Method for treating POLYQ disease and novel adipose tissue-derived mesenchymal stem cells

Abstract

The present invention provides the use of adipose tissue-derived stem cells expressing activin A or a combination of activin A and modified adipose tissue-derived stem cells for the treatment of polyQ diseases, such as spinocerebellar ataxia 3 (SCA 3), by reducing misfolded and aggregated polyQ proteins, such as the ataxia protein-3 protein. The present invention also provides compositions of polyQ proteins for reducing misfolding and aggregation, wherein the compositions comprise a population of modified adipose tissue-derived stem cells and activin A. The present invention also provides compositions of polyQ proteins for reducing misfolding and aggregation, wherein the compositions comprise activin A and a population of modified adipose tissue-derived stem cells.

Description

Technical Field

[0001] Cross-reference of related applications This application claims priority to Australian Provisional Application No. 2023901470, filed on 15 May 2023, the entire contents of which are incorporated herein by reference.

[0002] This invention relates to a method for treating POLYQ disease and novel adipose tissue-derived mesenchymal stem cells. Background Technology

[0003] Nine hereditary neurodegenerative disorders, collectively known as polyglutamine (polyQ) diseases, exist, including Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA), and spinocerebellar ataxia (SCA) 1, 2, 3, 6, 7, and 17. PolyQ diseases are caused by the amplification of polyglutamine (polyQ) in their respective disease proteins and share a common pathological feature: nuclear accumulation and inclusion body formation of the polyQ disease protein.

[0004] The most common pathogenic factor in polyQ diseases is the misfolding and aggregation of amplified polyQ proteins (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…”). SCA1Transgenic 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 disease.

[0005] There is a need to reduce mutant polyQ protein and effectively treat polyQ disease. This invention addresses this need, as well as other needs. Summary of the Invention

[0006] In one embodiment, the present invention provides a method for treating polyQ disease, comprising the steps of administering a therapeutically effective amount of the following to an individual in need: (a) activin A; and (b) a modified adipose tissue-derived stem cell (modified ADMSC) population having 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.

[0007] The present invention also provides a method for treating polyQ disease, comprising administering to an individual in need a therapeutically effective amount of ADMSCs expressing activin A as described herein, 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).

[0008] Methods for reducing mutant polyQ proteins (e.g., ATXN-3) in individuals are also provided, comprising the steps of administering a therapeutically effective amount of (a) activin A and (b) the modified ADMSCs described herein to the individual in need.

[0009] Methods for reducing mutant polyQ proteins (e.g., ATXN-3) in individuals with polyQ disease are also provided, comprising the steps of administering a therapeutically effective amount of ADMSCs expressing activin A as described herein to the individual in need.

[0010] Some implementations provide a segregation population of ADMSCs modified with activin A (ADMSCs expressing activin A), which express adipose tissue-derived stem cell markers including CD90, CD105, and CD73, but excluding CD45, CD34, CD11b, CD19, and HLA-DR, and maintain osteogenic, chondrogenic, and adipogenic differentiation capabilities.

[0011] Other embodiments provide a pharmaceutical composition comprising at least one activator A ADMSC described herein and a pharmaceutically acceptable carrier or excipient.

[0012] The terms “invention,” “the invention,” “this invention,” and “the present invention” as used in this patent are intended to broadly refer to all objects of this patent and the following claims. Statements containing these terms should be understood not to limit the objects described herein or the meaning or scope of the following claims. Embodiments of the invention covered by this patent are defined by the following claims, not by this summary. This summary provides a high-level overview of various aspects of the invention and introduces some concepts further described in the following embodiments section. This summary is not intended to identify key or essential features of the claimed objects, nor is it intended to be used alone to determine the scope of the claimed objects. The objects should be understood by referring to the appropriate portions of the specification, any or all drawings, and each claim.

