Treatment for polyglutamine (POLYQ) disease

Abstract

The present invention provides methods and articles of manufacture for use in stem cell therapy for the treatment of SCA diseases or conditions. In particular, the present invention provides a method for treating SCA comprising administering to a subject an effective amount of stem cells as a unit dose non-enterally or locally, where the administering is performed at one or more treatment cycles, where one treatment cycle comprises administering three unit doses at an administration interval of 2 to 6 weeks, respectively.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of May 26, 2017, application number: 201780091297.9, and invention name: "For the treatment of polyglutamine (POLYQ) diseases". Technical Field

[0002] The present invention relates to the field of neurodegenerative disease treatment. In particular, the present invention relates to a treatment regimen for polyglutamine (polyQ) diseases using stem cells. Background Art

[0003] Ataxia is a group of clinically and genetically heterogeneous neurodegenerative diseases that variably affect the cerebellum, brainstem and spinocerebellar pathways. Spinocerebellar ataxia (SCA) is a progressive, degenerative and fatal disease. SCA includes degeneration of neuronal tissue, with the primary sites of pathological changes present in the nuclei or neural pathways of the cerebellum, brainstem or spinal cord. The fatal condition of SCA comes not only from a large number of neuronal losses, but also from bedriddenness and respiratory failure during advanced disease. The most common SCA classification subtype is polyglutamine (polyQ)-mediated SCA, i.e., SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17.

[0004] US 7,067,545 provides a method for treating spinocerebellar degeneration, comprising administering to a patient suffering from the disease an effective dose of one or more components selected from D-cycloserine, D-serine esters, D-serine and salts thereof. US 9,125,924 relates to a method for alleviating the signs or symptoms of SCA by intravenously administering an aqueous formulation comprising trehalose.

[0005] However, there is no effective medical treatment or potential cure for SCA. Therefore, there is a need for treatments that can alleviate the signs and symptoms of SCA. Summary of the invention

[0006] The present invention provides a method for treating a polyglutamine (polyQ) disease in a subject, the method comprising parenterally or topically administering to the subject an effective amount of stem cells as a unit dose, wherein the administration is performed in one or more treatment cycles, wherein one treatment cycle comprises three unit doses given at dosing intervals of 2 to 6 weeks, respectively.

[0007] In some embodiments, polyQ diseases include, but are not limited to, spinocerebellar ataxia (SCA); Machado-Joseph disease (MJD / SCA3); Huntington's disease (HD); Dentatorubral pallidum Lewy body atrophy (DRPLA); and type 1 X-linked spinal bulbar muscular atrophy (SMAX1 / SBMA). In one embodiment, SCA is SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17.

[0008] In some embodiments, the mesenchymal stem cells are a mesenchymal stem cell population (MSC), an adipose tissue-derived stem cell population (ADMSC), an orbital adipose-derived stem cell population (OFSC), or a quadri-positive stromal cell (QPSC) population.

[0009] In some embodiments, the cells may be administered parenterally or by local therapy (such as intracerebral or intracranial administration).

[0010] In some embodiments, the unit dose is 0.5×10 5 Up to 5×10 10 cells / kg body weight.

[0011] In one embodiment, administration is carried out in one or more treatment cycles, wherein one treatment cycle comprises three unit doses given at a dosing interval of 2 to 6 weeks (ie, two weeks, three weeks, four weeks, five weeks or six weeks. In another embodiment, the interval is two weeks).

[0012] Simple diagram description

[0013] Figure 1 A to D show the appearance and behavior of SCA3 mice.

[0014] Figure 2 A to F show the immune tolerance of QPSC in a xenograft model. The figure shows the histopathological findings of the safety test after mice received 3 doses of QPSC. By H&E staining (400x), no brain ( Figure 2 A) Heart Figure 2 B) Kidneys Figure 2 C) Liver ( Figure 2 D) Lungs Figure 2 E) Pancreas ( Figure 2 F) or spleen ( Figure 2 G) significant lesions.

[0015] Figure 3A to 3C show weight loss in QPSC-stopped SCA3 mice. Body weight was recorded weekly before QPSC treatment and every two weeks after QPSC treatment. Animals were sacrificed one month after the third QPSC injection. Figure 3 A, Before QPSC treatment, SCA3 mice weigh less than wild-type mice. Figure 3 B and 3C, QPSC prevents weight loss in SCA3 mice.

[0016] Figure 4 A to Figure 4 C shows that QPSC alters the phenotype of SCA3 mice. Both WT mice and SCA3 transgenic mice (TG) received 3 intravenous QPSC administrations. Modified SHIRPA was performed before and after QPSC treatment. Figure 4 A Pelvic lift was used to illustrate that QPSC altered the phenotype of SCA3 mice. Figure 4 B and 4C, QPSC alters the phenotype of SCA3 mice in terms of grip strength.

