阅读说明：本技术 用于多麸酰氨酸(polyq)疾病的治疗 (Treatment of polyglutamine acid (POLYQ) diseases ) 是由 何慧君 张志刚 赖秀玉 王玮琦 于 2017-05-26 设计创作，主要内容包括：本发明提供用于干细胞疗法中,用于治疗SCA疾病或病症的方法及制品。特定言之,本发明提供一种用于治疗SCA的方法,所述方法包含向个体非经肠或局部地投与有效量的干细胞作为单位剂量,其中所述投与是以一或多个治疗周期进行,其中一个治疗周期包含分别以2至6周的给药间隔给与三个单位剂量。(The present invention provides methods and articles of manufacture for use in stem cell therapy for treating SCA diseases or disorders. In particular, the present invention provides a method for treating SCA, the method comprising parenterally or topically administering to a subject an effective amount of stem cells as a unit dose, wherein the administration is in one or more treatment cycles, wherein one treatment cycle comprises three unit doses administered at dosing intervals of 2 to 6 weeks each.)

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 in one or more treatment cycles, wherein one treatment cycle comprises administration of a multiple of the baseline BW of three unit doses at dosing intervals of 2 to 6 weeks, respectively.

2. The method as claimed in claim 1 wherein the polyQ disease is polyQ-mediated spinocerebellar ataxia (SCA), Machado-Joseph disease (MJD/SCA3), Huntington's Disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), or X-linked spinobulbar muscular atrophy type 1 (SMAX 1/SBMA).

3. The method as claimed in claim 1 wherein the SCA is SCA1, SCA2, SCA3, SCA6, SCA7 or SCA 17.

4. The method as claimed in claim 1 wherein the SCA is SCA2, SCA3 or SCA 6.

5. The method as claimed in claim 1 wherein said SCA is SCA 3.

6. The method of claim 1, wherein the mesenchymal stem cells are a population of Mesenchymal Stem Cells (MSCs), a population of adipose tissue-derived stem cells (ADMSCs), a population of orbital adipose-derived stem cells (OFSCs), or a population of Quadruperious Positive Stromal Cells (QPSCs).

7. The method as claimed in claim 1 wherein the QPSC population has a cellular homology of at least 70% and expresses cell markers CD273, CD46, CD55 and CXCR4 but does not express CD 45; wherein CD273 is strongly expressed with an intensity of more than 70%.

8. The method of claim 1, wherein the ADSCs are a population of OFSCs that express at least CD90, CD105, CD29, CD44, CD49b, CD49e, CD58, and HLA-ABC, but do not express CD133, CD31, CD106, CD146, CD45, CD14, CD 117.

9. The method as claimed in claim 1 wherein the parenteral administration is intramuscular, intravenous, intraarterial, or subcutaneous administration.

10. The method as claimed in claim 1 wherein the parenteral administration is intravenous administration.

11. The method as claimed in claim 1 wherein the local administration is intracerebral or intracranial administration.

12. The method as claimed in claim 1 wherein said local administration is intracranial administration.

13. The method as claimed in claim 1 wherein the unit dose is in the range of 0.5 x 10⁵To 5X 10¹⁰Individual cells/kg body weight range.

14. The method of claim 1 wherein after the first treatment period, wherein the subsequent treatment period will be performed if the individual maintains a total SARA score of greater than 5 minutes and one month.

15. The method as claimed in claim 1 wherein the dosing interval is once every two weeks.

16. The method of claim 1, such stem cells can be co-administered simultaneously or sequentially in any order with one or more additional therapeutic agents or in conjunction with another therapeutic intervention.

Technical Field

The present invention relates to the field of treatment of neurodegenerative disorders. In particular, the present invention relates to a therapeutic regimen for treating polyglutamine (polyQ) diseases using stem cells.

Background

Ataxia is a group of clinical and genetically heterogeneous neurodegenerative diseases that variably affect the cerebellar, brainstem and spinocerebellar diameters. Spinocerebellar ataxia (SCA) is a progressive, degenerative and fatal disease. SCA involves degeneration of neuronal tissue, where the major site of pathological change is present in the nuclei or neural pathways of the cerebellum, brainstem or spinal cord. The fatal condition of SCA results not only from massive neuronal loss, but also from bedridden and respiratory failure at advanced disease. The most common class of SCA subtypes are the polyglutamic acid (polyQ) -mediated SCA, i.e., SCA1, SCA2, SCA3, SCA6, SCA7, and SCA 17.