[0013] The present invention will become clearer when the following figures and embodiments are read. Attached Figure Description

[0014] The illustrative embodiments of the present invention are described in detail below with reference to the following figures: Figure 1 The bar graph shows how activin A reduces the level of mutant ataxia protein-3 in SH-SY5Y ATXN3 / Q75 cells in a dose-dependent manner.

[0015] Figure 2A and Figure 2B This study demonstrates the synergistic effect of various doses of modified ADMSCs and various doses of activin A in reducing mutant ataxia protein-3 (CI value less than 1 indicates synergistic effect).

[0016] Figure 3 A line graph showing that conditioned medium from ADMSCs expressing activin A reduced mutant ataxia protein-3 expression in a dose-dependent manner compared to the plasmid control group.

[0017] Figures 4A to 4C Bar graph showing the effect of different concentrations of activin A on the proliferation of modified ADMSCs seeded at different densities.

[0018] Figure 5A and Figure 5B Flow cytometry images of native ADMSCs isolated from adipose tissue. Detailed Implementation

[0019] As used herein, the articles “a” and “an” refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, “modified ADMSC” means one or more modified ADMSCs.

[0020] As used herein, “therapeutic effective dose” refers to a dose of the modified ADMSC or pharmaceutical composition disclosed herein that is sufficient to cause a reduction in mutant polyQ protein (e.g., ATXN-3) or to improve one or more symptoms and signs of polyQ disease, including (but not limited to) ataxia, involuntary movements, and cognitive impairment, which can be detected clinically or radiologically by various imaging modalities.

[0021] As used herein, the terms “treating,” “treated,” or “treatment” refer to palliative uses or outcomes, and / or slowing or inhibiting the progression of polyQ disease, the formation of mutant polyQ proteins, or intracellular inclusions in neurons.

[0022] The term "individual" can refer to a vertebrate that has or is suspected of having polyQ disease, or a vertebrate that is considered to require treatment for polyQ disease. Individuals include warm-blooded animals, such as mammals, primates, and preferably humans. Non-human primates are also considered individuals. The term "individual" includes domesticated animals (such as cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). Therefore, veterinary use and pharmaceutical preparations are considered in this document.

[0023] The expression level or surface density of a surface marker (such as CD273) on the surface of modified ADMSCs is "+" as long as the expression of the surface marker can be detected by any known method. In one exemplary embodiment, "+" indicates that the cell surface marker is detected above the isotype control in fluorescence activated cell sorting / flow cytometry or magnetic beads; or above the background in quantitative or semi-quantitative RT-PCR.

[0024] The expression level or surface density of a surface marker (such as CD45) is "-" if the expression of the surface marker is not detectable by any known means. In one exemplary embodiment, "-" indicates that the cell surface marker is not detectable above the isotype control in fluorescence-activated cell sorting / flow cytometry or magnetic beads; or is not detectable above the background in quantitative or semi-quantitative RT-PCR.

[0025] As used in this article, "substantially contains" means less than 2%, 1%, or 0.5%.

[0026] All figures in this article are to be understood as being modified by “approximately”. As used in this article, the term “approximately” is intended to cover a variation of ±10%.

[0027] 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".

[0028] Modified ADMSC The modified ADMSCs of this invention are derived from native mesenchymal stem cells isolated from adipose tissue. The native mesenchymal stem cells isolated from adipose tissue (native ADMSCs) are characterized by CD105. + CD73 + CD90 + CD45 - CD34 - CD11b - CD19 - HLA-DR - CD273 - It also maintains the ability to differentiate into osteoblasts, chondrocytes, and adipocytes. In one implementation scheme, the native ADMSCs are CD46. - CD55 - CXCR4 - .

[0029] If less than 2% of ADMSCs express CD45, CD34, CD11b, CD19, and HLA-DR, then the modified ADMSC population described in this paper is considered a purified population of modified ADMSCs.

