Treatment of polyglutamine diseases

Abstract

To provide methods and articles of manufacture for use in stem cell therapy, for the treatment of diseases or conditions of SCA.SOLUTION: The invention provides a method for treating polyglutamine disease in a subject, the method comprising parenterally or locally administering an effective amount of stem cells as a unit dosage to the subject. The administration is performed with one or more treatment cycles, wherein one treatment cycle comprises dosing three unit dosages each at a dosing interval of two to six weeks.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of treatment of neurodegenerative diseases, and in particular to a method for treating polyglutamine diseases using stem cells. [Background technology]

[0002] Ataxias are clinically and genetically heterogeneous neurodegenerative disorders that variably affect the cerebellum, brainstem, and spinocerebellar tracts. Spinocerebellar ataxias (SCAs) are progressive, degenerative, and fatal. SCAs involve degeneration of neural tissue, with lesions primarily located in the cerebellar nuclei or pathways, brainstem, or spinal cord. SCAs are fatal not only due to widespread neuronal loss but also due to bedriddenness and respiratory failure at the end of the disease. The most common attributable subtypes of SCAs are polyglutamine-mediated SCAs, namely, SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17.

[0003] Patent Document 1 provides a method for treating spinocerebellar degeneration (SCA). This method comprises administering to a patient suffering from SCA an effective dose of one or more selected from D-cycloserine, D-serine esters, D-serine, and salts thereof. Patent Document 2 relates to a method for alleviating the signs or symptoms of SCA by intravenously administering an aqueous medicament containing trehalose.

[0004] However, no effective drug therapy or potential cure for SCA has been disclosed.

[0005] Therefore, there is a need for treatments that alleviate the signs and symptoms of SCA. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 7,067,545 [Patent Document 2] U.S. Patent No. 9,125,924 Summary of the Invention

[0007] The present invention provides a method for treating a polyglutamine disease in a subject. The method comprises parenterally or topically administering to the subject an effective amount of stem cells as a unit dose. The administration is carried out in one or more treatment cycles. One treatment cycle comprises administering three unit doses at intervals of 2 to 6 weeks.

[0008] In certain embodiments, polyglutamine diseases include, but are not limited to, spinocerebellar ataxia (SCA), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), and X-linked spinal muscular atrophy (SMAX1 / SBMA). In one embodiment, the SCA is SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17.

[0009] In some embodiments, the mesenchymal stem cells are a mesenchymal stem cell (MSC) population, an adipose tissue-derived stem cell (ADSC) population, an orbital adipose-derived stem cell (OFSC) population, or a quadruple-positive stromal cell (QPSC) population.

[0010] In some embodiments, the cells may be administered parenterally or locally (such as intracerebrally or intracranially).

[0011] In some embodiments, the unit dose is 0.5×10 5 ~5×10 10 cells / weight (k g).