[0017] Figure 5 A to Figure 5 C shows that three doses of QPSC improve motor function in SCA3 mice without significant motor function deterioration. Both WT mice and SCA3 transgenic mice (TG) received 3 intravenous QPSC administrations. Modified SHIRPA and rotarod performance tests were performed before and after QPSC treatment. Figure 5 A and B: SCA3 mice have improved locomotion and negative geotaxis performance after QPSC treatment. Figure 5 C: Rotarod performance of SCA3 mice (Tg) was significantly improved after infusion of 3 doses of QPSC.

[0018] Figure 6 A to Figure 6 D shows that three doses of QPSC improve the motor function of SCA3 mice with significant motor function deterioration. Footprint analysis of mice was performed one month after the third QPSC injection. Figures A to D show the left front foot (LF) of SCA mice ( Figure 6 A) Right front foot (RF) Figure 6 B) Left hind foot (LH) Figure 6 C) and right hind foot (RH) ( Figure 6 D) The footprint span was reduced, and 3 doses of QPSC restored the abnormal span.

[0019] Figure 7 A to Figure 7 D shows that three doses of QPSC improve gait balance in SCA3 mice. Footprint analysis on mice was performed one month after the third QPSC injection. Figure 7 A to D: Figure 7 A and Figure 7 B shows the footprint span of the mouse's front paw (F) and hind paw (H), respectively. Figure 7 C and Figure 7 D shows the overlap of the footprints of the left (L) and right (R) feet of mice. Three doses of QPSC not only restored the abnormal span affected by SCA, but also maintained the footprint overlap close to 100%.

[0020] Figure 8 QPSCs were demonstrated to be able to localize intracranially via IV infusion. QPSCs were transplanted into wild-type mice via tail vein injection, and brain tissue was removed 7 days after transplantation for quantitative real-time 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 1) to 2.8% (mouse 4).

[0021] Fig. 9 A and 9B show that QPSCs can differentiate into Purkinje-type neurons in the cerebellum of SCA mice. Fig. 9 A: One month after three systemic (IV) administrations of QPSCs, some transplanted cells differentiated into Purkinje-like neurons with long axon structures (arrows) in the cerebellum of SCA mice. Fig. 9 B: No neuronal differentiation from QPSCs was found in the cerebellum of SCA mice injected intracranially (IC) three times.

[0022] Fig.10 A and 10B show that QPSC has strong immunomodulatory and anti-ROS capabilities. Fig.10 A: Human T cell proliferation was stimulated by CD3 / 28, and this stimulated proliferation was inhibited by co-culture with Stemchymal at all mixture ratios (*** indicates significant difference [P<0.05], n=3). Fig.10 B: The H2O2 resistance of QPSCs is 3-fold greater than that of human corneal epithelial cells (HCE-T; cells with relatively high resistance to oxidative stress).

[0023] Fig.11 A and 11B show that QPSCs inhibit oxidative stress-related motor function deterioration in SCA mice. Fig.11 A: SCA mice with low oxidative stress (low ROS levels) displayed superior rotarod performance than SCA mice with high oxidative stress (high ROS levels). Fig.11B: Motor performance deterioration progresses in SCA mice (Tg-Ctrl), while systemic QPSC transplantation maintains better rotarod performance in SCA mice with higher and lower oxidative loads (Tg-QPSC-High ROS, Tg-QPSC-Low ROS) compared to Tg-Ctrl, especially in the Tg-QPSC-Low ROS group (*P<0.05). Wild-type mice were used as normal controls (WT-Ctrl) for comparison.

[0024] Fig.12 A to 12C show that QPSC express multiple paracrine neurotrophic factors and tissue growth factors. Gene expression of neurotrophic factors including NT-3, NT-4, NGF, CNTF, BDNF and GDNF in QPSC was detected by quantitative PCR (qPCR) relative to the internal control 18srRNA gene (A). Tissue factors such as EGF, FGF-β and VEGF (B) and PDGF and TGF-β1 (C) in QPSC were also examined by ELISA, and the different concentrations of those factors between the intracellular and secreted parts were shown.

[0025] Fig.13 We show that QPSC paracrine protects astrocyte neurons from MPP-induced neuronal loss. Human astrocyte cell line SVG p12 was treated with 1.25 mM 1-methyl-4-phenylpyridine (MPP+) and co-cultured with QPSCs at different ratios. After 24 hours of treatment, the number of SVG p12 cells was counted. Fig.13 It was shown that after MPP treatment, the cell number of SVGp12 was significantly reduced and this phenomenon was reversed when SVG p12 cells were co-cultured with ten-fold amount of QPSCs.