[0030] Example 1 of this application illustrates one embodiment of a method for culturing the modified ADMSCs of the present invention, wherein native ADMSCs are cultured in MSC medium for approximately 12 hours to approximately 7 days, wherein the MSC medium comprises α-MEM, UltraGRO, and heparin. In another embodiment, the MSC medium comprises epidermal growth factor (EGF), fibroblast growth factor 2 (FGF-2), and an insulin-transferrin-selenium (ITS) supplement.

[0031] In one implementation, the modified ADMSCs are characterized by expressing CD273, CD46, CD55, and CXCR4, which are not present in naturally occurring or native ADMSCs. The phenotypic characteristic of the modified ADMSCs is CD273. + CD46 + CD55 + CXCR4 + CD105 + CD73 + CD90 + But not CD45 - CD34 - CD11b - CD19 - HLA-DR - Modified ADMSCs maintain the ability to differentiate into osteoblasts, chondrocytes, and adipocytes.

[0032] ADMSCs expressing activin A In one embodiment, one or more activin A plasmids are inserted into the modified ADMSCs described herein. The modified ADMSCs express activin A and are referred to as activin A-expressing ADMSCs.

[0033] The modified ADMSCs and ADMSCs expressing activin A of the present invention are not naturally occurring, because native ADMSCs do not express CD273.

[0034] Modified ADMSCs can originate from a single individual (i.e., autologous) or aggregate from multiple individuals (non-autologous allogeneic).

[0035] Pharmaceutical Composition In one embodiment, a pharmaceutical composition is provided comprising at least one modified ADMSC, activator A, and a pharmaceutically acceptable mediator, carrier, or excipient as described herein.

[0036] Advantageously, this combination has a synergistic effect on reducing mutant polyQ proteins (e.g., ATXN-3) or improving one or more symptoms and signs of polyQ disease.

[0037] Suitable carriers include, for example, water, saline, dextran, glycerol, ethanol, dimethyl sulfoxide (DMSO), trehalose, and combinations thereof. Suitable excipients include, for example, wetting agents, emulsifiers, or pH buffers. Pharmaceutically acceptable carriers may contain physiologically acceptable compounds that serve, for example, to stabilize or increase or decrease the absorption or clearance of the pharmaceutical compositions of the present invention. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or polydextrose; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposome carriers; or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives. Excipients may be nonionic surfactants, polyvinylpyrrolidone, human serum albumin, aluminum hydroxide, anesthetic agents, and various unmodified and derivatized cyclodextrins. In one embodiment, the nonionic surfactant may include polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Polyvinylpyrrolidone is preferably Plasdone C15, i.e., pharmaceutical-grade polyvinylpyrrolidone. The pharmaceutical composition containing this excipient or carrier is formulated using well-known conventional methods.

[0038] In one exemplary embodiment, the pharmaceutical composition is substantially free of bone marrow-derived interstitial stromal cells.

[0039] This invention also provides a method for treating polyQ diseases by administering a therapeutically effective amount of the pharmaceutical composition described herein or ADMSCs expressing activin A as described herein to an individual in need. PolyQ diseases include Huntington's disease (HD), dentate nucleus-globus pallidus Lewy body atrophy (DRPLA), spinobulbar muscular atrophy (SBMA), and six spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17).

[0040] In some embodiments, a method for reducing mutant polyQ proteins (e.g., ATXN-3) is provided by administering a therapeutically effective amount of the pharmaceutical composition described herein or ADMSCs expressing activin A as described herein to an individual in need. In one embodiment, the polyQ protein of SCA3 is ATXN-3.

[0041] In another embodiment of the invention, the use of modified ADMSCs and activin A combination or ADMSCs expressing activin A as described herein is provided for the manufacture of a medicament for treating polyQ disease or reducing polyQ protein in the disease.

[0042] The routes of administration for 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, local, subcutaneous, intradermal, percutaneous, subcutaneous, non-intestinal, rectal, spinal, intrathecal, intracardiac, intracranial, or inter-vertebral 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.

[0043] The modified ADMSCs of the present invention can be administered simultaneously with, before or after activator A to treat polyQ disease.

