modified ADMSCs and activin A combination, or ADMSCs expressing activin A of the present invention are administered by intravenous injection or infusion. The modified ADMSCs of the present invention can be administered simultaneously with, before, or after activin A to treat polyQ diseases. The ADMSCs expressing activin A or pharmaceutical composition described herein can be administered in a single dose treatment or in multiple dose treatments over a schedule and time period according to the age, weight, and condition of the individual, the specific composition used, the administration route, whether the ADMSCs expressing activin A or the pharmaceutical composition of the present invention are for prophylactic or curative purposes, etc. In some embodiments, the ADMSCs expressing activin A or the pharmaceutical composition of the present invention are administered once a year, once every 6 months, once every 4 months, once every 3 months, once a month, twice a month, three times a month, once every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, once every other day (qod), once a day (qd), twice a day (qid), or three times a day (tid). Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for human use. In one embodiment, the dosage is in an ED including minimal or no toxicity. 50 within the cyclic concentration range. The dosage can vary within this range depending on the dosage form used and the route of administration employed. In another embodiment, the therapeutically effective dosage can initially be estimated by cell culture analysis. Dosages can be formulated using animal models to achieve a cyclic plasma concentration range, including the IC measured in cell cultures 50 (i.e., the concentration at which the antibody achieves half-maximal inhibition of symptoms). Sonderstrup, Springer, Sem. Immunopathol. 25: 35-45, 2003. Nikula et al., Inhal. Toxicol. 4(12): 123-53, 2000. The pharmaceutical composition is formulated to contain a therapeutically effective amount of the modified ADMSCs and activin A described herein or ADMSCs expressing activin A, wherein the amount depends on the individual to be treated and the condition to be treated. The specific dosage for any particular individual depends on a variety of factors, including the activity of the specific modified monocytes or modified ADMSCs, age, weight, general health, sex, diet, time of administration, route of administration and rate of excretion, drug combination, and the severity of the particular disease being treated. An exemplary non-limiting range of the therapeutically or prophylactically effective amount of the modified ADMSCs / ADMSCs expressing activin A of the present invention is at least about 1 × 10 4 cells / dose to about 1×10 10 cells / dose. Other dosages are also possible, including (but not limited to): 1 × 10 5 、2 × 10 5 、3 × 10 5 、4 × 10 5 、5 × 10 5 、6 × 10 5 、7 × 10 5 、8 × 10 5 、9 × 10 5 、1 × 10 6 、2 × 10 6 、3 × 10 6 , 4 × 10 6 , 5 × 10 6 , 6 × 10 6 , 7 × 10 6 , 8 × 10 6 , 9 × 10 6 , 1 × 10 7 , 2 × 10 7 , 3 × 10 7 , 4 × 10 7 , 5 × 10 7 , 6 × 10 7 , 7 × 10 7 , 8 × 10 7 , 9 × 10 7 , 1 × 10 8 , 2 × 10 8 , 3 × 10 8 , 4 × 10 8 , 5 × 10 8 , 6 × 10 8 , 7 × 10 8 , 8 × 10 8 , 9 × 10 8 or 1 × 10 9 , 2 × 10 9 , 3 × 10 9 , 4 × 10 9 , 5 × 10 9 , 6 × 10 9 , 7 × 10 9 , 8 × 10 9 , 9 × 10 9 . The following examples for implementing specific aspects of the present invention are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Examples Example 1: Modified ADMSC Native ADMSC was isolated from the stromal vascular fraction of human adipose tissue and characterized by CD105 + , CD73 + , CD90 + , CD45 - , CD34 - , CD11b - , CD19 - , HLA-DR - , CD. Native ADMSC was cultured in MSC medium containing α-MEM (purchased from Gibco, USA), 5% Helio Bioscience UltraGRO (purchased from AventaCell BioMedical Corp, USA), and 2 U / ml heparin (purchased from MP Biomedicals, USA), and subcultured when the cells reached 80% confluence. Before trypsin digestion, the MSC medium was removed and the cells were washed with 1×DPBS (Gibco, 14190-144). The cells were incubated with 0.25% trypsin-EDTA (Gibco, 25200072) for 3 minutes at room temperature. After cell separation, MSC medium was added to neutralize the trypsin. The cultured cells were transferred to a centrifuge tube and centrifuged at 330 g for 5 minutes. The cell pellet was resuspended in MSC medium and the cells were cultured in a 5% CO 2 incubator at 37°C. After 12 passages, the cultured ADMSCs (hereinafter referred to as modified ADMSCs) were analyzed using flow cytometry. Table 1 below shows the percentage of modified ADMSCs expressing specific biomarkers. Table 1. In addition, the modified ADMSCs retained chondrogenic, osteogenic, and adipogenic differentiation capabilities. Example 2: Maintenance of SH-SY5Y ATXN3 / Q75 cells SH-SY5Y ATXN3 / Q75 cells and the culture system were obtained from Dr. Guey-Jen Lee-Chen at National Normal University, Taiwan. SH-SY5Y ATXN3 / Q75 cells were cultured in a medium containing DMEM (Dulbecco's Modified Eagle Medium) / Hams F-12 (DMEM / F12) (Corning, 16-405-CV) supplemented with 10% FBS (Corning, 35-010-CV), 5 μg / ml blasticidin (InvivoGen, ant-bl), and 100 μg / ml hygromycin (InvivoGen, ant-hg). When the cells reached 80% confluence, the SH-SY5Y ATXN3 / Q75 cells were cultured. Before trypsin digestion, the medium was removed and the cells were washed with 1×DPBS (Gibco, 14190-144) and incubated with 0.25% trypsin-EDTA (Gibco, 25200072) for 3 minutes at room temperature. After cell dissociation, DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blasticidin, and 100 μg / ml hygromycin was added to neutralize the trypsin. The SH-SY5Y ATXN3 / Q75 cells were transferred to a centrifuge tube and centrifuged at 330 g for 5 minutes. The cell pellet was resuspended in DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blasticidin, and 100 μg / ml hygromycin, and the cells were cultured at 37°C in a 5% CO2 incubator. Within 7 days, the cells would become 80% confluent and could be used for other experiments. Example 3: Effect of modified ADMSCs on the SCA3 diseased cell model SH-SY5Y ATXN3 / Q75 cells Dr. GJ Lee (National Taiwan Normal University) generated an SCA3 cell model in which the accumulation of polyQ amplified ataxin-3 to form aggregates in neuronal cells, a phenomenon commonly observed in the brains of SCA3 patients [6]. Different ratios of modified ADMSCs and SH-SY5Y ATNX3 / Q75 cells (0:1, 1:20, 1:15, and 1:10) were examined to determine the dose-dependent efficacy of modified ADMSCs. Briefly, 9×10 3 modified ADMSCs, 1.2×10 4 modified ADMSCs, and 1.8×10 4 ADMSCs were seeded into the upper compartment of a six-well transwell system (Falcon, 353090) and cultured in MSC medium containing α-MEM, 5% Helio Bioscience UltraGRO, and 2 U / ml heparin. 2.0×10 5 SH-SY5Y ATXN3 / Q75 cells were placed in the lower compartment of a six-well transwell system (Falcon, 353502) and cultured in a medium containing DMEM / F12, 10% FBS, and 10 μM retinoic acid (Sigma, R2625). The next day, the modified ADMSC-seeded inserts were transferred to the six-well plate in the lower compartment where SH-SY5Y ATXN3 / Q75 cells were cultured in a medium containing DMEM / F12 and 10% FBS. SH-SY5Y ATXN3 / Q75 cells were treated with 5 μg / ml doxycycline (Sigma, D9891) to induce the expression of mutant ataxin-3. 