[0026] Fig.14 A to 14C show that QPSC rescues Purkinje neuron loss in the cerebellum of SCA3 mice. After sacrifice, mouse cerebellum was collected. The collected tissues were fixed and embedded in paraffin for further histopathological analysis. Tissue sections were stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining was performed for Purkinje cells (anti-calbindin, ab11426, abcam). Fig.14 A. It was observed that Purkinje cells were smaller and deformed in the cerebellum of SCA3 mice with significant motor degeneration when compared to the cerebellum of wild-type mice. Fig.14 B and Fig.14 C, The number of Purkinje cells was significantly reduced in the cerebellum of SCA3 mice, and three doses of QPSCs prevented the loss of Purkinje neurons caused by SCA. DETAILED DESCRIPTION

[0027] The present invention provides methods and articles for use in stem cell therapy for treating diseases or conditions of polyQ diseases. PolyQ diseases are a group of neurodegenerative disorders caused by expansion of cytosine-adenine-guanine (CAG) repeat sequences encoding long polyQ chains in individual proteins. PolyQ diseases are characterized by pathological expansion of CAG trinucleotide repeats in the translational regions of unrelated genes. Translated polyQ accumulates in degenerating neurons, leading to dysfunction and degeneration of specific neuronal subpopulations. The present invention unexpectedly discovered a therapeutic regimen utilizing stem cells that provides an effective therapy for restoring function of degenerated and / or damaged neurons in polyQ diseases.

[0028] Unless otherwise noted, technical terms are used according to common usage.

[0029] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise.

[0030] As used herein, the terms "and" and "or" may be used to refer to either conjunctive or disjunctive properties. That is, the two terms should be understood as equivalent to "and / or" unless otherwise specified.

[0031] As used herein, the term "treatment", "treat" or "treating" refers to the complete or partial improvement or alleviation of a disease or condition or disorder, or a symptom, adverse effect or consequence, or a phenotype associated therewith. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, and improving the disease state.

[0032] As used herein, the term "delaying disease progression" means delaying, hindering, slowing, stabilizing, inhibiting and / or delaying the development of a disease. Depending on the disease being treated and / or the individual's medical history, this delay may have different lengths of time.

[0033] As used herein, in the context of administration, the term "effective amount" of an agent (eg, a pharmaceutical formulation, cell, or composition) refers to an amount effective to achieve the desired result at the required dosage / amount and for the required period of time.

[0034] As used herein, the term "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation or cells) refers to an amount effective to achieve the desired therapeutic result (such as for treating a disease, condition or disorder, and / or a pharmacokinetic or pharmacodynamic effect of a treatment) at the required dosage and for the required period of time.

[0035] As used herein, "first dose" is used to describe the timing of a given dose prior to administration of a consecutive or subsequent dose. The term does not necessarily imply that the individual has never received a dose of cell therapy before or even that the individual has never received a dose of the same cells before.

[0036] As used herein, the term "subsequent dose" refers to a dose administered to the same individual following a previous dose (eg, a first dose) during which no intervening doses were administered to the individual.

[0037] As used herein, the term "subject" is a mammal, such as a human or other animal, and typically a human. In some embodiments, the subject has been treated with a therapeutic agent targeting a disease or condition prior to administration.

[0038] As used herein, the term "pharmaceutical formulation" refers to a preparation which is in a form which permits the biological activity of the active ingredients contained therein to be effective, and which contains no additional components which are unacceptably toxic to the subject to which the formulation is administered.

[0039] As used herein, the term "pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0040] In one aspect, the present invention provides a method for treating a polyglutamine (polyQ) disease in a subject, the method comprising parenterally or topically administering to the subject an effective amount of stem cells as a unit dose, wherein the administration is performed in one or more treatment cycles, wherein one treatment cycle comprises three unit doses administered at dosing intervals of 2 to 6 weeks, respectively.

[0041] In some embodiments, polyQ diseases include, but are not limited to, spinocerebellar ataxia (SCA); Machado-Joseph disease (MJD / SCA3); Huntington's disease (HD); dentatorubral atrophy with Lewy bodies (DRPLA); and X-linked spinal bulbar muscular atrophy type 1 (SMAX1 / SBMA).

[0042] In some embodiments, the SCA is a polyglutamine (polyQ)-mediated SCA, and the SCA is preferably SCA1, SCA2, SCA3, SCA6, SCA7 or SCA17. More preferably, the SCA is SCA3.

[0043] In some embodiments, mesenchymal stem cells are mesenchymal stem cell groups (MSCs), adipose tissue-derived stem cells (ADMSCs), orbital adipose-derived stem cells (OFSCs), or quadripotential positive stromal cells (QPSCs). In one embodiment, QPSCs are QPSCs described in U.S. Application No. 14 / 615,737, which have at least 70% cell homology and express cell markers CD273, CD46, CD55, and CXCR4, but do not express CD45; wherein CD273 is strongly expressed with an intensity of more than 70%. In one embodiment, ADSCs are OFSCs described in U.S. 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, CD117. Stem cells are preferably QPSC groups.