[0044] The ADMSCs expressing activin A or the pharmaceutical composition described herein can be administered as a single dose or multiple doses over a period of time, depending on the individual's age, weight, condition, the specific composition used, the route of administration, and whether the ADMSCs expressing activin A or the pharmaceutical composition of the present invention is for preventative or curative purposes. In some embodiments, the ADMSCs expressing activin A or the pharmaceutical composition of the present invention can be 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 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).

[0045] Data obtained from cell culture analysis and animal studies can be used to formulate dosage ranges for human use. In one embodiment, the dosage is within the range of minimally toxic or non-toxic EDTA. 50 The dose is within the range of circulating concentrations. The dosage may vary within this range depending on the dosage form and route of administration. In another embodiment, the therapeutically effective dose may initially be estimated by cell culture analysis. Dosage can be formulated using animal models to obtain the range of circulating plasma concentrations, including the IC50 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.

[0046] The pharmaceutical composition is formulated to contain a therapeutically effective amount of the modified ADMSCs and activin A or activin A-expressing ADMSCs described herein, wherein the amount depends on the individual to be treated and the disease condition to be treated. The specific dose 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 status, sex, diet, time of administration, route of administration and excretion rate, drug combination, and the severity of the specific disease to which therapies have been previously performed. An illustrative, non-limiting range for a therapeutic or prophylactic effective amount of the modified ADMSCs / activin A-expressing ADMSCs of the present invention is at least about 1 × 10⁻⁶. 4 Cells / dose to approximately 1×10⁻⁶ 10 Cells / dose. Other doses are also possible, including (but not limited to): 1 × 10⁻⁶ cells / dose. 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 8Or 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 .

[0047] The following examples for implementing specific aspects of the invention are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0048] Example Example 1: Modified ADMSC Native ADMSCs were isolated from the stromal vascular portion of human adipose tissue and characterized as CD105. + CD73 + CD90 + CD45 - CD34 - CD11b - CD19 - HLA-DR - Native ADMSCs were cultured in MSC medium containing α-MEM (available from Gibco, USA), 5% Helio Bioscience UltraGRO (available from AventaCell BioMedical Corp, USA), and 2 U / ml heparin (available from MP Biomedicals, USA), and were subcultured when cells reached 80% confluence. Before trypsin digestion, the MSC medium was removed and cells were washed with 1×DPBS (Gibco, 14190-144). Cells were cultured at room temperature with 0.25% trypsin-EDTA (Gibco, 25200072) for 3 minutes. After cell separation, MSC medium was added to neutralize the trypsin. The cultured cells were transferred to centrifuge tubes and centrifuged at 330 g for 5 minutes. The cell aggregates were resuspended in MSC medium and cultured at 37°C in a 5% CO2 incubator.

[0049] After 12 passages, cultured ADMSCs (hereinafter referred to as modified ADMSCs) were analyzed using flow cytometry. Table 1 below shows the modified ADMSCs expressing specific biomarkers.

[0050] Table 1.

[0051] In addition, the modified ADMSCs retain the ability to form cartilage, osteogenes, and adipogenic differentiation.

[0052] Example 2: Maintenance of SH-SY5Y ATXN3 / Q75 cells The SH-SY5Y ATXN3 / Q75 cell line and culture system were obtained from Dr. Guey-Jen Lee-Chen of National Taiwan Normal University. SH-SY5Y ATXN3 / Q75 cells were cultured in a medium containing DMEM (Dalberg modified Eagle medium) / Hams F-12 (DMEM / F12) (Corning, 16-405-CV) supplemented with 10% FBS (Corning, 35-010-CV), 5 μg / ml blastomycin (InvivoGen, ant-bl), and 100 μg / ml hygromycin (InvivoGen, ant-hg). SH-SY5Y ATXN3 / Q75 cells were cultured when they reached 80% confluence. Before trypsin digestion, the culture medium was removed, and the cells were washed with 1×DPBS (Gibco, 14190-144) and incubated at room temperature with 0.25% trypsin-EDTA (Gibco, 25200072) for 3 minutes. After cell separation, DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blastcin, and 100 μg / ml hygromycin was added to neutralize the trypsin. SH-SY5Y ATXN3 / Q75 cells were transferred to centrifuge tubes and centrifuged at 330 g for 5 minutes. The cell aggregates were resuspended in DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blastcin, and 100 μg / ml hygromycin, and cultured in a 5% CO2 incubator at 37°C. Within 7 days, the cells reached 80% confluence and were ready for other experiments.

