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

Abstract

This 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 treating poly-Q disease, such as spinocerebellar ataxia type 3 (SCA3), by reducing misfolded and aggregated poly-Q proteins, such as ataxin 3 protein. It also provides a population of modified adipose tissue-derived stem cells and a composition containing activin A for reducing misfolded and aggregated poly-Q proteins.

Description

[Technical Field]

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

[0002] Polyglutamine (poly-Q) diseases are a collective term for nine hereditary neurodegenerative disorders, including Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), and spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, and 17. Poly-Q diseases are caused by polyglutamine (poly-Q) elongation in the respective disease proteins and share common pathological features: nuclear accumulation of poly-Q disease proteins and formation of inclusion bodies.

[0003] The most common pathogenesis cascade of polyQ disease is the aggregation of misfolded and elongated 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 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 curative treatment that can cure or slow the exacerbations of polyQ disease.

[0004] There is a need to reduce mutated polyQ proteins and effectively treat polyQ disease. This invention addresses this and various other needs. [Overview of the project]

[0005] In one embodiment, the present invention provides a method for treating polyQ disease, comprising the step of administering to a subject in need of the treatment (a) activin A in a therapeutically effective amount and (b) a population of modified adipose tissue-derived stem cells (modified ADMSCs) that have the phenotypic characteristics of CD273, CD46, CD55, CXCR4, CD90, CD105, and CD73, but do not express CD45, CD34, CD11b, CD19, and HLA-DR, and maintain osteogenic, chondrogenic, and adipogenic differentiation potential.

[0006] Furthermore, the present invention provides a method for treating polyQ disease, comprising the step of administering a therapeutically effective amount of the activin A-expressing ADMSC described herein to a subject in need thereof, wherein the activin A-expressing ADMSC has the phenotypic characteristics of CD273, CD46, CD55, CXCR4, CD90, CD105, and CD73 (but does not express CD45, CD34, CD11b, CD19, and HLA-DR).

[0007] The present invention also provides a method for reducing mutant polyQ protein (e.g., ATXN-3) in a subject, comprising the steps of administering a therapeutically effective amount of (a) activin A and (b) a modified ADMSC as described herein to a subject in need thereof.

[0008] Furthermore, the present invention provides a method for reducing mutant poly-Q protein (e.g., ATXN-3) in subjects with poly-Q disease, comprising the step of administering a therapeutically effective amount of activin A-expressing ADMSC described herein to a subject in need.

[0009] In some embodiments, activin A-expressing ADMSCs express adipose tissue-derived stem cell markers including CD90, CD105, and CD73, but do not express CD45, CD34, CD11b, CD19, and HLA-DR, and provide an isolated population of modified ADMSCs expressing activin A (activin A-expressing ADMSCs) that maintain osteogenic, chondrogenic, and adipogenic differentiation potential.

[0010] In other embodiments, a pharmaceutical composition is provided comprising at least one activin A ADMSC described herein and a pharmaceutically acceptable carrier or excipient.

[0011] The terms “invention,” “the invention,” “this invention,” and “the present invention” as used in this patent are intended to broadly refer to all of the subject matter of this patent and the claims below. It should be understood that any use of such terms does not limit the subject matter of this specification, nor the meaning and scope of the claims below. Embodiments of the invention as encompassed by this patent are defined by the claims below, not by this abstract. This abstract is an overview of various aspects of the invention and introduces some of the concepts further described in the section on embodiments for carrying out the invention below. This abstract is not intended to identify key essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, all drawings, and the appropriate parts of each claim.

[0012] The present invention will become more apparent when understood in conjunction with the following accompanying drawings and embodiments for carrying out the invention.