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

However, there is no effective medical treatment or potential cure for SCA. Thus, there is a need for therapeutic methods for alleviating the symptoms and symptoms of SCA.

Disclosure of Invention

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 in one or more treatment cycles, wherein one treatment cycle comprises three unit doses administered at dosing intervals of 2 to 6 weeks each.

In some embodiments, polyQ diseases include, but are not limited to, spinocerebellar ataxia (SCA); Machado-Joseph disease (Machado-Joseph disease, MJD/SCA 3); huntington's Disease (HD); dentate globus pallidus Louis atrophy (DRPLA); and type 1X-linked spinal bulbar muscular atrophy (SMAX 1/SBMA). In one embodiment, the SCA is SCA1, SCA2, SCA3, SCA6, SCA7, or SCA 17.

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

In some embodiments, the cells can be administered by parenteral administration or local therapeutic administration (such as intracerebral or intracranial administration).

In some embodiments, the unit dose is 0.5 × 10⁵To 5X 10¹⁰Individual cells/kg body weight range.

In one embodiment, administration is performed in one or more treatment cycles, wherein a treatment cycle comprises three unit doses administered at dosing intervals of 2 to 6 weeks (i.e., two, three, four, five or six weeks, in another embodiment, two weeks apart).

Brief description of the drawings

Fig. 1A-D show the appearance and behavior of SCA3 mice.

Figures 2A to F show the immunological tolerance of QPSC in xenograft models. Histopathological findings of safety tests performed after mice received 3 doses of QPSC are shown. No significant lesions of brain (fig. 2A), heart (fig. 2B), kidney (fig. 2C), liver (fig. 2D), lung (fig. 2E), pancreas (fig. 2F) or spleen (fig. 2G) were observed in QPSC-treated mice by H & E staining (400 ×).

Figures 3A to 3C show that QPSC stopped weight loss in SCA3 mice. Body weights were recorded weekly prior to QPSC treatment and biweekly after QPSC treatment. Animals were sacrificed one month after the third QPSC injection. Figure 3A, prior to QPSC treatment, the body weight of SCA3 mice was lighter than wild-type mice. Figures 3B and 3C, QPSC prevented SCA3 mice weight loss.

Figures 4A-4C show that QPSC alters the phenotype of SCA3 mice. Both WT mice and SCA3 transgenic mice (TG) received intravenous QPSC administration 3 times. Modified SHIRPA was performed before and after QPSC treatment. FIG. 4A illustrates, using pelvic pull-up, that QPSC alters the phenotype of SCA3 mice. FIGS. 4B and 4C, QPSC altered the phenotype of SCA3 mice in terms of grip strength.

Figures 5A-5C show that three doses of QPSC increased motor function in SCA3 mice without significant motor function deterioration. Both WT mice and SCA3 transgenic mice (TG) received intravenous QPSC administration 3 times. Modified SHIRPA and rotarod performance tests were performed before and after QPSC treatment. FIGS. 5A and B: the SCA3 mice had improved motor and negative tropism performance following QPSC treatment. FIG. 5C: the rotarod performance of SCA3 mice (Tg) was significantly improved after infusion of 3 doses of QPSC.

Figures 6A-6D show that three doses of QPSC increased motor function in SCA3 mice with significant motor function deterioration. Footprint analysis for mice was performed one month after the third QPSC injection. Graphs a-D show that left forefoot (L.F.) (fig. 6A), right forefoot (r.f.) (fig. 6B), left hind foot (L.H.) (fig. 6C), and right hind foot (r.h.) (fig. 6D) of SCA mice exhibited reduced footprint spans, and 3 doses of QPSC restored abnormal spans.

Figures 7A-7D show that three doses of QPSC improved gait balance in SCA3 mice. Footprint analysis for mice was performed one month after the third QPSC injection. Fig. 7A to D: fig. 7A and 7B show footprint spans of mouse forefoot (F) and hind (H), respectively, while fig. 7C and 7D show footprint overlaps of mouse left (L) and right (R) feet. The three dose QPSC not only restores the abnormal span affected by SCA, but also maintains close to 100% overlap of footprints.