5 μg / ml doxycycline and 10 μM retinoic acid were added every three days for one week. Example 4: Effect of Activin A on the SCA3 Diseased Cell Model (SH-SY5Y ATXN3 / Q75 Cells) The effects of different concentrations of Activin A (0.2 to 5 ng / ml) (Biolegened, 592002) on the SCA3 diseased cell model (SH-SY5Y ATXN3 / Q75 cells) were examined. First, SH-SY5Y ATXN3 / Q75 cells were seeded at 20,833 cells / cm 2Cells were seeded at a density of in a six-well cell culture dish (Corning, 353046), where the DMEM / F12 medium was supplemented with 10% FBS, 5 μg / ml blasticidin, 100 μg / ml hygromycin, and 10 μM retinoic acid. The next day, SH-SY5Y ATXN3 / Q75 cells were treated with 5 μg / ml doxycycline, 10 μM retinoic acid, and different doses (0.2, 1.0, and 5.0 ng / ml) of activin A. 5 μg / ml doxycycline and 10 μM retinoic acid were added every three days for one week. Results: Activin A decreased the expression of mutant ataxin-3 in the SCA3 cell model. Figure 1 shows that activin A decreased the level of mutant ataxin-3 in SH-SY5Y ATXN3 / Q75 cells in a dose-dependent manner. Example 5: Combined effects of modified ADMSCs and activin A on the SCA3 diseased cell model SH-SY5Y ATXN3 / Q75 cells 9×10 3 modified ADMSCs, 1.2×10 4 modified ADMSCs, and 1.8×10 4 modified ADMSCs were seeded in the upper compartment of a six-well transwell system and cultured in MSC medium containing α-MEM, 5% Helio Bioscience UltraGRO, and 2 U / ml heparin. 2.0×10 5 SH-SY5Y ATXN3 / Q75 cells were placed in the lower compartment of a six-well transwell system and cultured in DMEM / F12 medium supplemented with 10% FBS and 10 μM retinoic acid. The next day, the modified ADMSC-seeded insert was transferred to a six-well dish in the lower compartment, where SH-SY5Y ATXN3 / Q75 cells were cultured in DMEM / F12 medium supplemented with 10% FBS. SH-SY5Y ATNX3 / Q75 cells and modified ADMSCs were treated with various concentrations of activin A (0.2, 1.0, and 5.0 ng / ml). 5 μg / ml doxycycline and 10 μM retinoic acid were added every three days for one week. Pulse Shape Analysis (PulSA): On day 7, SH-SY5Y ATXN3 / Q75 cells with the different treatment conditions described above were collected and resuspended in DPBS containing 2% FBS for flow cytometry (Guava ® Analyze using the easyCyte™. Based on the analysis results of Guava software, measure the level of mutant ataxin-3. Use the combination index (CI) method to evaluate the effects of modified ADMSCs and / or activin A (Chou, T.C., Cancer Res, 2010. 70(2): pp. 440 - 6). Results: Figures 2A and 2B show the synergistic effect of the combination of various doses of modified ADMSCs and various doses of activin A in reducing mutant ataxin-3 (CI value less than 1 indicates synergy). Example 6: Modified adipose tissue-derived mesenchymal stem cells expressing activin A (ADMSCs expressing activin A) One day before transfection, culture 4.8×10 4 modified ADMSCs in 1.5 ml of MSC medium, which contains MEM, 5% Helio Bioscience UltraGRO and 2 U / ml heparin per well, so that the modified ADMSCs reach 50 - 60% confluence at the time of transfection. Prepare diluted plasmid DNA, activin A expression plasmid (Sino Biological, HG10429-UT) or control plasmid (Sino Biological, CV011) in Opti-MEM I medium (Thermo Fisher Scientific, 11058021) and Lipofectamine Stem Cells (Thermo Fisher Scientific, STEM00001) separately in different eppendorf tubes, and incubate at room temperature for 5 minutes. After incubation, combine the diluted plasmid DNA with the diluted Lipofectamine Stem Cells, mix gently and incubate at room temperature for 10 minutes to allow the formation of plasmid DNA-Lipofectamine Stem Cell complexes. Add 150 μl of plasmid DNA-Lipofectamine Stem Cell complexes to each well containing modified ADMSCs and MSC medium, and gently mix the mixture by rocking the