[0013] Exemplary embodiments of the present invention are described in detail below with reference to the following drawings. [Brief explanation of the drawing]

[0014] [Figure 1] This bar graph illustrates that activin A dose-dependently reduces mutant ataxin 3 levels in SH-SY5Y ATXN3 / Q75 cells. [Figure 2A] Figures 2A and 2B show the synergistic effects of various doses of modified ADMSC and various doses of activin A in reducing mutant attaxin 3 (CI values ​​less than 1 indicate synergistic effects). [Figure 2B] Figures 2A and 2B show the synergistic effects of various doses of modified ADMSC and various doses of activin A in reducing mutant attaxin 3 (CI values ​​less than 1 indicate synergistic effects). [Figure 3]This line graph shows that mutant ataxin 3 expression decreases in a dose-dependent manner when activin A-expressing ADMSCs are cultured in a controlled medium compared to the plasmid control group. [Figure 4A] Figures 4A to 4C are bar graphs showing the effects of various concentrations of activin A on the growth of modified ADMSCs seeded at various densities. [Figure 4B] Figures 4A to 4C are bar graphs showing the effects of various concentrations of activin A on the growth of modified ADMSCs seeded at various densities. [Figure 4C] Figures 4A to 4C are bar graphs showing the effects of various concentrations of activin A on the growth of modified ADMSCs seeded at various densities. [Figure 5A] Figures 5A and 5B are flow cytometry images of native ADMSCs isolated from adipose tissue. [Figure 5B] Figures 5A and 5B are flow cytometry images of native ADMSCs isolated from adipose tissue. [Modes for carrying out the invention]

[0015] As used herein, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects of the article. For example, “a modified ADMSC” means one modified ADMSC or two or more modified ADMSCs.

[0016] "Therapeutic dose" means, as used herein, a dose of the modified ADMSC or pharmaceutical composition disclosed herein that is sufficient to produce a reduction in mutant poly-Q protein (e.g., ATXN-3) or improvement in one or more symptoms and signs of poly-Q disease, including but not limited to ataxia, involuntary movements, and cognitive impairment, which are clinically or radiologically detectable by various imaging means.

[0017] The term “treating, treated, or treatment,” as used herein, refers to symptomatic use or results, and / or slowing or inhibiting the progression of polyQ disease, mutant polyQ proteins, or the formation of intracellular inclusions in nerve cells.

[0018] The term "subject" may refer to vertebrates that have or are suspected of having PolyQ disease, or vertebrates that appear to require treatment for PolyQ disease. Subjects include warm-blooded animals, e.g., mammals, primates, and more preferably humans. Non-human primates are also subject. The term "subject" includes domesticated animals, e.g., cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). Veterinary use and medical prescriptions are discussed herein.

[0019] The expression level or surface density of a surface marker on the surface of modified ADMSCs, such as CD273, is "+" if the expression of the surface marker is detectable by any conventional means. In one exemplary embodiment, "+" means that the cell surface marker is detectably present compared to an isotype control in fluorescence-activated cell classification / flow cytometry or magnetic beads, or is detectable beyond the background in quantitative or semi-quantitative RT-PCR.

[0020] The expression level or surface density of a surface marker, such as CD45, is "-" if the expression of the surface marker is undetectable by any conventional means. In one exemplary embodiment, "-" means that the cell surface marker is either undetectably absent compared to an isotype control in fluorescence-activated cell classification / flow cytometry or magnetic beads, or undetectable beyond the background in quantitative or semi-quantitative RT-PCR.

[0021] As used herein, “substantially not containing” means less than 2%, 1%, or 0.5%.

[0022] All numbers in this specification can be understood as being modified by "about". When used in this specification, the term "about" is intended to encompass a variation of ±10%.

[0023] 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 ADMSC of the present invention is derived from natural mesenchymal stem cells isolated from adipose tissue. Natural mesenchymal stem cells isolated from adipose tissue (natural ADMSC) are CD105 , ,

[0025] , - , - , , - , <000001​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0026] In one embodiment, modified ADMSCs are characterized by the expression of CD273, CD46, CD55, and CXCR4, which are not present in naturally occurring or native ADMSCs. The phenotypic characteristics of modified ADMSCs are CD273 + CD46 + CD55 + CXCR4 + CD105 + CD73 + CD90 + However, CD45 - CD34 - CD11b - CD19 - HLA-DR - No. Modified ADMSCs maintain their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells. Activin A-expressing ADMSC In one embodiment, one or more activin A plasmids are inserted into the modified ADMSC described herein. The modified ADMSC is capable of expressing activin A and is known as an activin A-expressing ADMSC.