[0044] In some embodiments, the cells can be administered parenterally or locally (such as intracerebral or intracranial administration). Parenteral infusions include intramuscular, intravenous, intraarterial, or subcutaneous administration. Parenteral administration is preferably intravenous injection.

[0045] In some embodiments, the unit dose is 0.5×10 5 Up to 5×10 10 In some embodiments, the unit dose is in the range of 0.5×10 5 Up to 5×10 9 , 0.5×10 5 Up to 5×10 8 , 0.5×10 5 Up to 5×10 7 , 0.5×10 5 Up to 5×10 6 , 1.0×10 5 Up to 5×10 10 , 1.0×10 5 Up to 5×10 9 , 1.0×10 5 Up to 5×10 8 , 1.0×10 5 Up to 5×10 7 or 1.0×10 5 Up to 5×10 6 cells / kg body weight.

[0046] In one embodiment, administration is carried out in one or more treatment cycles, wherein one treatment cycle comprises three unit doses given at a dosing interval of 2 to 6 weeks (ie, two weeks, three weeks, four weeks, five weeks or six weeks. In another embodiment, the interval is two weeks). The number of treatment cycles of the present invention is determined according to the scale for the assessment and rating of ataxia (SARA) (Subramony SH., SARA--a new clinical scale for the assessment and rating of ataxia. Nat Clin Pract Neurol. 2007; 3(3):136-7; Kim BR, Lim JH, Lee S, Park S, Koh SE, Lee IS, JungH, Lee J. Usefulness of the Scale for the Assessment and Rating of Ataxia (SARA) in Ataxic Stroke Patients. Ann Rehabil Med. 2011; 35: 772-780; and Tan S, Niu HX, Zhao L et al., Reliability and validity of the Chinese version of the Scale for Assessment and Rating of Ataxia. Chin Med J. 2013; 126(11):2045-8). SARA is a clinical scale based on the semiquantitative assessment of the level of cerebellar ataxia (spinocerebellar, Friedreich and sporadic ataxia) impairment. SARA is an expression scale based on 8 items, with a total score of 0 (no ataxia) to 40 (most severe ataxia). These scores are based on patient performance in gait, stride, sitting posture, speech disorders, finger chase, nose-finger test, rapid alternating hand movements and heel-shin sliding. After the first treatment cycle, if the individual maintains a total SARA score above 5 points for one month, the second and subsequent treatment cycles will be performed.

[0047] 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 in two weeks, three weeks, four weeks, five weeks, or six weeks. In one embodiment, the dosing interval is once every two weeks. In one embodiment, biweekly dosing means that the unit dose of stem cells is administered once every two weeks, that is, administered once during a 14-day period, preferably on the same day every two weeks. In a biweekly dosing regimen, a unit dose is administered generally about every 14 days.

[0048] In the context of stem cell therapy, administration of a unit dose comprises administration of a given amount or number of cells in a single composition and / or in a single, uninterrupted administration (eg, a single injection or continuous infusion).

[0049] In some embodiments, the cells are administered as part of a combination therapy, such as simultaneously or sequentially in any order with another therapeutic intervention. In some embodiments, stem cells are co-administered simultaneously or sequentially in any order with one or more additional therapeutic agents or in combination with another therapeutic intervention. In some cases, these additional therapeutic agents or another therapy are co-administered with the cells in close enough time so that these additional therapeutic agents or another therapy enhance the effect of the cell population, or vice versa. In some embodiments, stem cells are administered before the one or more additional therapeutic agents. In some embodiments, stem cells are administered after the one or more additional therapeutic agents.

[0050] The stem cells used in the methods of the present invention are formulated in the form of a pharmaceutical composition or formulation, such as a unit dosage composition comprising a number of cells for administration of a given dose or a portion thereof. Pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0051] The choice of carrier is determined in part by the particular stem cells and / or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative.

[0052] Pharmaceutically acceptable carriers are substantially nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride; benzethonium chloride; chloride); phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben 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 dextrins; 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 nonionic surfactants such as polyethylene glycol (PEG).

[0053] In some aspects, a buffer is included in the composition. 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 known.

[0054] The formulation may include an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for a particular indication, disease or condition to be treated with stem cells.

[0055] In some embodiments, the pharmaceutical composition comprises stem cells in an amount effective to treat a disease or condition, such as a therapeutically effective amount. In some embodiments, the efficacy of the treatment is monitored by regularly assessing the treated individual. The desired dose can be delivered by administering the cells in a single bolus injection, by administering the cells in multiple bolus injections, or by administering the cells in a continuous infusion.

[0056] The stem cells and compositions can be administered using standard administration techniques, formulations and / or devices. Administration of stem cells can be autologous or allogeneic.