Figure 8 shows that qpsc capable of intracranial localization via IV infusion was implanted into wild type mice via tail vein injection, and brain tissue was removed 7 days after implantation for quantitative real-time RT-PCR analysis the ratio of human DNA (detected by human β 2 microglobulin) to mouse DNA (detected by mouse 18s rRNA) was about 0.8% (mouse No. 1) to 2.8% (mouse No. 4).

FIGS. 9A and 9B show QPSCs capable of differentiating into Purkinje-type neuronal cells in the cerebellum of SCA mice. FIG. 9A: one month after three systemic (IV) administrations of QPSC, some transplanted cells were transformed into purkinje-type neuronal cells with long-axis process structures in the cerebellum of SCA mice (arrows). FIG. 9B: neuronal differentiation from QPSC was not found in the cerebellum of SCA mice injected three times Intracranial (IC).

FIGS. 10A and 10B show that QPSC has strong immunomodulatory and anti-ROS ability. FIG. 10A: human T cell proliferation is stimulated by CD3/28, and this stimulated proliferation is throughInhibition by co-culture with Stemchymal at all mixture ratios (. indicates a significant difference [ P ]<0.05]And n is 3). FIG. 10B: anti-H of QPSC₂O₂H-resistance compared to human corneal epithelial cells (HCE-T; cells with relatively high resistance to oxidative stress)₂O₂The capacity is 3 times greater.

Figures 11A and 11B show that QPSC inhibits oxidative stress-related motor function degeneration in SCA mice. FIG. 11A: the SCA mice with low oxidative stress (low ROS content) show superior rotarod performance than the SCA mice with high oxidative stress (high ROS content). FIG. 11B: motor function performance deterioration progressed in SCA mice (Tg-Ctrl), while systemic QPSC transplantation maintained better rotarod performance in SCA mice with higher and lower oxidative load (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 compared as normal controls (WT-Ctrl).

FIGS. 12A-12C show that QPSC expresses 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 is 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- β 1(C) in QPSC were also examined by ELISA and indicate different concentrations of those factors between the intracellular and secreted portions.

Figure 13 shows that QPSC paracrine spares astrocytic neurons from MPP-induced neuronal loss. The human astrocyte strain SVG p12 was treated with 1.25mM 1-methyl-4-phenylpyridine (MPP +) and co-cultured with different ratios of QPSC simultaneously. After 24 hours of treatment, the number of cells of SVG p12 was counted. Figure 13 shows 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 times the amount of QPSC.

Figures 14A-14C show that QPSC rescued the loss of purkinje neurons in the cerebellum of SCA3 mice. After sacrifice, mouse cerebellum was collected. The collected tissues were fixed and embedded with paraffin for further histopathological analysis. Tissue sections were stained with Hematoxylin and Eosin (HE) and Immunohistochemical (IHC) staining was performed on purkinje cells (anti-calcium binding protein, ab11426, abcam). Figure 14a. it was observed that the sizes of purkinje cells in the cerebellum of SCA3 mice with marked motor function deterioration were small and deformed when compared to the cerebellum of wild type mice. Fig. 14B and 14C, the number of purkinje cells was significantly reduced in the cerebellum of SCA3 mice, while the QPSC at three doses prevented purkinje neuron loss due to SCA.

Detailed Description

The present invention provides methods and articles of manufacture for use in stem cell therapy for treating a disease or condition of a polyQ disease. polyQ diseases are a group of neurodegenerative disorders caused by the amplification of cytosine-adenine-guanine (CAG) repeats encoding long polyQ strands in individual proteins. PolyQ disease is characterized by the pathological amplification of CAG trinucleotide repeats in the translational regions of unrelated genes. Translated polyQ accumulates within degenerated neurons, leading to dysfunction and degeneration of specific subsets of neurons. The present invention surprisingly finds a therapeutic regimen utilizing stem cells that provides an effective therapy for restoring the function of degenerated and/or damaged neurons in polyQ disease.

Unless otherwise indicated, technical terms are used in accordance with well-known usage.

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

As used herein, the terms "and" or "may be used to refer to either connectivity or separability. That is, the two terms should be understood as being equivalent to "and/or" unless otherwise indicated.