[0027] Since natural ADMSCs do not express CD273, the modified ADMSCs and activin A-expressing ADMSCs of the present invention are not naturally occurring.

[0028] Modified ADMSCs can originate from a single individual, i.e., be autogenic, or they can be stored from multiple individuals (non-autogenic and homogeneous). Pharmaceutical composition In one embodiment, a pharmaceutical composition is provided comprising at least one modified ADMSC described herein, activin A, and a pharmaceutically acceptable medium, carrier, or excipient.

[0029] 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.

[0030] Suitable media include, for example, water, physiological saline, dextrose, glycerol, ethanol, dimethyl sulfoxide (DMSO), trehalose, and combinations thereof. Suitable excipients include, for example, wetting agents, emulsifiers, or pH buffers. Pharmaceutically acceptable carriers may include, for example, physiologically acceptable compounds that stabilize the pharmaceutical composition of the present invention or act to increase or decrease its absorption or clearance rate. Physiologically acceptable compounds may include, for example, carbohydrates, e.g., glucose, sucrose, or dextran; antioxidants, e.g., ascorbic acid or glutathione; chelating agents; low molecular weight proteins; surfactants; liposome carriers; or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives. Excipients may include nonionic surfactants, polyvinylpyrrolidone, human serum albumin, aluminum hydroxide, anesthetic substances, and various unmodified and derivatized cyclodextrins. In one embodiment, the nonionic surfactant may include polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. The polyvinylpyrrolidone may preferably be Plasdone C15, which is pharmaceutical-grade polyvinylpyrrolidone. Pharmaceutical compositions containing such excipients or carriers are formulated by well-known conventional methods.

[0031] In an exemplary embodiment, the pharmaceutical composition substantially does not contain bone marrow-derived mesenchymal stromal cells.

[0032] Furthermore, the present invention provides a method for treating poly-Q disease by administering a therapeutically effective amount of the pharmaceutical composition or activin A-expressing ADMSC described herein to a subject in need. Poly-Q disease includes Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), and six spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17).

[0033] In some embodiments, a method is provided for reducing mutant polyQ protein (e.g., ATXN-3) by administering a therapeutically effective amount of the pharmaceutical composition or activin A-expressing ADMSC described herein to a subject in need. In one embodiment, the polyQ protein is ATXN-3 of SCA3.

[0034] Further embodiments of the present invention provide combinations of modified ADMSCs and activin A as described herein, or the use of activin A-expressing ADMSCs, for the manufacture of pharmaceuticals for the treatment of polyQ disease or for the reduction of diseased polyQ protein.

[0035] The administration routes of the pharmaceutical compositions of the present invention, including but not limited to intravenous, intramuscular, subcutaneous, oral, topical, subcutaneous, intradermal, transdermal, subdermal, parenteral, rectal, spinal, intrathecal, intraventricular, intracranial, or intervertebral administration, include, but are not limited to, intravenous, intramuscular, subcutaneous, oral, topical, subcutaneous, intradermal, transdermal, subdermal, parenteral, rectal, spinal, intrathecal, intraventricular, intracranial, or intervertebral administration. In one embodiment, the pharmaceutical composition of the present invention, the combination of modified ADMSC and activin A, or activin A-expressing ADMSC is administered by intravenous injection or infusion.

[0036] The modified ADMSC of the present invention can be administered simultaneously with, before, or after activin A to treat polyQ disease.

[0037] The activin A-expressing ADMSCs or pharmaceutical compositions described herein can be administered by single-dose or multi-dose therapy on a schedule over a period appropriate to the age, weight, and symptoms of the target patient, the specific composition used, and the route of administration, and whether the activin A-expressing ADMSCs or pharmaceutical compositions of the present invention are to be used for purposes such as prevention or cure. In some embodiments, the activin A-expressing ADMSCs or pharmaceutical compositions of the present invention are administered once a year, once every six months, once every four months, once every three 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, every other day (qod), once a day (qd), twice a day (qid), or three times a day (tid).