[0053] Example 3: Effects of modified ADMSCs on SH-SY5Y ATXN3 / Q75 cells, a lesion cell model of SCA3. Dr. GJ Lee (National Taiwan Normal University) developed an SCA3 cell model in which polyQ accumulation amplifies ataxia protein-3 to form aggregates in neuronal cells, a phenomenon commonly observed in the brains of SCA3 patients [6]. Different ratios of modified ADMSCs with 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. In short, 9 × 10 3 Modified ADMSCs, 1.2 × 10 4 Modified ADMSCs and 1.8 × 104 ADMSCs were seeded in 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.

[0054] 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 medium containing DMEM / F12, 10% FBS, and 10 μM retinoic acid (Sigma, R2625). The following day, the modified ADMSC seeding insert was transferred to the six-well plates in the lower compartment, with SH-SY5Y ATXN3 / Q75 cells cultured in medium containing DMEM / F12 and 10% FBS. SH-SY5Y ATXN3 / Q75 cells were treated with 5 μg / ml doxycycline (Sigma, D9891) to induce expression of mutant ataxia-3. 5 μg / ml doxycycline and 10 μM retinoic acid were added every three days for one week.

[0055] Example 4: Effects of activin A on the SCA3 lesion 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 lesion cell model (SH-SY5Y ATXN3 / Q75 cells) were examined. First, SH-SY5Y ATXN3 / Q75 cells were cultured at 20,833 cells / cm². 2 Cells were seeded at a density in six-well cell culture dishes (Corning, 353046) with DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blastomycin, 100 μg / ml hygromycin, and 10 μM retinoic acid. The following 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. Doxycycline and 10 μM retinoic acid were added every three days for one week.

[0056] Results: Activin A reduced the expression of mutant ataxia protein-3 in the SCA3 cell model. Figure 1 The study showed that activin A reduced the level of mutant ataxia protein-3 in SH-SY5Y ATXN3 / Q75 cells in a dose-dependent manner.

[0057] Example 5: Combined effect of modified ADMSCs and activin A on SH-SY5Y ATXN3 / Q75 cells, a lesion cell model of SCA3. 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 × 10⁶ ADMSCs were then seeded. 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 following day, the modified ADMSC seeding insert was transferred to the six-well plates in the lower compartment, with SH-SY5Y ATXN3 / Q75 cells cultured in DMEM / F12 medium supplemented with 10% FBS. SH-SY5Y ATXN3 / Q75 cells and modified ADMSCs were treated with various concentrations of activin A (0.2, 1.0, and 5.0 ng / ml). Doxycycline (5 μg / ml) and 10 μM retinoic acid were added every three days for one week.

[0058] 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). ® The analysis was performed using easyCyte™. Results were analyzed using Guava software, and the level of mutant ataxia protein-3 was measured.

[0059] The effects of modified ADMSCs and / or activin A were assessed using the combination index (CI) method (Chou, TC, Cancer Res, 2010. 70(2): 440-6).

[0060] result: Figure 2A and Figure 2B This study demonstrates the synergistic effect of various doses of modified ADMSCs and various doses of activin A in reducing mutant ataxia-3 (CI value less than 1 indicates synergistic effect).