[0012] In one embodiment, administration occurs in one or more treatment cycles. One treatment cycle comprises administering three unit doses at a 2-6 week (i.e., 2, 3, 4, 5, or 6 week) interval between doses. In a further embodiment, the interval is two weeks. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the appearance and behavior of SCA3 mice. [Figure 2] Figure 1 shows the immune tolerance of QPSCs in a xenograft model. The figure shows histopathological findings from a safety study in mice after administration of three doses of QPSCs. Almost no lesions were observed in the brain (A), heart (B), kidney (C), liver (D), lung (E), pancreas (F), or spleen (G) of QPSC-treated mice (HE staining (400x magnification)). [Figure 3] Figure 1 shows that QPSCs prevent weight loss in SCA3 mice. Body weights were recorded weekly before QPSC treatment and biweekly after treatment. Mice were sacrificed one month after the third QPSC injection. (A) Before treatment with QPSCs, SCA3 mice weighed less than wild-type mice. (B) and (C) show that QPSCs prevent weight loss in SCA3 mice. [Figure 4] QPSCs alter the phenotype of SCA3 mice. Wild-type (WT) and SCA3 transgenic (TG) mice were both intravenously administered with QPSCs three times. Modified SHIRPA was performed before and after QPSC treatment. (A) QPSCs alter the phenotype of SCA3 mice in pelvic elevation. (B) and (C) QPSCs alter the phenotype of SCA3 mice in the grip strength test. [Figure 5] This figure shows that three doses of QPSC improved the motor function of SCA3 mice with minimal motor impairment. Both wild-type (WT) mice and SCA3 transgenic (TG) mice received three intravenous injections of QPSC. Modified SHIRPA and rotarod tests were performed before and after QPSC treatment. (A) and (B) show that the mice's motor and negative geotaxis abilities improved after QPSC treatment. (C) shows that the rotarod performance of SCA3 mice (Tg) was significantly improved after injection of three doses of QPSC. [Figure 6] Figure 1 shows that three doses of QPSCs improve the motor function of SCA3 mice, which had severe motor impairment. One month after the third QPSC injection, mice were subjected to footprint analysis. The results show that the reduced stride length of the left front paw (A), right front paw (B), left hind paw (C), and right hind paw (D) of SCA3 mice was improved by three doses of QPSCs. [Figure 7] This figure shows that three doses of QPSC improved the walking balance of SCA3 mice. One month after the third QPSC injection, the mice underwent footprint analysis. (A) and (B) show the positions of the front and back paw prints, respectively, and (C) and (D) show the overlap of the right and left paw prints, respectively. Three doses of QPSC not only improved the stride length reduced by SCA3, but also maintained nearly 100% overlap of the footprints. [Figure 8] This figure shows the intracranial localization ability of QPSCs after intravenous injection. QPSCs were transplanted into wild-type mice via tail vein injection, and 7 days after transplantation, brain tissue was excised for quantitative real-time reverse transcription PCR (RT-PCR) analysis. The ratio of human DNA (detected by human β2 microglobulin) to mouse DNA (detected by mouse 18s rRNA) was approximately 0.8% (mouse No. 1) to 2.8% (mouse No. 4). [Figure 9] These figures demonstrate that QPSCs have the ability to differentiate into Purkinje neuron-like cells in the cerebellum of SCA mice. (A) One month after systemic administration of QPSCs via three intravenous (IV) injections, some of the transplanted cells differentiated into Purkinje neuron-like cells with axonal structures (arrows) in the cerebellum of SCA mice. (B) No neuronal differentiation from QPSCs was observed in the cerebellum of SCA mice, even after three intracranial (intradermal) injections. [Figure 10]These figures show that QPSCs have strong immunomodulatory and anti-ROS (reactive oxygen species) capabilities. (A) Human T cell proliferation was stimulated by CD3 / 28, and the stimulated proliferation was blocked by co-culture with StemKymal® at all mixing ratios (*** indicates significant difference [P<0.05], n=3). (B) The anti-HO ability of QPSCs is three-fold greater than that of human corneal epithelial cells (HCE-T), which are relatively resistant to oxidative stress. [Figure 11] Figure 1 shows that QPSCs prevent the oxidative stress-associated decline in motor function in SCA mice. (A) SCA mice with low oxidative stress (low ROS) exhibited better rotarod performance than SCA mice with high oxidative stress (high ROS). (B) While the decline in motor function in SCA mice (Tg control) progressed, the rotarod performance of SCA mice with high oxidative stress (Tg(QPSC high ROS)) and low oxidative stress (Tg(QPSC low ROS)) that received systemic transplants of QPSCs was higher in the Tg(QPSC low ROS) group than in the Tg control group (*P<0.05). Wild-type mice were used as normal controls (WT(control)). [Figure 12] Figure 1 shows that QPSCs express paracrine neurotrophic factors and tissue growth factors. (A) Gene expression of neurotrophic factors, including NT-3, NT-4, NGF, CNTF, BDNF, and GDNF, in QPSCs was detected by quantitative PCR against the internal control gene 18srRNA. Tissue factors such as EGF, FGF-β, and VEGF (B), and PDGF and TGF-β1 (C) in QPSCs were examined by ELISA, and the concentration differences between the intracellular and secreted fractions of these factors were shown. [Figure 13]This figure shows that QPSC paracrine responses prevent 1-methyl-1-4-phenylpyridine (MPP+)-induced astrocyte neuronal loss. SVG p12, a human astrocyte cell line, was treated with 1.25 mM MPP+ and co-cultured with different ratios of QPSCs. Twenty-four hours after treatment, SVG p12 cell numbers were counted. MPP+ treatment significantly reduced SVG p12 cell numbers, a phenomenon that was reversed when co-cultured with 10x the amount of QPSCs. [Figure 14-1] FIG. 1 shows that QPSCs prevent Purkinje neuron loss in SCA3 mice. [Figure 14-2] This figure shows that QPSCs prevent the loss of Purkinje neurons in SCA3 mice. Cerebellum was harvested after mouse death. The harvested tissue was fixed and embedded in paraffin for further histopathological analysis. Tissue sections were stained with hematoxylin and eosin (HE), and targeted Purkinje cells were immunohistochemically stained (IHC) using anti-calbindin antibody, ab11426, Abcam. (A) SCA3 mice, which exhibit severe motor dysfunction, have smaller and more deformed Purkinje cells than wild-type mice. (B) and (C) show a significant reduction in Purkinje cell number in the cerebellum of SCA3 mice, but three doses of QPSCs prevented the loss of Purkinje neurons in SCA3 mice. DETAILED DESCRIPTION OF THE INVENTION