plate back and forth. Incubate the cells in a CO 2 incubator at 37°C for 24 hours. Subsequently, supplement the ADMSCs expressing activin A with MSC medium. Culture the ADMSCs expressing activin A under standard conditions (5% CO 2 ; 37°C) for 24 hours. Collect the conditioned medium samples, centrifuge to remove cell debris, and then freeze in aliquots at -20°C. Effect of conditioned medium on SH-SY5Y ATXN3 / Q75 cells with SCA3 lesions: SH-SY5Y ATXN3 / Q75 cells were treated with different doses of conditioned medium (secreted from ADMSCs expressing activin A as described above), and the expression level of mutant ataxin-3 was analyzed. First, SH-SY5Y ATXN3 / Q75 cells were seeded in six-well cell culture dishes at a density of 20,833 cells / cm 2 in DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blasticidin, 100 μg / ml hygromycin, and 10 μM retinoic acid. The next day, SH-SY5Y ATXN3 / Q75 cells were treated with 5 μg / ml doxycycline, 10 μM retinoic acid, and different doses (3%, 10%, 30%, 50%, and 70%) of conditioned medium. 5 μg / ml doxycycline and 10 μM retinoic acid were added every three days for one week. On day 7, SH-SY5Y ATXN3 / Q75 cells with different treatment conditions were collected and analyzed by flow cytometry. Results: Overexpression of activin A in modified ADMSCs enhanced its ability to reduce the expression of mutant ataxin-3 in the SCA3 cell model. SH-SY5Y ATXN3 / Q75 cells were treated with ADMSC conditioned medium expressing activin A at different volumes. Figure 3 shows that compared with the plasmid control group, the conditioned medium from ADMSCs expressing activin A reduced the expression of mutant ataxin-3 in a dose-dependent manner. Example 7: Effect of activin A on modified adipose tissue-derived mesenchymal stem cells (ADMSCs) The effect of activin A on the proliferation of modified ADMSCs was evaluated. The method was as follows: The proliferation of ADMSCs was tested using CCK-8 (DOJINDO, Japan). Cells were seeded in the wells of a 96-well microtiter plate and incubated at 37 °C for 16 to 18 hours. Fresh medium containing activin A was replaced at different concentrations and cultured at 37 °C for 3 days. CCK-8 solution (10 μL / well) was added, and then cultured for another 3 hours. The absorbance was measured at 450 nm. High seeding density: 4,762 cells / cm 2 (equivalent to the co-culture ratio of modified ADMSCs to SH-SY5Y ATNX3 / Q75 = 1:10 in this application); Medium seeding density: 3,175 cells / cm 2 (Equivalent co-culture ratio of modified ADMSC to SH-SY5Y ATNX3 / Q75 = 1:15); Low seeding density: 2,381 cells / cm 2 (Equivalent to co-culture ratio of modified ADMSC to SH-SY5Y = 1:20). Results: Figures 4A to 4C show that different doses of activin A do not affect the proliferation of modified ADMSC at different densities. These data confirm that the synergistic effect of combining modified ADMSC and activin A on reducing the level of mutant ataxin-3 is not due to an increase in the number of modified ADMSC. Example 8: Characterization of native ADMSC Isolate the stromal vascular fraction cell population (1.00×10 8 ) from the adipose tissue of healthy adult donors and process as follows: The collected stromal vascular fraction is first centrifuged at 100 RCF for 3 - 10 minutes to remove unwanted aqueous blood and digested with collagenase at 37°C for 0.5 - 6 hours. The aforementioned incubation time length and additional shaking depend on the tissue state and size. The isolated native ADMSC are then washed with PBS and centrifuged at 400 RCF for 3 - 10 minutes twice. After the pellet is dispersed, the native ADMSC are counted and seeded at a density of less than 5×10 6 cells / cm 2 onto cell culture flasks. Flow cytometry