[0057] Sterile injectable solutions can be prepared by incorporating the cells into a solvent, such as mixing the cells with a suitable carrier, diluent or excipient (such as sterile water, saline, glucose, dextrose or the like). Depending on the route of administration and the desired formulation, the composition may contain auxiliary substances such as wetting agents, dispersants or emulsifiers (e.g., methylcellulose), pH buffers, gelling or viscosity enhancing additives, preservatives, flavoring agents and / or coloring agents. In some aspects, reference may be made to standard texts to prepare suitable formulations.

[0058] Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol and sorbic acid. Prolonged absorption of the injectable pharmaceutical form may be achieved by the use of absorption delaying agents, such as aluminum monostearate and gelatin.

[0059] Examples

[0060] I. Animal Model Experiments

[0061] Materials and Methods

[0062] Animals and experimental design

[0063] Mice without significant motor deterioration

[0064] The MJD84.2 (B6; CBA-Tg (ATXN3*) 84.2Cce / IbezJ) mouse has been established as a disease model for human Machado-Joseph disease, also known as spinocerebellar ataxia type 3 (MJD / SCA3). In this study, MJD84.2 animals ranging in age from 20 to 34 weeks were studied. The animals were subjected to behavioral assays including the modified SHIRPA, footprint assay, and rotarod test. Three injections of the test article were performed at two-week intervals in mice at 21, 23, and 25 weeks of age. The study design is outlined below.

[0065]

[0066] Mice with significant motor deterioration

[0067] A total of thirteen SCA3 Tg / 0 mice (B6; CBA-Tg (ATXN3*)84.2Cce / IbezJ) and eight C57BL / 60 / 0 wild-type mice were originally obtained from JAX Laboratories. Animals were randomly assigned to four experimental groups: (1) SCA3+ cells; (2) SCA3+PBS; (3) Wt+ cells; (4) Wt+PBS. Three injections of test substances were performed at two-week intervals after SCA3 Tg / 0 mice developed an obvious disease phenotype. The study design is summarized as follows.

[0068]

[0069] Mesenchymal stem cells

[0070] The QPSC in this study is human ADMSC (Stemchymal®), a cell product manufactured by Steminent Biotherapeutics Inc. (SBI). In a cell factory established according to the PIC / S Good Manufactory Practice guideline, the SBI standard operating procedures are followed to culture ADMSC in vitro for expansion and quality control. In brief, adipose tissue is collected from healthy donors and immediately transported to the SBI processing facility at low temperature (0-5°C). ADMSCs are separated, purified and maintained in SBI's unique culture medium during culture expansion. The 12th generation ADMSCs are QPSCs that express CD273, CD46, CD55 and CXCR4 at high levels, which are then packaged in cryopreservation bags, and the product (Stemchymal®) is sent for quality inspection and cryopreservation. The quality control of Stemchymal® consists of in-process controls and product release tests, which include (but are not limited to) viability, sterility, mycoplasma testing, endotoxin assessment, MSC phenotype (positive for CD 73, CD 90 and CD 105, negative for CD 34, CD 45, CD11b, CD 19 and HLA-DR) and tri-lineage differentiation capacity (osteoblastic differentiation, chondrogenic differentiation and adipogenic differentiation).

[0071] Cell administration

[0072] For studies using mice without significant motor deterioration:

[0073] 2.5×10 7 Stemchymal cells / kg body weight ® Thaw, prepare and load into 1 ml insulin syringe (29 1 / 2G). Inject cells slowly (15 to 20 seconds duration) within one hour of thawing.

[0074] For studies using mice with significant motor deterioration:

[0075] Mice were randomly divided into four groups: (1) SCA3+ cells; (2) SCA3+PBS; (3) Wt+ cells; (4) Wt+PBS. 7 Stemchymal cells / kg body weight ®Intravenously infused into each mouse in Group 1 and Group 3. A total of 125 μl of the cell suspension (1:1 cryogenic solution (Biolife)) or PBS (Gibco) was administered to each mouse.

[0076] For both studies, animals were monitored for 4 hours after injection and observed daily. Cells were administered once every two weeks for a total of three times.

[0077] Data collection and analysis

[0078] Mice were sacrificed one month after the last test article injection. The weight of mice and latency to fall were recorded throughout the study. After the test article injection, mouse footprints were also analyzed for gait performance. Mouse tissues (cortex, cerebellum, heart, kidney, liver, spleen, lung and tail) were collected for future histopathological analysis and biodistribution studies.

[0079] statistics

[0080] Data are presented as mean ± SEM. Results of the rotarod and paw print tests were analyzed using Student's t-test with a significance threshold of p < 0.05.