[0014] Methods and products for use in stem cell therapy to treat polyglutamine diseases are provided. Polyglutamine diseases are a group of neurodegenerative disorders caused by the expansion of cytosine-adenine-guanine (CAG) repeats that encode long polyglutamine tracts within respective proteins. Polyglutamine diseases are characterized by pathological expansion of CAG trinucleotide repeats in the translated regions of unrelated genes. The translated polyglutamine aggregates into degenerated neurons, causing dysfunction and degeneration of specific neuronal subpopulations. The present invention provides a stem cell-based therapeutic regimen for the treatment of degeneration and / or remodeling of cells in polyglutamine diseases. has led to the surprising discovery that it may provide an effective treatment for restoring function to damaged neurons.

[0015] Unless otherwise noted, technical terminology is used conventionally.

[0016] As used herein, the singular articles refer to both the singular and the plural, unless otherwise specified.

[0017] As used herein, the terms "and" and "or" can refer to either the conjunctive or disjunctive meanings, i.e., both terms should be understood to be equivalent to "and / or" unless otherwise specified.

[0018] As used herein, the term "treatment" or "treating" means to completely or partially improve or alleviate a disease, condition, or disorder, or a symptom, side effect, or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, preventing direct or indirect pathological consequences of a disease, slowing the rate of disease progression, and alleviating the condition.

[0019] As used herein, the phrase "delaying disease progression" means to prevent, slow, arrest, halt, and / or arrest the progression of the disease. The length of time for this delay may vary depending on the history of the disease and / or the person being treated.

[0020] As used herein, the term "effective amount" of an agent, e.g., a pharmaceutical composition, cell, or composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result.

[0021] As used herein, the term "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation, or cells, means an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result and / or pharmacokinetic or pharmacodynamic effect of treatment for a disease, illness, or disorder.

[0022] As used herein, the term "first administration" refers to the timing of a given administration prior to subsequent or sequential administrations. This term does not necessarily imply that the subject has not previously received a cell therapy or the same cells.

[0023] As used herein, the term "subsequent administration" refers to an administration given to the same subject after a previous, e.g., initial, administration, without any intervening administrations.

[0024] As used herein, the term "subject" refers to a mammal, such as a human or other animal, primarily a human. In some embodiments, the subject has been treated with a therapeutic agent directed at a disease or disorder prior to administration.

[0025] As used herein, the term "pharmaceutical formulation" refers to a preparation in a form that effectively utilizes the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that are toxic and unacceptable to the subject to which it is administered.

[0026] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0027] One aspect of the present invention provides a method for treating a polyglutamine disease in a subject. The method comprises parenterally or topically administering to the subject an effective amount of stem cells as a unit dose. The administration is carried out in one or more treatment cycles, each cycle comprising three unit doses administered at intervals of 2 to 6 weeks.

[0028] In certain embodiments, polyglutamine diseases include, but are not limited to, spinocerebellar ataxia (SCA), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), and X-linked spinal muscular atrophy (SMAX1 / SBMA).

[0029] In some embodiments, the SCA is polyglutamine-mediated SCA, and preferably the SCA is SCA1, SCA2, SCA3, SCA6, SCA7, or SCA 17. More preferably, the SCA is SCA3.

[0030] In some embodiments, the mesenchymal stem cells are a mesenchymal stem cell (MSC) population, an adipose tissue-derived stem cell (ADSC) population, an orbital adipose-derived stem cell (OFSC) population, or a quadruple-positive stromal cell (QPSC) population.