As used herein, the terms "treatment", "treating" or "treatment" refer to a complete or partial improvement or reduction of a disease or condition or disorder, or a symptom, adverse effect or consequence, or phenotype associated therewith. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, and ameliorating the disease state.

As used herein, the term "delay of disease progression" means delaying, hindering, slowing, arresting, stabilizing, inhibiting and/or delaying the progression of the disease. This delay may be of varying length depending on the disease being treated and/or the individual's medical history.

As used herein, the term "effective amount" of an agent (e.g., a pharmaceutical formulation, cell, or composition) in the context of administration refers to an amount effective to achieve the desired result at the desired dose/amount and for the desired period of time.

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

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

As used herein, the term "subsequent dose" refers to a dose administered to the same subject after a previous dose (e.g., the first dose), during which no intervening dose is administered to the subject.

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 that targets the disease or condition prior to administration.

As used herein, the term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain additional components that have unacceptable toxicity to the individual to whom the formulation is administered.

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

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 carried out in one or more treatment cycles, wherein one treatment cycle comprises three unit doses administered at intervals of 2 to 6 weeks each.

In some embodiments, polyQ diseases include, but are not limited to, spinocerebellar ataxia (SCA); Machado-Joseph disease (MJD/SCA 3); huntington's Disease (HD); dentate globus pallidus Louis atrophy (DRPLA); and type 1X-linked spinal bulbar muscular atrophy (SMAX 1/SBMA).

In some embodiments, the SCA is a polyglutamic acid (polyQ) -mediated SCA, preferably the SCA1, SCA2, SCA3, SCA6, SCA7, or SCA 17. More preferably, the SCA is SCA 3.

In some embodiments, the mesenchymal stem cells are a mesenchymal stem cell population (MSC), an adipose tissue-derived stem cell (ADMSC) population, an orbital adipose-derived stem cell (OFSC) population, or a tetrapotent positive stromal cell (QPSC) population. In one embodiment, QPSC is a QPSC described in U.S. application No. 14/615,737, having at least 70% cellular homology and expressing the cell markers CD273, CD46, CD55 and CXCR4, but not CD 45; wherein CD273 is strongly expressed with an intensity of more than 70%. In one embodiment, ADSCs are those OFSCs described in us 20120288480 that express at least CD90, CD105, CD29, CD44, CD49b, CD49e, CD58, and HLA-ABC, but do not express CD133, CD31, CD106, CD146, CD45, CD14, CD 117. The stem cells are preferably QPSC populations.

In some embodiments, the cells can be administered by parenteral administration or local therapeutic administration (such as intracerebral or intracranial administration). Parenteral infusion includes intramuscular, intravenous, intraarterial, or subcutaneous administration. Parenteral administration is preferably intravenous injection.

In some embodiments, the unit dose is 0.5 × 10⁵To 5X 10¹⁰Individual cells/kg body weight range. In some embodiments, the unit dose is0.5×10⁵To 5X 10⁹、0.5×10⁵To 5X 10⁸、0.5×10⁵To 5X 10⁷、0.5×10⁵To 5X 10⁶、1.0×10⁵To 5X 10¹⁰、1.0×10⁵To 5X 10⁹、1.0×10⁵To 5X 10⁸、1.0×10⁵To 5X 10⁷Or 1.0X 10⁵To 5X 10⁶Individual cells/kg body weight range.

In one embodiment, administration is performed in one or more treatment cycles, wherein a treatment cycle comprises three unit doses administered at dosing intervals of 2 to 6 weeks (i.e., two, three, four, five or six weeks, in another embodiment, two weeks apart). The number of treatment cycles of the present invention IS determined according to the economic Assessment and Rating Scale (SARA-A new clinical Scale for the Assessment and Rating of Ataxia, SARA) of the present invention (Subremony SH., SARA-A new clinical Scale for the Assessment and Rating of Ataxia. Nat Clin practice. 2007; 3(3) 136-7; Kim BR, Lim JH, Lee S, Park S, Chih SE, Lee IS, Jung H, LeJ. useful of the Scale for the Assessment and Rating of Ataxia (SARA) Innove tissue services. Ann Rehabil. 2011; 35: 780; 35: 2045; and Tan S, Niu, Zo L et al, HX of the repair and Rating of the sample tissue grade, Ax.8. Ax Rehabil. 2011. 35: 2045: 2048). SARA is a clinical scale based on a semi-quantitative assessment of the level of cerebellar ataxia (spinocerebellum, Friedreich, and sporadic ataxia) injury. SARA is a scale based on the expression of 8 items, giving a total score of 0 (no ataxia) to 40 (most severe ataxia). These scores are based on patient performance of gait, stride, sitting posture, speech impairment, finger tracking (finger chase), nose-finger testing, rapid hand-alternating movements, and heel-to-shin sliding. After the first treatment cycle, if the individual maintains a total SARA score above 5 minutes for one month, a second and subsequent treatment cycles will be performed.