[0081] Movement coordination and balance analysis

[0082] Motor coordination and balance were evaluated in a rotating rod apparatus (MK-670, Muromachi Kikai Co., Ltd., Japan). Mice were placed on a rotating rod at a constant speed (4 rpm) that was accelerated to 40 rpm over a 5 min period. The waiting time to fall or complete a full passive rotation (complete rotation attached to the rod) was recorded. Mice were subjected to each test 3 times with a 15 min rest between each trial. The average waiting time for each mouse in each test was calculated. The test results were statistically analyzed using a t-test.

[0083] SHIRPA Test

[0084] The modified SHIRPA test was performed on mice at 20, 24 and 28 weeks of age. The SHIRPA protocol was modified from the modified SHIRA protocol of RIKEN BRC. The test items and scoring criteria are listed in the table below.

[0085]

[0086] Mice were scored based on their behavior. The total number of mice in the study group was counted as 100%. Results are presented as a percentage of the number of mice in certain scoring levels.

[0087] Footprint analysis

[0088] The mouse footprints were analyzed approximately one month after the last administration of cells. Referring to the paper published in 2015 (1), the mouse paws were dipped in ink (front feet: red; rear feet: green) so that the mice left footprints when walking along the corridor or running to the target box. The mice were placed on a piece of paper (50 cm long, 10 cm wide) in front of the tunnel. Stride, swing, span length, and overlap of front and rear paws were measured to indicate gait (see the figure below). All mice were measured three times before being killed.

[0089] MTT assay

[0090] CD3 + T cell isolation

[0091] Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood from healthy donors by Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation. Subsequently, CD3 was purified from PBMCs by positive selection using magnetic particles coupled to anti-human CD3 antibodies (BD Biosciences) following the manufacturer's instructions. + T lymphocytes.

[0092] T cell proliferation assay

[0093] Purified human CD3 T cells were stimulated in 96-well plates with plate-bound anti-CD3 monoclonal antibodies (2 μg / ml) and anti-CD28 monoclonal antibodies (2 μg / ml) (BD Biosciences). + T cells (1×10 5 Cells) were co-cultured with different numbers of ADMSCs 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 hours, 5-bromo-2-deoxyuridine (BrdU) was added to each well and the plate was incubated for another 18 hours to measure T cell proliferation. The amount of BrdU incorporated into T cells was measured using a Cell Proliferation ELISA BrdU kit (Roche) according to the manufacturer's instructions.

[0094] Immunohistochemistry (IHC)

[0095] To evaluate the neuroprotective effect of QPSCs, QPSCs were injected into the cerebellum via the tail vein of C57BL / 6J SCA2 transgenic mice (IV hMSC-Tg group) or via the foramen magnum (IC hMSC-Tg group). A specific antibody reactive with human β2 microglobulin (Abcam, code: ab15976) was selected to visualize human cells in murine brain tissue by IHC. Murine sections (4 μm) were cut and mounted on microscope slides. The sections were rehydrated by rinsing twice in xylene, 100% ethanol, 95% ethanol, and 80% ethanol at 5 min intervals. After paraffin removal, sections were treated with 3% H2O2 to inactivate peroxidase, heated in 10 mM citrate buffer (containing 0.05% Tween20) for antigen retrieval, and blocked with 1% blocking solution (1% BSA and 0.1% Triton X-100 (in PBS), Chang et al., Journal of Biomedical Science 2011, 18:54 http: / / www.jbiomedsci.com / content / 18 / 1 / 54, page 3 of 9). Sections were incubated with specific anti-human b2 microglobulin polyclonal antibodies (Abcam) diluted (1:400) in blocking solution at room temperature for 40 min. After extensive washing three times with PBS, sections were incubated with secondary antibodies diluted (1:1000) in blocking solution at room temperature for 40 min. The primary antibody was detected using the EnVision detection system (DAKO) and visualized with diaminobenzidine (DAB; DAKO). Counter staining was performed with aqueous hematoxylin (SigmaAldrich).For direct comparisons, all slides were processed in batches to minimize variability.

[0096] Safety Testing

[0097] animal

[0098] C57BL / 6 mice received 3 doses of QPSC injected intravenously via the tail vein using an insulin syringe 1 / 2cc 30G x 3 / 8'' needle (Terumo, or BD Bioscience). Prior to injection, animals were warmed for 15 to 20 minutes by a heating pad placed under the cage to dilate their tail vein. Prior to necropsy, all animals were anesthetized with urethane (2 g / kg body weight, Sigma-Aldrich), followed by blood collection from the submandibular vein or via cardiac puncture.

[0099] Blood sample collection

[0100] For hematology analysis, whole blood samples were collected in blood collection tubes containing EDTA (BD Bioscience, catalog number 365974). For blood chemistry analysis, whole blood samples were collected in blood collection tubes containing plasma separators (BD Bioscience, catalog number 365967). After standing the plasma tubes at room temperature for 20 min, plasma was then separated by centrifugation at 6000 rpm for 5 min at 4°C.