[0031] In one embodiment, the QPSCs are those described in U.S. Patent Application No. 14 / 615,737, have at least 70% cellular homogeneity, express the cell markers CD273, CD46, CD55, and CXCR4, but not CD45, and are positively expressed at an intensity greater than 70%.

[0032] In one embodiment, the ADSCs are OFSCs as described in U.S. Patent Publication No. 20120288480, which express at least CD90, CD105, CD29, CD44, CD49b, CD49e, CD58, and HLA-ABC, but do not express CD133, CD31, CD106, CD146, CD45, CD14, or CD117. Preferably, the stem cells are a QPSC population.

[0033] In some embodiments, the cells may be administered parenterally or locally (intracerebrally or intracranially). Parenteral administration includes intramuscular, intravenous, intraarterial, or subcutaneous administration. Preferably, parenteral administration is intravenous injection.

[0034] In some embodiments, the unit dose is 0.5×10 5 ~5×10 10 In some embodiments, the unit dose is 0.5 x 10 cells / kg body weight. 5 ~5×10 9 , 0.5×10 5 ~5×10 8 , 0.5×10 5 ~5×10 7 , 0.5×10 5 ~5×10 6 , 1.0×10 5 ~5×10 10 , 1.0×10 5 ~5×10 9 , 1.0×10 5 ~5×10 8 , 1.0×10 5 ~5×10 7 , or 1.0×10 5 ~5×10 6 cells / body weight (kg).

[0035] In one embodiment, administration occurs in one or more treatment cycles. One treatment cycle comprises administering three unit doses at a 2-6 week (i.e., 2, 3, 4, 5, or 6 week) interval between doses. In a further embodiment, the interval is two weeks. The number of treatment cycles in the present invention is determined by the SARA (Status of Ataxia Assessment). (Subramony SH, SARA - a new clinical rating scale for ataxia. Nat Clin Pract Neurol. 2007; 3(3):136-7; Kim BR, Lim JH, Lee S, Park S, Koh SE, Lee IS, Jung H, Lee J. Usefulness of the SARA (Status of Ataxia Assessment) in acute stroke patients. Ann Rehabil Med. 2011; 35:772-780; Tan S, Niu HX, Zhao L et al. Reliability and validity of the Chinese version of the SARA. Chin Med J. 2013; 126(11):2045-8). SARA is a clinical scale based on semiquantitative assessment of cerebellar ataxias (spinocerebellar, Friedreich's, and sporadic ataxias) at the level of injury. The SARA is an eight-component scale with a total score ranging from 0 (no ataxia) to 40 (severe ataxia). Scores are based on the patient's performance in walking, standing, sitting, speech impairment, finger tracking, nose-to-finger test, hand pronation-supination, and heel-shin test. If the subject's one-month total SARA score is greater than 5 after the first treatment cycle, subsequent treatment cycles are initiated.

[0036] The unit dose of stem cells is administered at intervals of 2 to 6 weeks. An interval of 2 to 6 weeks means that the unit dose of stem cells is administered once every 2, 3, 4, 5, or 6 weeks. In one embodiment, the administration interval is every other week. In one embodiment, biweekly administration means that the unit dose of stem cells is administered once every two weeks, i.e., once every 14 days, preferably on the same day of the week every two weeks. In a biweekly dosing regimen, the unit dose is generally administered approximately every 14 days.

[0037] In stem cell therapy, administration of a unit dose includes administration of a predetermined amount or number of cells as a single composition and / or a single continuous administration, such as, for example, a single injection or continuous infusion.

[0038] In some embodiments, the cells may be administered, for example, as part of a combination therapy, simultaneously with other therapeutic interventions or sequentially in any order. In some embodiments, the stem cells are administered simultaneously with or in combination with one or more additional therapeutic agents or other therapeutic interventions, simultaneously or sequentially in any order. In some embodiments, the additional therapeutic agent or other therapy is introduced sufficiently close in time to the administration of the cells to enhance the effect of the cell population, or the cell population is administered sufficiently close in time to the introduction of the additional therapeutic agent or other therapy to enhance the effect of the additional therapeutic agent or other therapy. In some embodiments, the stem cells are administered before one or more additional therapeutic agents. In some embodiments, the stem cells are administered after one or more additional therapeutic agents.

[0039] The stem cells used in the methods of the present invention are formulated as pharmaceutical compositions or formulations, such as a unit dose composition containing a number of cells for administration at a predetermined dose or a fraction thereof. Pharmaceutical compositions and formulations generally contain one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition contains at least one additional therapeutic agent.