[0038] Data obtained from cell culture assays and animal studies can be used to formulate various dosages for human use. In one embodiment, the dosage is ED with little or no toxicity. 50 The blood concentration ranges include a variety of levels. The dosage may vary within this range depending on the dosage form used and the route of administration. In another embodiment, the therapeutically effective dose may be estimated first from a cell culture assay. The dose is formulated in an animal model and determined by cell culture in an IC. 50 A range of plasma concentrations including (i.e., concentrations that achieve 50% inhibition of symptoms) can be obtained. (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, or activin A-expressing ADMSCs, as described herein, the amount of which depends on the target and the symptom being treated. Specific dose levels for any particular target depend on a variety of factors, including the activity of the specifically modified monocytes or ADMSCs, age, body weight, general health, sex, diet, administration time, route of administration, and excretion rate, concomitant medications, and the severity of the specific disease being treated. An unlimiting range of examples of therapeutically or prophylactically effective amounts of the modified ADMSCs / activin A-expressing ADMSCs of the present invention is at least about 1 × 10⁶ per dose. 4 ~Approximately 1 x 10 per dose 10 Other dosages are also possible, such as 1 × 10⁻⁶. 5 , 2×10 5 , 3 x 10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1 x 10 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1 x 10 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 2×10 8 , 3 x 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 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 This includes, but is not limited to, the following:

[0039] The following embodiments of specific aspects for carrying out the present invention are provided for illustrative purposes only and are not intended in any way to limit the scope of the invention.

[0040] Examples Example 1 Qualified ADMSC CD105 + CD73 + CD90 + CD45 - CD34 - CD11b - CD19 - HLA-DR - Natural ADMSCs, characterized as CD, were isolated from stromal vascular cells of human adipose tissue. Natural ADMSCs were cultured in MSC culture medium containing alpha-MEM (Gibco, commercially available from the USA), 5% Helio Bioscience UltraGRO (AventaCell BioMedical Corp, commercially available from the USA), and 2 U / ml heparin (MP Biomedicals, commercially available from the USA). Once the cells reached an 80% concentration, they were subcultured. Before trypsin treatment, the MSC culture 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) at room temperature for 3 minutes. After the cells detached, MSC culture 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 precipitate was resuspended in MSC culture medium, and the cells were incubated at 37°C in a 5% CO2 incubator.

[0041] After 12 passages, 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.

[0042] [Table 1]

[0043] In addition, modified ADMSCs retain their chondroplastic, osteogenic, and adipogenic differentiation potential.

[0044] Example 2 Maintenance of SH-SY5Y ATXN3 / Q75 cells SH-SY5Y ATXN3 / Q75 cells 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 (Dulbeccoo's modified Eagle medium) / Hams F-12 (DMEM / F12) (Corning, 16-405-CV), and 10% FBS (Corning, 35-010-CV), 5 μg / ml blastosidine (InvivoGen, ant-bl), and 100 μg / ml hygromycin (InvivoGen, ant-hg) were added. SH-SY5Y ATXN3 / Q75 cells were subcultured once the cell population reached 80%. Before trypsin treatment, the culture medium was removed, the cells were washed with 1× DPBS (Gibco, 14190-144), and incubated with 0.25% trypsin-EDTA (Gibco, 25200072) at room temperature for 3 minutes. Once the cells detached, the trypsin was neutralized by adding DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blastosidine, and 100 μg / ml hygromycin. The SH-SY5Y ATXN3 / Q75 cells were transferred to a centrifuge tube and centrifuged at 330 g for 5 minutes. The cell precipitate was resuspended in DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blastosidine, and 100 μg / ml hygromycin, and the cells were incubated at 37°C in a 5% CO2 incubator. The cells reached an 80% concentration within 7 days and were ready for further experiments.