[0101] Gross necropsy and tissue collection

[0102] After blood sampling, the animal organs were collected and divided into two parts: (1) half of the organs were stored in a -80°C freezer and then transferred and stored in a liquid nitrogen container for biodistribution analysis; (2) the other half was fixed (4% paraformaldehyde, Sigma-Aldrich) and paraffin-embedded for histopathological analysis.

[0103] Quantitative PCR

[0104] Following the manufacturer's instructions, total RNA was extracted from QPSC or murine tissue using a Total RNA Miniprep Purification Kit (GMbiolab catalog number TR01). Subsequently, cDNA synthesis was performed using a two-step MMLV RT-PCR kit (GMbiolab catalog number RP012-M). Quantitative PCR was performed using The Fast SYBR® Green Master Mix (Thermo catalog number 4385612) to analyze the relative expression of selected genes.

[0105] ELISA

[0106] To measure the intracellular and secreted levels of EGF, FGF-b, VEGF, PDGF and TGF-b1 in QPSCs, samples of cell lysates and modified culture medium were prepared as follows: QPSCs were lysed by using a freeze-thaw method, and the supernatant of the cell lysate was collected after ultracentrifugation. And for modified culture medium collection, the culture medium was collected after 3 days of QPSC culture. Finally, the concentrations of the above growth factors were determined by ELISA (R&D system) according to the manufacturer's instructions.

[0107] Neuronal cell co-culture assay

[0108] Human astrocyte cell line SVG p12 was treated with 1250 μM 1-methyl-4-phenylpyridine (MPP+) and co-cultured with different ratios of QPSC (SVG p12: QPSC=1:0.1-1:10). After 24 hours, the number of SVG p12 cells was counted.

[0109] Example 1 QPSC changes the phenotype of SCA3

[0110] QPSCs alter the phenotype of SCA3 mice. Figure 1 As shown, SCA3 mice exhibited a slightly wider base compared to wild-type mice and after QPSC treatment, the appearance of SCA3 mice looked similar to wild-type mice. In various functional tests, such as the modified SHIRPA ( Figure 4 , Figure 5 ),footprint( Figure 6 , Figure 7 ) and analysis of the performance of the rotating rod ( Figure 5 C), similar improvements were observed.

[0111] Example 2: Treatment of the present invention stops weight loss without side effects on organ tissues

[0112] QPSC also stopped weight loss in SCA3 during disease progression ( Figure 3 ). Nevertheless, three doses of QPSC did not affect the complete blood count (Table 1) or blood biochemistry (Table 2) profiles of SCA3 subjects. Tables 1 and 2 show that the complete blood count / biochemistry profiles of 25-30 week old wild-type mice were not different between three doses of QPSC and saline (three doses were administered at one week intervals). Histopathological analysis showed normal findings in various important organ tissues after three doses of QPSC injection ( Figure 2 ).

[0113] Table 1

[0114]

[0115] Data are presented as mean ± SD

[0116] Table 2

[0117]

[0118] Data are presented as mean ± SD

[0119] Example 3 Immunomodulatory and anti-ROS capabilities and expression of multiple neurotrophic factors and growth factors in mice treated with the present invention

[0120] In vitro studies have shown that QPSCs not only have immunomodulatory and anti-ROS capabilities ( Fig.10 ) and also has the ability to express multiple neurotrophic factors and growth factors ( Fig.12 ). In vivo studies have shown that after QPSC treatment, SCA mice have improved rotarod performance under oxidative stress ( Fig.11In addition, QPSCs can also be used in vitro ( Fig.13 ) and in vivo ( Fig.14 ) prevent neuronal loss. Although questions have been raised regarding the potential for cell migration across the blood-brain barrier (BBB), the ability of QPSCs to localize intracranially via intravenous infusion has been demonstrated ( Figure 8 and Fig. 9 ).

[0121] Therefore, it is reasonable to conclude that QPSCs can reach the cerebellum through the BBB and protect the neuronal cells of SCA individuals from damage by ROS and immune overreaction via intravenous infusion. QPSCs also secrete multiple neurotrophic and growth factors to maintain the number of neuronal cells, thereby delaying the progression of poly-Q diseases such as polyglutamine spinocerebellar ataxia, Machado-Joseph disease, Huntington's disease, DRPLA and SMAX1 / SBMA.

[0122] II. Human Clinical Trials

[0123] The human clinical trial is designed to study the therapeutic efficacy and safety of Stemchymal® infusion for the treatment of polyglutamine-mediated diseases (such as polyglutamine spinocerebellar ataxia, Machado-Joseph disease, Huntington's disease, DRPLA and SMAX1 / SBMA) through a randomized, double-blind, placebo-controlled study design. Eligible individuals will receive Stemchymal® via intravenous infusion.