[0040] The choice of carrier will depend in part on the particular stem cells and / or the method of administration. Accordingly, there are a variety of suitable formulations. For example, pharmaceutical compositions may contain preservatives.

[0041] Pharmaceutically acceptable carriers are generally non-toxic to the subject in the amounts and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol; and m-cresol); low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or polyethylene The composition may comprise a non-ionic surfactant such as ethylene glycol (PEG). In some aspects, the composition may comprise a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. Methods for preparing administrable pharmaceutical compositions are well known.

[0042] The formulation may comprise an aqueous solution. The formulation or composition may comprise multiple active ingredients beneficial for the particular indication, symptom, or condition being treated with stem cells.

[0043] In some embodiments, the pharmaceutical composition comprises an amount of stem cells effective to treat a disease or condition, e.g., a therapeutically effective amount. In some embodiments, the effectiveness of the treatment is monitored by periodic evaluation of the treated subject. The desired dose may be provided by a single bolus of cells, multiple boluses, or continuous infusion.

[0044] The stem cells and compositions may be administered using standard administration techniques, formulations, and / or devices. Administration of the stem cells may be autologous or xenogeneic.

[0045] Sterile injectable solutions can be prepared by incorporating the cells into a solvent mixed with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-promoting additives, preservatives, flavoring agents, and / or coloring agents, depending on the route of administration or the preparation desired. In some aspects, standard textbooks may be consulted for appropriate formulations.

[0046] Various additives may be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of injectable pharmaceutical preparations can be achieved by using absorption delaying agents, such as aluminum monostearate and gelatin.

[0047] [Example] I. Animal Model Testing material and method Animals and experimental design.

[0048] Mice with almost no decline in motor function MJD84.2(B6;CBA-Tg(ATXN3 *MJD84.2Cce / IbezJ mice were used as a disease model for Machado-Joseph disease (MJD / SCA3), also known as spinocerebellar ataxia type 3 in humans. MJD84.2 mice aged 20–34 weeks were used for the experiments. Behavioral analyses, including modified-SHIRPA, footprint analysis, and the rotarod test, were performed on these mice. Mice aged 21, 23, and 25 weeks were injected with the test substance three times, every two weeks. The experimental design is outlined below.

[0049] [Table 1]

[0050] Mice with severely impaired motor function Thirteen SCA3 Tg / 0 mice (B6; CBA-Tg(ATXN3 * )84.2Cce / IbezJ) and eight C57BL / 6 0 / 0 wild-type mice, all obtained from The Jackson Laboratory. Mice were randomly divided into four experimental groups: (1) SCA3+ cells, (2) SCA3+ phosphate-buffered saline (PBS), (3) wild-type (Wt)+ cells, and (4) Wt+ PBS. After the SCA3 Tg / 0 mice exhibited a significant disease phenotype, they received three injections of the test substance, spaced 2 weeks apart. The experimental design is outlined below.

[0051] [Table 2]

[0052] Mesenchymal stem cells The QPSCs used in this experiment were human ADSCs (Stemkaimal®), a cell product manufactured by Steminent Biotherapeutics Inc. ADSCs were culture-expanded in vitro in a cell factory constructed in accordance with PIC / S GMP guidelines, and quality control was performed according to Steminent's standard operating procedures. Briefly, adipose tissue was collected from healthy donors and immediately transported to Steminent's processing facility, which was maintained at a low temperature (0–5°C). After isolation and purification, ADSCs were maintained in Steminent's medium during culture expansion. Passage 12 ADSCs became QPSCs that highly expressed CD273, CD46, CD55, and CXCR4 and were packaged in cryopreservation bags. The product (Stemkaimal®) was sent for quality certification and cryopreservation. Stemkaimal® quality control consisted of in-process control and product release testing. Quality controls include, but are not limited to, viability testing, sterility testing, mycoplasma testing, endotoxin testing, MSC phenotyping (positive for CD73, CD90, and CD105, negative for CD34, CD45, CD11b, CD19, and HLA-DR), and tri-lineage differentiation potential (osteogenesis, chondrogenesis, and adipogenesis).