[0061] Example 6: Modified adipose-derived stem cells (ADMSCs) expressing activin A (ADMSCs expressing activin A) The day before transfection, 4.8 × 10 4 Modified ADMSCs were cultured in 1.5 ml MSC medium per well containing MEM, 5% Helio Bioscience UltraGRO, and 2 U / ml heparin, ensuring 50-60% confluence of the modified ADMSCs at transfection. Diluted plasmid DNA, activin A expression plasmid (Sino Biological, HG10429-UT) or control plasmid (Sino Biological, CV011), and lipoamine stem cells (Thermo Fisher Scientific, STEM00001) in Opti-MEM I medium (Thermo Fisher Scientific, 11058021) were prepared separately in different eppendorf immersion chambers and incubated at room temperature for 5 minutes. After incubation, the diluted plasmid DNA was combined with the diluted lipoamine stem cells, gently mixed, and incubated at room temperature for 10 minutes to allow the plasmid DNA-lipoamine stem cell complex to form. 150 μl of the plasmid DNA-lipoamine stem cell complex was added to each well containing the modified ADMSCs and MSC medium, and the mixture was gently mixed by oscillating the plate back and forth. Cells were cultured in a CO2 incubator at 37°C for 24 hours. ADMSCs expressing activin A were then supplemented with MSC medium. ADMSCs expressing activin A were cultured for 24 hours under standard conditions (5% CO2; 37°C). Conditioned medium samples were collected, centrifuged to remove cell debris, and then aliquoted and frozen at -20°C.

[0062] The role of conditioned medium in SCA3 lesion cells SH-SY5Y ATXN3 / Q75 cells: 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 ataxia protein-3 was analyzed.

[0063] First, SH-SY5Y ATXN3 / Q75 cells were introduced at a concentration of 20,833 cells / cm². 2Cells were seeded at a density in six-well cell culture dishes, with DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blastomycin, 100 μg / ml hygromycin, and 10 μM retinoic acid. The following day, SH-SY5Y ATXN3 / Q75 cells were treated with 5 μg / ml doxycycline and 10 μM retinoic acid, and with different doses (3%, 10%, 30%, 50%, and 70%) of conditioned medium. Doxycycline and retinoic acid were added every three days for one week. On day 7, SH-SY5Y ATXN3 / Q75 cells treated with different conditions were collected and analyzed by flow cytometry.

[0064] Results: Overexpression of activin A in modified ADMSCs enhanced its ability to reduce the expression of mutant ataxia-3 in the SCA3 cell model.

[0065] SH-SY5Y ATXN3 / Q75 cells were treated with ADMSC conditioned medium containing different volume fractions of activin A. Figure 3 The results showed that, compared with the plasmid control group, conditioned medium from ADMSCs expressing activin A reduced mutant ataxia protein-3 expression in a dose-dependent manner.

[0066] Example 7: Effects of activin A on modified adipose tissue-derived stem cells (ADMSCs) The effect of activin A on the proliferation of modified ADMSCs was evaluated. The method was as follows: ADMSC proliferation was assessed using CCK-8 (DOJINDO, Japan). Cells were seeded into the wells of 96-well microtiter plates and incubated at 37°C for 16–18 hours. Fresh medium containing activin A was then added at different concentrations, and the cells were incubated at 37°C for 3 days. CCK-8 solution (10 μL / well) was added, followed by another 3 hours of incubation. Absorbance was measured at 450 nm. High seeding density: 4,762 cells / cm². 2 (In this application, the equivalent co-culture ratio of modified ADMSCs to SH-SY5Y ATNX3 / Q75 is 1:10); Medium seeding density: 3,175 cells / cm² 2 (Equivalent to a co-culture ratio of modified ADMSCs to SH-SY5YATNX3 / Q75 = 1:15); Low seeding density: 2,381 cells / cm² 2 (Equivalent to a co-culture ratio of modified ADMSCs to SH-SY5Y = 1:20).

[0067] result: Figures 4A to 4CThe data showed that different doses of activin A did not affect the proliferation of modified ADMSCs at different densities. These data confirm that the synergistic effect of combined modified ADMSCs and activin A in reducing mutant ataxia-3 levels is not attributable to an increase in the number of modified ADMSCs.