[0045] Example 3 Effects of modified ADMSCs on SH-SY5Y ATXN3 / Q75 cells, a cell model of SCA3-affected cells. Dr. GJ Lee (National Taiwan Normal University) generated an SCA3 cell model in which poly-Q-extended attaxin 3 accumulates and aggregates in neurons, a phenomenon commonly observed in the brains of SCA3 patients [6]. The dose-dependent efficacy of modified ADMSCs was determined by investigating modified ADMSCs in various ratios versus SH-SY5Y ATNX3 / Q75 cells (0:1, 1:20, 1:15, and 1:10). In short, modified ADMSCs were 9 × 10⁶ 3, modified ADMSC 1.2×10 4 , and ADMSC 1.8×10 4 The cells were seeded in the upper compartment of a 6-well Transwell system (Falcon, 353090) and cultured in MSC culture medium containing alpha-MEM, 5% Helio Bioscience UltraGRO, and 2 U / ml heparin.

[0046] SH-SY5Y ATXN3 / Q75 2.0×10 5 Cells were placed in the lower compartment of a 6-well Transwell system (Falcon, 353502) and cultured in a medium containing DMEM / F12, 10% FBS, and 10 μM retinoic acid (Sigma, R2625). The following day, the insert seeded with modified ADMSCs was transferred to the lower compartment of a 6-well plate, and these 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 expression of mutant ataxin 3. 5 μg / ml doxycycline and 10 μM retinoic acid were added every 3 days for 1 week.

[0047] Example 4 The effect of activin A on an SCA3-affected cell model (SH-SY5Y ATXN3 / Q75 cells) The effects of various concentrations of activin A (0.2-5 ng / ml) (Biolegened, 592002) on an SCA3-affected cell model (SH-SY5Y ATXN3 / Q75 cells) were investigated. First, 20,833 cells / cm³ of SH-SY5Y ATXN3 / Q75 cells were introduced. 2In a 6-well plate cell culture dish (Corning, 353046) with DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blasticidin, 100 μg / ml hygromycin, and 10 μM retinoic acid at a density of , it was seeded. The next day, SH-SY5Y ATXN3 / Q75 cells were treated with 5 μg / ml doxycycline, 10 μM retinoic acid, and various 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.

[0048] Results: Activin A decreased the expression of mutant ataxin 3 in the SCA3 cell model. Figure 1 shows that activin A dose-dependently decreased the mutant ataxin 3 level in SH-SY5Y ATXN3 / Q75 cells.

[0049] Example 5 Combined effect of modified ADMSC and activin A on SCA3-affected cell model SH-SY5Y ATXN3 / Q75 cells Modified ADMSC 9×10 3 Modified ADMSC 1.2×10 4 And modified ADMSC 1.8×10 4 Were seeded into the upper compartment of a 6-well transwell system and cultured in MSC culture medium containing alpha MEM, 5% Helio Bioscience UltraGRO, and 2 U / ml heparin. SH-SY5Y ATXN3 / Q75 2.0×10 5Cells were placed in the lower compartment of a 6-well Transwell system and cultured in DMEM / F12 medium supplemented with 10% FBS and 10 μM retinoic acid. The following day, the insert seeded with modified ADMSCs was transferred to the lower compartment of a 6-well plate, and these SH-SY5Y ATXN3 / Q75 cells were 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). 5 μg / ml doxycycline and 10 μM retinoic acid were added every 3 days for 1 week.

[0050] Pulse waveform analysis (PulSA) On day 7, SH-SY5Y ATXN3 / Q75 cells were collected under the various treatment conditions described above, resuspended in DPBS containing 2% FBS, and analyzed by flow cytometry (Guava® easyCyte®). The results were analyzed using Guava software to determine the level of mutant attaxin 3.

[0051] The effects of modified ADMSC and / or activin A were evaluated using the combination index (CI) method (Chou, TC, Cancer Res, 2010.70(2):p.440-6).