[0053] Cell administration - Tests using mice with almost no decline in motor function 2.5×10 7 StemKymal® cells / kg body weight were thawed and prepared and loaded into a 1 ml insulin syringe (29 1 / 2 G). Cells were injected slowly (15-20 seconds) within 1 hour of thawing.

[0054] -Examination using mice with severely impaired motor function Mice were randomly divided into four experimental groups: (1) SCA3+ cells, (2) SCA3+PBS, (3) wild-type (Wt)+ cells, and (4) Wt+PBS. Mice in groups 1 and 3 each received 2.5 × 10 7StemKymal® cells / kg body weight (125 μl total cell suspension (1:1 in Cryosolution (Biolife) or PBS (Gibco)) was administered intravenously to each mouse.

[0055] In both experiments, mice were monitored for four hours each day after injection, with a total of three cell injections administered every two weeks.

[0056] Data collection and analysis Mice were sacrificed one month after the last test substance injection. The weight of the mice and the time spent before falling were recorded throughout the experiment. After the test substance injection, footprint analysis was also performed to assess the walking ability of the mice. Mouse tissues (cortex, cerebellum, heart, kidney, liver, spleen, lung, and tail) were collected for further histopathological analysis and biodistribution testing.

[0057] statistics Data are presented as mean ± standard error. The results of the rotarod test and footprint analysis were analyzed using Student's t-test (significance threshold p<0.05).

[0058] Motor coordination and balance assessment Motor coordination and balance were assessed using a rotarod apparatus (MK-670, Muromachi Kikai Co., Ltd., Japan). Mice were placed on the rotarod apparatus at a constant speed (4 rpm) that accelerated to 40 rpm over a 5-minute period. The time it took to fall or complete the full rotation (holding on to the rod until the complete rotation) was recorded. Mice were given three trials for each test, with a 15-minute rest period between trials. The average time spent on each test was calculated for each mouse. Statistical analysis of the test results was performed using Student's t-test.

[0059] SHIRPA test The modified SHIRPA test was performed on mice aged 20, 24, and 28 weeks. The SHIRPA protocol was a modified version of the RIKEN BioResource Research Center's modified SHIRPA protocol. The test items and scoring criteria are shown in Table 3 below.

[0060] [Table 3]

[0061] Mice were scored based on their behavior. The total number of mice in the test group was calculated as 100%. Results were expressed as a percentage of the number of mice at a particular score level.

[0062] Footprint Analysis After the final cell injection, the mice's footprints were analyzed approximately one month later. According to a publication published in January 2015, the mice's paws were dipped in ink (red ink for the front paws and green ink for the hind paws). The mice then left footprints while walking or running along the pathway to the goal box. The mice were placed on a piece of paper (50 cm long, 10 cm wide) in front of the tunnel. Their walking pattern was measured by stride length, sway, step length, and overlap of the front and hind paws (see the diagram below). All mice were killed after running three times.

[0063] MTT assay CD3 + T cell isolation Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using Histopaque 1077 (Sigma-Aldrich). CD3 was then isolated from PBMCs by positive selection using anti-human CD3 antibody-conjugated magnetic particles (BD Biosciences) according to the manufacturer's instructions. + T lymphocytes were purified.

[0064] T cell proliferation assay Purified human CD3 + T cells (1×10 5T cells (ADSCs) were stimulated in 96-well plates with plate-bound anti-CD3 (2 μg / ml) and anti-CD28 (2 μg / ml) monoclonal antibodies (BD Biosciences) and cocultured with different numbers of ADSCs in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U / ml penicillin, 100 U / ml streptomycin, and 25 mM HEPES. After 48 h, 5-bromo-2-deoxyuridine (BrdU) was added to each well, and the plates were incubated for an additional 18 h to measure T cell proliferation. The amount of BrdU in T cells was measured using a BrdU cell proliferation ELISA kit (Roche) according to the manufacturer's instructions.