[0124] In one example for polyglutamine spinocerebellar ataxia, the individual undergoing the trial has genotype-confirmed spinocerebellar ataxia type 2 or spinocerebellar ataxia type 3. The individual's baseline SARA score is in the range of 5 to 15 points.

[0125] 2.5×10 7 Stemchymal cells / kg body weight ® Thaw, prepare and load into syringe. Inject cells slowly within one hour of thawing. ® The cells are administered to each subject via intravenous infusion three times at intervals of once every two weeks. After one or more treatment cycles, the subject's SARA score decreases and the SCA2 or SCA3 condition improves.

[0126] 1. A method for treating a polyglutamine (polyQ) disease in a subject, the method comprising parenterally or topically administering to the subject an effective amount of stem cells as a unit dose, wherein the administration is performed in one or more treatment cycles, wherein a treatment cycle comprises three unit doses of a multiple of a baseline BW given at dosing intervals of 2 to 6 weeks, respectively.

[0127] 2. A method as described in claim 1, wherein the polyQ disease is polyQ-mediated spinocerebellar ataxia (SCA), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentate-rubral-pallidular Lewy body atrophy (DRPLA) or type 1 X-linked spinal bulbar muscular atrophy (SMAX1 / SBMA).

[0128] 3. The method of claim 1, wherein the SCA is SCA1, SCA2, SCA3, SCA6, SCA7 or SCA17.

[0129] 4. The method of claim 1, wherein the SCA is SCA2, SCA3 or SCA6.

[0130] 5. The method of claim 1, wherein the SCA is SCA3.

[0131] 6. The method of item 1, wherein the mesenchymal stem cells are a mesenchymal stem cell (MSC) population, an adipose tissue-derived stem cell (ADMSC) population, an orbital adipose-derived stem cell (OFSC) population, or a quadri-positive stromal cell (QPSC) population.

[0132] 7. A method as described in claim 1, wherein the QPSC population has at least 70% cell homology and expresses cell markers CD273, CD46, CD55 and CXCR4 but does not express CD45; wherein CD273 is more strongly expressed with an intensity of more than 70%.

[0133] 8. A method as described in item 1, wherein these ADSCs are an OFSC population that expresses at least CD90, CD105, CD29, CD44, CD49b, CD49e, CD58 and HLA-ABC but does not express CD133, CD31, CD106, CD146, CD45, CD14, and CD117.

[0134] 9. The method of claim 1, wherein the parenteral administration is intramuscular, intravenous, intraarterial or subcutaneous.

[0135] 10. The method of claim 1, wherein the parenteral administration is intravenous administration.

[0136] 11. The method of claim 1, wherein the local administration is intracerebral or intracranial administration.

[0137] 12. The method of claim 1, wherein the local administration is intracranial administration.

[0138] 13. A method as in claim 1, wherein the unit dose is 0.5×10 5 Up to 5×10 10 cells / kg body weight.

[0139] 14. The method of claim 1, wherein after the first treatment cycle, if the individual maintains a total SARA score above 5 for one month, the subsequent treatment cycle will be performed.

[0140] 15. The method of claim 1, wherein the dosing interval is once every two weeks.

[0141] 16. The method of claim 1, wherein the stem cells may be co-administered with one or more additional therapeutic agents or in combination with another therapeutic intervention simultaneously or sequentially in any order.

Claims

1. Use of stem cells in the preparation of a medicament for treating a polyglutamine (polyQ) disease in an individual, wherein the stem cells are adipose tissue-derived stem cells (ADMSCs) that express CD273, CD46, CD55 and CXCR4 but do not express CD45.

2. The method of claim 1, wherein the polyglutamine disease is polyglutamine-mediated spinocerebellar ataxia (SCA), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral atrophy with Lewy bodies (DRPLA), or type 1 X-linked spinal bulbar muscular atrophy (SMAX1 / SBMA).

3. The use according to claim 2, wherein the SCA is SCA1, SCA2, SCA3, SCA6, SCA7 or SCA17.

4. The use according to claim 2, wherein the SCA is SCA2, SCA3 or SCA6.

5. The use according to claim 2, wherein the SCA is SCA3. The use according to claim 1 , wherein CD273 is expressed at an intensity exceeding 70%.

7. The use of claim 1, wherein the medicament comprises an effective amount of the stem cells administered parenterally or topically to the individual.

8. The use according to claim 7, wherein the parenteral administration is intramuscular, intravenous, intraarterial or subcutaneous administration.

9. The use according to claim 7, wherein the parenteral administration is intravenous administration.

10. The use according to claim 7, wherein the local administration is intracerebral or intracranial administration.

11. The use according to claim 7, wherein the local administration is intracranial administration.

12. The use according to claim 1, wherein the stem cells are used together with one or more additional therapeutic agents or another therapeutic intervention in the preparation of a medicament.

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