[0052] Results: Figures 2A and 2B show the synergistic effects of various doses of modified ADMSC combined with various doses of activin A in reducing mutant attaxin 3 (CI values ​​less than 1 indicate synergistic effects).

[0053] Example 6 Modified adipose tissue-derived stem cells (ADMSCs) expressing activin A (activin A-expressing ADMSCs) One day before transfection, modified ADMSC 4.8×10 4The modified ADMSCs were cultured in 1.5 ml per well of MSC culture medium containing alpha-MEM, 5% Helio Bioscience UltraGRO, and 2 U / ml heparin, so that the concentration of modified ADMSCs reached 50–60% at the time of transfection. Diluted plasmid DNA, activin A expression plasmid (Sino Biological, HG10429-UT) or control plasmid (Sino Biological, CV011), and Lipofectamine stem (Thermo Fisher Scientific, STEM00001) were prepared separately in Opti-MEM I Medium (Thermo Fisher Scientific, 11058021) in different Eppendorf tubes and incubated at room temperature for 5 minutes. After incubation, the diluted plasmid DNA was combined with the diluted Lipofectamine stem, gently mixed, and incubated at room temperature for 10 minutes to form a complex of plasmid DNA and Lipofectamine stem. 150 μl of a plasmid DNA-Lipofectamine stem complex was added to each well containing modified ADMSCs and MSC culture medium, and the mixture was gently mixed by shaking the plate back and forth. The cells were incubated at 37°C in a CO2 incubator for 24 hours. Subsequently, activin A-expressing ADMSCs were supplemented with MSC culture medium. The activin A-expressing ADMSCs were cultured for 24 hours under standard conditions (5% CO2; 37°C). Conditional medium samples were collected, centrifuged to remove cell debris, and then frozen in fixed volumes at -20°C.

[0054] Effects of conditional media in the SCA3-affected cell model SH-SY5Y ATXN3 / Q75 cells SH-SY5Y ATXN3 / Q75 cells were treated with various doses of conditional media secreted from the above-mentioned activin A-expressing ADMSCs, and the expression levels of mutant ataxin 3 were analyzed.

[0055] First, we introduced 20,833 cells / cm³ of SH-SY5Y ATXN3 / Q75 cells. 2SH-SY5Y ATXN3 / Q75 cells were seeded at a density in 6-well cell culture dishes containing DMEM / F12 medium supplemented with 10% FBS, 5 μg / ml blastosidine, 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, as well as conditional media at various doses (3%, 10%, 30%, 50%, and 70%). 5 μg / ml doxycycline and 10 μM retinoic acid were added every 3 days for 1 week. On day 7, SH-SY5Y ATXN3 / Q75 cells under various treatment conditions were collected and analyzed by flow cytometry.

[0056] Results: High expression of activin A in modified ADMSCs enhanced the ability to reduce mutant attaxin 3 expression in the SCA3 cell model.

[0057] SH-SY5Y ATXN3 / Q75 cells were treated with activin A-expressing ADMSC-conditioned medium in various volume fractions. Figure 3 shows that mutant ataxin 3 expression is dose-dependently reduced by activin A-expressing ADMSC-conditioned medium compared to expression in the plasmid control group.

[0058] Example 7 The effect of activin A on modified adipose tissue-derived stem cells (ADMSCs) The effect of activin A on the proliferation of modified ADMSCs was evaluated.

[0059] method ADMSC proliferation was examined using CCK-8 (DOJINDO, Japan). Cells were seeded in the wells of a 96-well microtiter plate and incubated at 37°C for 16-18 hours. The fresh culture medium containing various concentrations of activin A was then replaced and the cells were incubated at 37°C for 3 days. After adding CCK-8 solution (10 μL per well), the cells were incubated for a further 3 hours. Absorbance was measured at 450 nm. High seeding density: 4,762 cells / cm² 2(In this application, the co-culture ratio of modified ADMSC to SH-SY5Y ATNX3 / Q75 = equivalent to 1:10); medium seeding density: 3,175 cells / cm 2 (equivalent to the co-culture ratio of modified ADMSC to SH-SY5Y ATNX3 / Q75 = 1:15); low seeding density: 2,381 cells / cm 2 (equivalent to the co-culture ratio of modified ADMSC to SH-SY5 = 1:20).