[0065] Immunohistochemistry (IHC) To evaluate the neuroprotective effects of QPSCs, C57BL / 6J SCA2 transgenic mice were injected with QPSCs via the tail vein (IV hMSC-Tg group) or into the cerebellar region via the foramen magnum (IC hMSC-Tg group). A specific antibody reacting with human β2-microglobulin (Abcam, code: ab15976) was selected to reveal human cells in mouse brain tissue by histological staining. Mouse sections (4 μm) were mounted on microscope slides. The sections were rehydrated by rinsing twice in xylene, 100% ethanol, 95% ethanol, and 80% ethanol for 5 min each. After deparaffinization, sections were treated with 3% H2O2 to inactivate peroxidase, heated in 10 mM citrate buffer (with 0.05% Tween 20) for antigen retrieval, and blocked with 1% blocking solution (1% BSA and 0.1% Triton X-100) (Chang et al., Journal of Biomedical Science 2011, 18:54, http: / / www.jbiomedsci.com / content / 18 / 1 / 54, page 3 of 9PBS). Sections were incubated with a specific anti-human β2-microglobulin polyclonal antibody (Abcam) diluted in blocking solution (1:400) for 40 minutes at room temperature. After three extensive washes with PBS, sections were incubated with a secondary antibody diluted in blocking solution (1:1000) for 40 minutes at room temperature. Primary antibodies were detected using the EnVision detection system (DAKO) and visualized with diaminobenzidine (DAB; DAKO). Counterstaining was performed with aqueous hematoxylin (Sigma-Aldrich). For direct comparison, all slides were processed in a single batch to minimize variability.

[0066] Safety Testing ·animal C57BL / 6 mice were intravenously injected with three doses of QPSCs using a 1 / 2 cc insulin syringe and a 30G × 3 / 8" needle (Terumo or BD Biosciences). Before injection, mice were warmed for 15–20 min on a heating pad placed under their cage to allow the tail vein to dilate. Before necropsy, all animals were anesthetized with urethane (2 g / kg body weight, Sigma-Aldrich), and blood was collected from the submandibular vein or by cardiac puncture.

[0067] Collection of blood samples For hematology analysis, whole blood samples were collected into EDTA-containing blood collection tubes (BD Biosciences, Catalog No. 365974). For blood chemistry analysis, whole blood samples were collected into serum separator-containing blood collection tubes (BD Biosciences, Catalog No. 365967). The blood collection tubes were then left standing at room temperature for 20 minutes and centrifuged at 6000 rpm for 5 minutes at 4°C to separate the serum.

[0068] Gross necropsy and tissue sampling After blood collection, the organs of the mice were harvested and each was divided into two parts. (1) Half of the organs were stored in a -80°C freezer and then transferred to a liquid nitrogen container for biodistribution testing. (2) The other half was fixed (4% paraformaldehyde, Sigma-Aldrich) and embedded in paraffin for histopathological analysis.

[0069] quantitative PCR Total RNA was extracted from QPSCs or mouse tissues using a total RNA miniprep purification kit (GM Biolabs, catalog no. TR01) according to the manufacturer's instructions. cDNA was then synthesized using a two-step MMLV RT-PCR kit (GM Biolabs, catalog no. RP012-M). Quantitative PCR for relative expression analysis of selected genes was performed using Fast SYBR® Green Master Mix (Thermo, catalog no. 4385612).

[0070] ELISA To measure the intracellular and secreted concentrations of EGF, FGF-b, VEGF, PDGF, and TGF-b1 in QPSCs, cell lysates and conditioned media were prepared as follows.

[0071] QPSCs were lysed by freeze-thawing, and the cell lysate supernatant was collected after ultracentrifugation. After 3 days of QPSC culture, the medium was harvested for conditioned medium collection. Finally, the concentrations of the above growth factors were determined by ELISA according to the manufacturer's instructions (R&D Systems).

[0072] Neuronal co-culture assay SVG p12, a human astrocyte cell line, was treated with 1250 μM 1-methyl-4-phenylpyridine (MPP+) and co-cultured with QPSCs at different ratios (SVG p12:QPSC = 1:0.1 to 1:10). After 24 hours, the number of SVG p12 cells was counted.

[0073] Example 1: QPSCs alter the SCA3 phenotype QPSC altered the phenotype of SCA3 mice. As shown in Figure 1, the base of the neck of SCA3 mice was slightly thicker than that of wild-type mice, and after treatment with QPSC, the SCA3 mice appeared similar to wild-type mice. Similar improvements were observed in various functional tests, including modified SHIRPA (Figures 4 and 5), footprint analysis (Figures 6 and 7), and the rotarod test (Figure 5(C)).