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 within two, three, four, five or six weeks. In one embodiment, the dosing interval is once every two weeks. In one embodiment, once every two weeks means that the unit dose of stem cells is administered once every two weeks, i.e., once during a 14 day period, preferably on the same day every two weeks. In a once-every-two-week dosing regimen, unit doses are generally administered about every 14 days.

In the case 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 a single uninterrupted administration (e.g., a single injection or continuous infusion).

In some embodiments, the cells are administered as part of a combination therapy, such as concurrently with another therapeutic intervention or sequentially in any order. In some embodiments, the stem cells are co-administered simultaneously or sequentially in any order with one or more additional therapeutic agents or in conjunction with another therapeutic intervention. In some cases, the additional therapeutic agent or the additional therapy is co-administered with the cells in sufficient proximity such that the additional therapeutic agent or the additional therapy enhances the effect of the cell population, or vice versa. In some embodiments, the stem cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the stem cells are administered after the one or more additional therapeutic agents.

The stem cells used in the methods of the invention are formulated in pharmaceutical compositions or formulations, such as unit dosage compositions comprising the number of cells to be administered in a given dose or portion thereof. Pharmaceutical compositions and formulations typically include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one additional therapeutic agent.

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

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, 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 octadecyl dimethyl benzyl ammonium chloride; hexa hydroxy quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); a low molecular weight polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamyl acid, 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 a non-ionic surfactant, such as polyethylene glycol (PEG).

In some aspects, a buffer is included in the composition. Suitable buffering agents 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 of preparing administrable pharmaceutical compositions are known.

The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for a particular indication, disease or condition being treated with stem cells.

In some embodiments, the pharmaceutical composition comprises stem cells in an amount effective to treat the disease or condition (such as a therapeutically effective amount). In some embodiments, treatment efficacy is monitored by periodically evaluating the treated individuals. 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 by continuous infusion.

The stem cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the stem cells may be autologous or heterologous.

Sterile injectable solutions can be prepared by incorporating the cells into a solvent, such as by 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 compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring and/or coloring agents. In some aspects, reference may be made to standard text to prepare suitable formulations.

Various additives may be added that enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, and sorbic acid). Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of absorption delaying agents, such as aluminum monostearate and gelatin.

Examples of the invention

I. Animal model test

Materials and methods

Animal and experimental design

Mice without significant motor function deterioration

MJD84.2 (B6; CBA-Tg (ATXN 3): 84.2Cce/IbezJ) mice have been established as a disease model for human Machado-Joseph disease, also known as spinocerebellar ataxia type 3 (MJD/SCA 3). In this study, MJD84.2 animals ranging in age from 20 to 34 weeks were studied. Behavioral analysis, including modified SHIRPA, footprint analysis, and swing-bar tests were performed on these animals. Three test injections were performed at two week intervals on 21, 23 and 25 week-old mice. The study design is summarized below.

Mice with significant motor function degeneration

A total of thirteen SCA3 Tg/0 mice (B6; CBA-Tg (ATXN 3) 84.2Cce/IbezJ) and 8C 57BL/60/0 wild-type mice were initially all from the JAX laboratory. Animals were randomly enrolled into four experimental groups: (1) SCA3+ cells; (2) SCA3+ PBS; (3) wt + cells; (4) wt + PBS. Three test injections were performed at two week intervals after the SCA3 Tg/0 mice were determined to develop a significant disease phenotype. The study design is summarized below.