[0060] Results: Figures 4A - 4C show that various doses of activin A do not affect the proliferation of modified ADMSC at various densities. Such data confirm that the synergistic efficacy of the combination of modified ADMSC, which reduces mutant ataxin 3 levels, and activin A is not due to an increase in the number of modified ADMSC.

[0061] Example 8 Characterization of native ADMSC A cell population of stromal vascular cells (1.00×10 8 ) was isolated from the adipose tissue of healthy adult donors and processed as follows.

[0062] First, the collected stromal vascular cell population was centrifuged at 100 RCF for 3 - 10 minutes to remove unnecessary watery blood and digested with collagenase at 37°C for 0.5 - 6 hours. The aforementioned incubation time and the length of the accompanying shaking depend on the condition and size of the tissue. Next, the isolated native ADMSC was washed with PBS and centrifuged at 400 RCF for 3 - 10 minutes twice. After dispersion of the pellet, the isolated native ADMSC was counted and seeded in a cell culture flask at a density of less than 5×10 6 cells / cm 2 The surface markers of native ADMSC in the stromal vascular cell population were analyzed using flow cytometry analysis without further culturing.

[0063] The positive / specific staining region was defined using an isotype control. In short, the positive / specific staining region was defined by identifying nonspecific staining with unrelated immunoglobulins labeled with fluorescent dyes similar to the staining antibody. In this test, the positive threshold defined by the isotype control was 10. 2 Set it.

[0064] Based on flow cytometry images, natural ADMSCs are CD45 - CD34 + CD90 + CD73 + This exhibits the expression of [specific characteristic], which is a characteristic of natural ADMSCs. See Figure 5A.

[0065] Results: CD273 expression in natural ADMSCs was further investigated using flow cytometry. Figure 5B shows that natural ADMSCs do not express CD273 (i.e., CD273 expression). - ) is an example.

[0066] References 1. McLoughlin, HS, LR Moore, and HL Paulson, Pathogenesis of SCA3 and implications for other polyglutamine diseases. Neurobiol Dis, 2020. 134: p. 104635. 2. Rub, 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.

Claims

1. A method for treating PolyQ disease, the therapeutically effective amount (a) Activin 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, A method comprising the step of administering it to a subject in need.

2. A method for treating poly-Q disease, comprising the step of administering a therapeutically effective amount of activin A-expressing ADMSC, wherein the activin A-expressing ADMSC is (a) Expresses CD273, CD46, CD55, CXCR4, CD105, CD73, and CD90, but does not express CD45, CD34, CD11b, CD19, and HLA-DR. (b) A method for expressing activin A.

3. The method according to 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 spinal-bulbar muscular atrophy X-linked type 1 (SMAX1 / SBMA).

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

5. A method for reducing mutant polyQ protein in a target, wherein the therapeutically effective amount (a) Activin A and (b) Modified adipose tissue-derived stem cells (ADMSCs) or activin A-expressing ADMSCs, A method comprising the step of administering it to a subject in need.

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

7. (a) Expresses CD273, CD46, CD55, CXCR4, CD105, CD73, and CD90, but does not express CD45, CD34, CD11b, CD19, and HLA-DR. (b) expressing activin A, An isolated population of activin A-expressing ADMSCs.

8. The isolated population of activin A-expressing ADMSCs according to claim 7, wherein the ADMSC comprises at least one activin A expression plasmid.

9. The activin A-expressing ADMSC described in claim 7, Pharmacologically acceptable excipients, A pharmaceutical composition containing the above.

10. The pharmaceutical composition according to claim 9, further comprising activin A.

11. A method for reducing mutant polyQ protein in a subject, comprising the step of administering a therapeutically effective amount of activin A-expressing ADMSC to a subject in need thereof.

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

Images