[0074] Example 2: The treatment of the present invention prevents weight loss without adverse effects on organ tissues Although QPSC prevented weight loss in SCA3 mice during disease progression (Figure 3), three doses of QPSC (three QPSC administrations) did not affect the complete blood count (CBC) (Table 4) or blood chemistry (Table 5) profiles of individual SCA3 mice. Tables 4 and 5 show that the complete blood count / blood chemistry profiles of 25-30 week-old wild-type mice administered three doses of QPSC weekly were no different from those administered three doses of saline weekly. Histopathological analysis showed normal findings in various vital organ tissues after injection of three doses of QPSC (Figure 2).

[0075] [Table 4]

[0076] Data are shown as mean ± standard deviation.

[0077] [Table 5]

[0078] Data are presented as mean ± standard deviation.

[0079] Example 3: Immunomodulatory and anti-ROS capacity, and neurotrophic and growth factor expression in mice using the treatment of the present invention.

[0080] In vitro studies have demonstrated that QPSCs not only possess immunomodulatory and anti-ROS capabilities (Figure 10), but also express neurotrophic and growth factors (Figure 12). In vitro studies have demonstrated improved rotarod performance in SCA mice subjected to oxidative stress after treatment with QPSCs (Figure 11). Furthermore, QPSCs may prevent neuronal loss both in vitro (Figure 13) and in vivo (Figures 14-1 and 14-2). Although the ability of cells to migrate across the blood-brain barrier (BBB) ​​has been questioned, the ability of QPSCs to localize intracranially following intravenous injection has been demonstrated (Figures 8 and 9).

[0081] Therefore, we can conclude that QPSCs penetrate the BBB via intravenous injection and reach the cerebellum, protecting neurons in SCA patients from damage caused by ROS and immune overreaction. Furthermore, QPSCs express neurotrophic and growth factors to maintain a large number of neurons, thereby slowing the progression of polyglutamine diseases such as polyglutamine spinocerebellar ataxia, Machado-Joseph disease, Huntington's disease, DRPLA, and SMAX1 / SBMA.

[0082] II. Human Clinical Trials A randomized, double-blind, placebo-controlled study was conducted to examine the therapeutic efficacy and safety of StemKymal® injection for the treatment of polyglutamine-mediated diseases (such as spinocerebellar ataxia, Machado-Joseph disease, Huntington's disease, DRPLA, and SMAX1 / SBMA). Eligible subjects were administered StemKymal® intravenously.

[0083] In one example of a polyglutamine spinocerebellar ataxia, the subject has genotypically confirmed spinocerebellar ataxia type 2 or spinocerebellar ataxia type 3. The subject's baseline SARA score is 5-15.

[0084] 2.5×10 7 StemKymal® cells / kg body weight were thawed, prepared, and loaded into a syringe. The cells were slowly infused within one hour of thawing. Each subject received three intravenous injections of StemKymal® every two weeks to administer the cells. After one or more treatment cycles, the subjects' SARA scores decreased and their SCA2 or SCA3 status improved.

Claims

1. 1. A composition for treating a polyglutamine disease in a subject, comprising: A composition comprising an effective amount of adipose tissue-derived stem cells (ADSCs) that express the cell markers CD273, CD46, CD55, CXCR4, CD73, CD90 and CD105.

2. The composition described in claim 1, wherein the polyglutamine disease is spinocerebellar ataxia (SCA), dentatorubral-pallidoluysian atrophy (DRPLA), or X-linked spinal and bulbar muscular atrophy (SMAX1 / SBMA).

3. The composition described in claim 1, wherein the polyglutamine disease is spinocerebellar ataxia (SCA).

4. The composition described in claim 3, wherein the SCA is SCA1, SCA2, SCA6, SCA7, or SCA17.

5. The composition described in claim 4, wherein the SCA is SCA2.

6. The composition described in claim 4, wherein the SCA is SCA6.

7. The composition of claim 1 , wherein the ADSCs are administered parenterally or topically.

8. The composition of claim 7 , wherein the parenteral administration is intramuscular, intravenous, intraarterial, or subcutaneous administration.

9. The composition of claim 7 , wherein the local administration is intracerebral or intracranial administration.

10. The ADSCs are administered in one or more treatment cycles, each treatment cycle comprising three unit doses administered at a 2-6 week interval, each unit dose being 0.5 x 10 ５ ~5 x 10 ７ The composition of claim 1, wherein the ADSC is 0.01 mg / kg body weight.

11. 10. The composition of claim 1, wherein the ADSCs can be administered simultaneously or sequentially in any order with one or more additional therapeutic agents or in combination with other therapeutic interventions.

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