Mesenchymal stem cells

QPSC in this study is human ADMSC

Namely a cell product manufactured by SteminentBiotherapeutics Inc (SBI). ADMSC were expanded and quality controlled ex vivo in a cell factory built according to the PIC/S good factory Practice guidelines (GoodManufactory Practice guidelines) following the SBI standard procedures. Briefly, adipose tissue was collected from healthy donors and immediately transported to the SBI processing facility at low temperature (0-5 ℃). The ADMSCs were isolated, purified and maintained in media unique to SBI during culture expansion. Generation 12 ADMSCs are QPSCs expressing CD273, CD46, CD55 and CXCR4 at high levels, which are then packaged in cryopreservation bags, and the product

And (5) carrying out quality verification and low-temperature preservation.Quality control of (a) consists of in-process control and product release tests including, but not limited to, viability, sterility, mycoplasma test, endotoxin assessment, MSC phenotype (positive for CD73, CD90, and CD105, negative for CD 34, CD45, CD11b, CD 19, and HLA-DR), and trilineage differentiation capacity (osteogenic, chondrogenic, and adipogenic).

Administration of cells

Study with mice without significant motor function deterioration:

mixing 2.5X 10⁷Per cell/kg body weight

Thawed, ready and loaded into a 1ml insulin syringe (291/2G). Cells were injected slowly (15 to 20 seconds duration) within one hour of thawing.

For studies using mice with significant motor function degeneration:

mice were randomized into four groups: (1) SCA3+ cells; (2) SCA3+ PBS; (3) wt + cells; (4) wt + PBS. Mixing 2.5X 10⁷Per cell/kg body weight

Intravenous infusion was performed into each mouse in groups 1 and 3. A total of 125. mu.l of cell suspension (1:1 cryo solution (Biolife)) or PBS (Gibco) was administered to each mouse.

For both studies, animals were monitored 4 hours after injection and daily observations. Cells were given every two weeks for a total of three times.

Data collection and analysis

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

Statistics of

Data are presented as mean ± SEM. The results of the swing and footprint tests were analyzed using the schraden t test (Student's t-test) and with a significance threshold of p < 0.05.

Exercise coordination and balance analysis

Coordination of movement and balance was evaluated in a swing rod device (MK-670, Muromachi Kikai co., ltd., Japan). The mouse was placed on a rotating lever at constant speed (4rpm) that was accelerated to 40rpm over a 5min period. The waiting time for the drop or for the complete passive rotation (full rotation attached to the rod) is recorded. Mice were subjected to 3 separate tests with 15min rest between each test. The average waiting time for each mouse in each test was calculated. The test results were statistically analyzed using the t-test.

SHIRPA test

The modified SHIRPA test was performed at mice ages 20, 24, and 28 weeks. The SHIRPA regimen is a modified SHIRA regimen that is modified from RIKENBRC. The test items and scoring criteria are listed in the table below.

Mice were scored according to their behavior. The total number of mice in the study group was 100%. Results are presented as a percentage of the number of mice at some scoring level.

Footprint analysis

The mouse footprints were analyzed approximately one month after the last cell administration. Referring to the paper published at 2015(1), the mouse soles of feet were dipped in ink (front leg: red; back leg: green) so that the mice left a trace of the footprint as they walked down the hallway or run to the target bin. The mice were placed on a piece of paper (50cm long, 10cm wide) in front of the tunnel. Measuring stride length, swing, span length, and forefoot and hindfoot overlap indicates gait (see below). All mice were subjected to three rounds of measurements prior to sacrifice.

MTT assay

CD3⁺T cell isolation

Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from heparinized whole blood from healthy donors by Histopaque-1077(Sigma-Aldrich) density gradient centrifugation. Subsequently, CD3 was purified from PBMC by positive selection using anti-human CD3 antibody-coupled magnetic particles (BD Biosciences) following the manufacturer's instructions⁺T lymphocytes.

T cell proliferation assay

In 96-well plates, disk-bound anti-CD 3 monoclonal antibody (2. mu.g/ml) and anti-CD 28 monoclonal antibody (2. mu.g/ml) (BD Biosciences) were used for spikingHuman CD3 purified by agitation⁺T cell (1X 10)⁵Individual cells) and co-cultured with varying amounts of ADMSC in medium containing 10% Fetal Bovine Serum (FBS), 2mM l-glutaminate, 100U/ml penicillin, 100U/ml streptomycin and 25mM HEPES RPMI-1640 (Gibco). After 48 hours, 5-bromo-2-deoxyuridine (BrdU) was added to each well and the plates were incubated for an additional 18 hours to measure T cell proliferation. The amount of BrdU incorporated into T cells was measured using a cell Proliferation elisa (cell Proliferation elisa) BrdU kit (Roche) according to the manufacturer's instructions.