analysis is used to analyze the surface markers of native ADMSC in the stromal vascular fraction without any further culture. A positive / specific staining region is set using an isotype control. Briefly, non-related immunoglobulins labeled with a fluorescent dye similar to the staining antibody are used to identify non-specific staining to establish the positive / specific staining region. In this study, 10 2 is set as the positive threshold value set by the isotype control. Based on the flow cytometry images, native ADMSC exhibit CD45 - CD34 + CD90 + CD73 + expression, which are characteristics of native ADMSC, see Figure 5A. Results: Further examination of CD273 expression in native ADMSCs was performed using flow cytometry. Figure 5B shows that native ADMSCs do not express CD273 (i.e., CD273 - ). References 1. McLoughlin, H.S., L.R. Moore and H.L. Paulson, Pathogenesis of SCA3 and implications for other polyglutamine diseases. Neurobiol Dis, 2020. 134: p. 104635. 2. Rüb, U., et al., Clinical features, neurogenetics and neuropathology of the polyglutamine spinocerebellar ataxias type 1, 2, 3, 6 and 7. Prog Neurobiol, 2013. 104: p. 38-66. 3. Da Silva, J.D., A. Teixeira-Castro and P. Maciel, From Pathogenesis to Novel Therapeutics for Spinocerebellar Ataxia Type 3: Evading Potholes on the Way to Translation. Neurotherapeutics, 2019. 16(4): p. 1009-1031. 4. Morianos, I., et al., Activin-A in the regulation of immunity in health and disease. Journal of Autoimmunity, 2019. 104: p. 102314. 5. Lin, C.H., et al., Novel Lactulose and Melibiose Targeting Autophagy to Reduce PolyQ Aggregation in Cell Models of Spinocerebellar Ataxia 3. CNS Neurol Disord Drug Targets, 2016. 15(3): p. 351-9. None The illustrative embodiments of the present invention are described in detail below with reference to the following figures: FIG. 1 is a bar graph showing that activin A reduces the level of mutant ataxin-3 in SH-SY5Y ATXN3 / Q75 cells in a dose-dependent manner. FIGS. 2A and 2B show the synergistic effect of the combination of various doses of modified ADMSCs and various doses of activin A in reducing mutant ataxin-3 (CI value less than 1 indicates synergy). FIG. 3 is a line graph showing that the conditioned medium from ADMSCs expressing activin A reduces the expression of mutant ataxin-3 in a dose-dependent manner compared to the plasmid control group. FIGS. 4A to 4C are bar graphs showing the effect of different concentrations of activin A on the proliferation of modified ADMSCs seeded at different densities. FIGS. 5A and 5B are flow cytometry images of native ADMSCs isolated from adipose tissue.

Claims

1. Use of a composition in the preparation of a medicament for treating polyQ disease, wherein the composition comprises: (a) activin A; and (b) adipose tissue-derived stem cells (ADMSCs) expressing CD273, CD46, CD55, CXCR4, CD105, CD73, and CD90, but not expressing CD45, CD34, CD11b, CD19, and HLA-DR, wherein the polyQ disease is spinocerebellar ataxia (SCA); and the SCA is SCA3 or SCA6.

2. Use of adipose tissue-derived stem cells (ADMSCs) expressing activin A in the preparation of a medicament for treating polyQ disease, wherein the ADMSCs express: (a) CD273, CD46, CD55, CXCR4, CD105, CD73 and CD90, but not CD45, CD34, CD11b, CD19 and HLA-DR; and (b) activin A, wherein the polyQ disease is spinocerebellar ataxia (SCA); and the SCA is SCA3 or SCA6.

3. Use of a composition in the preparation of a medicament for reducing mutant polyQ protein in an individual, wherein the composition comprises: (a) activin A; and (b) adipose tissue-derived stem cells (ADMSCs) or ADMSCs expressing activin A, wherein the mutant polyQ protein is ATXN-3.

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