[0068] Example 8: Features of native ADMSC A stromal vascular cell population (1.00 × 10⁻⁶) was isolated from adipose tissue of a healthy adult donor. 8 And process it as follows: The collected stromal vascular fraction was first centrifuged at 100 RCF for 3–10 minutes to remove unwanted watery blood, and then digested with collagenase at 37°C for 0.5–6 hours. The aforementioned incubation time and additional shaking depend on the tissue condition and size. The isolated native ADMSCs were then washed with PBS and centrifuged twice at 400 RCF for 3–10 minutes. After dispersion of the aggregated particles, the native ADMSCs were counted and measured at a value less than 5 × 10⁻⁶. 6 cells / cm 2 The sample was coated at a density onto a cell culture flask. Flow cytometry was used to analyze surface markers of native ADMSCs in the stromal vascular fraction without any further culture.

[0069] Positive / specific staining zones were established using isotype controls. In short, non-specific staining was established by recognizing non-specific immunoglobulins using fluorescent dyes labeled with similar staining antibodies. In this study, 10 2 The positive threshold value was set by the isotype control.

[0070] Based on flow cytometry imaging, native ADMSCs showed CD45 - CD34 + CD90 + CD73 + Expression, which is a characteristic of native ADMSCs, see [link to relevant documentation]. Figure 5A .

[0071] Results: Flow cytometry was used to further examine CD273 expression in native ADMSCs. Figure 5B This shows that native ADMSCs do not express CD273 (i.e., CD273). - ).

[0072] 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。

[0073] 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。

[0074] 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。

[0075] 4. Morianos, I., et al., Activin - A in the regulation of immunity in health and disease. Journal of Autoimmunity, 2019. 104: p. 102314。

[0076] 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。

Claims

1. A method for treating polyQ disease, comprising the step of administering a therapeutically effective amount of the following substance to an individual in need: (a) Activator A; and (b) Modified adipose tissue-derived stem cells (modified ADMSCs) that express CD273, CD46, CD55, CXCR4, CD105, CD73 and CD90, but do not express CD45, CD34, CD11b, CD19 and HLA-DR.

2. A method for treating polyQ disease, comprising the step of administering a therapeutically effective amount of ADMSCs expressing activin A, said ADMSCs expressing activin A expressing: (a) CD273, CD46, CD55, CXCR4, CD105, CD73, and CD90, but not CD45, CD34, CD11b, CD19, and HLA-DR; and (b) Activator A.

3. The method of claim 1 or 2, wherein the polyQ disease is spinocerebellar ataxia (SCA), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), or X-linked spinal and bulbar muscular atrophy (SMAX1 / SBMA).

4. The method of claim 3, wherein the SCA is SCA3.

5. A method for reducing mutant polyQ protein in an individual, comprising the step of administering a therapeutically effective amount of the following substance to the individual in need: (a) Activator A; and (b) Modified adipose tissue-derived stem cells (ADMSCs) or ADMSCs expressing activin A.

6. The method of claim 5, wherein the polyQ protein is ATXN-3.

7. A segregating population of ADMSCs expressing activin A, wherein the ADMSCs expressing activin A express... (a) CD273, CD46, CD55, CXCR4, CD105, CD73, and CD90, but not CD45, CD34, CD11b, CD19, and HLA-DR; and (b) Activator A.

8. The segregating population of ADMSCs expressing activin A as described in claim 7, wherein the ADMSCs contain at least one activin A expression plasmid.

9. A pharmaceutical composition comprising: The modified adipose-derived stem cells (ADMSCs) expressing activin A as described in claim 7; and Pharmaceutically acceptable excipients.

10. The pharmaceutical composition of claim 9, further comprising activator A.

11. A method for reducing mutant polyQ protein in an individual, comprising the step of administering a therapeutically effective amount of ADMSCs expressing activin A to the individual in need.

12. The method of claim 11, wherein the polyQ protein is ATXN-3.

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