Immunohistochemistry (IHC)

To evaluate the neuroprotective effect of QPSC, QPSC were injected into the cerebellar site via the tail vein of C57BL/6J SCA2 transgenic mice (IVhMSC-Tg panel) or via the occipital macropores (IC hMSC-Tg panel). specific antibodies (Abcam, code: ab15976) reactive with human β 2 microglobulin were selected, human cells in murine brain tissue were displayed by IHC.murine sections (4 μm) were cut and mounted onto microscope slides.sections were rehydrated by two washes in xylene, 100% ethanol, 95% ethanol and 80% ethanol at 5min intervals.after paraffin removal, sections were rehydrated with 3% H₂O₂Treatment to inactivate peroxidase, heating in 10mM citrate buffer (containing 0.05% Tween20) for antigen retrieval, and blocking with 1% blocking solution (1% BSA and 0.1% Triton X-100 in PBS), Chang et al, Journal of biological Science 2011,18:54http:// www.jbiomedsci.com/content/18/1/54, p 3, p 9). Sections were incubated with specific anti-human b2 microglobulin polyclonal antibody (Abcam) diluted (1:400) in blocking solution for 40min at room temperature. After washing well three times with PBS, sections were incubated with secondary antibody diluted in blocking solution (1:1000) for 40min 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 (SigmaAldrich). For direct comparison, all slides were processed in bulk to minimize variability.

Safety test

Animal(s) production

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

Blood sample collection

For hematological analysis, whole blood samples were collected in blood collection tubes containing EDTA (BD Bioscience, catalog No. 365974). For blood chemistry analysis, whole blood samples were collected in blood collection tubes (BDBioscience, catalog No. 365967) containing plasma separators. After standing the plasma tube at room temperature for 20min, the plasma was subsequently separated by centrifugation at 6000rpm for 5min at 4 ℃.

Gross necropsy and tissue collection

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

Quantitative PCR

Total RNA was extracted from QPSC or murine tissue using a Total RNA miniprep purification Kit (Total RNA miniprep purification Kit; GMbiolab catalog number TR01) following the manufacturer's instructions. Subsequently, cDNA synthesis was performed using a two-step MMLV RT-PCR kit (GMbiolab catalog number RP 012-M). Using The FastQuantitative PCR was performed on GreenMaster Mix (Thermo catalog No. 4385612) to analyze the relative expression of selected genes.

ELISA

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

Neuronal cell co-culture assay

Human astrocyte strain SVG p12 was treated with 1250 μ M1-methyl-4-phenylpyridine (MPP +) and co-cultured with varying ratios of QPSC (SVG p12: QPSC ═ 1:0.1-1: 10). After 24 hours, the number of cells of SVG p12 was counted.

Example 1 QPSC altered the phenotype of SCA3

QPSC altered the phenotype of SCA3 mice. As shown in figure 1, the SCA3 mouse exhibited a slightly wider base (base) compared to the wild-type mouse and the appearance of the SCA3 mouse after QPSC treatment appeared similar to the wild-type mouse. Similar improvement results were observed in various functional tests, such as modified SHIRPA (fig. 4, 5), footprint (fig. 6, 7), and swing arm performance analysis (fig. 5C).

Example 2 weight loss stopped by treatment of the invention without adverse effects on organ tissues

QPSC also stopped the weight loss of SCA3 during disease progression (fig. 3). Nevertheless, the QPSC of 3 doses did not affect the morphology of the complete blood count (table 1) or blood biochemistry (table 2) of SCA3 individuals. Tables 1 and 2 show that the complete blood count/biochemical profile of 25-30 week old wild-type mice did not differ between the three doses of QPSC and saline (three doses administered at one week intervals). Histopathological analysis showed normal findings of various vital organ tissues after three doses of QPSC injection (figure 2).

TABLE 1

Data are presented as mean ± SD

TABLE 2

Data are presented as mean ± SD

Example 3 immunomodulating and anti-ROS Capacity and expression of multiple neurotrophic factors and growth factors in mice treated with the invention

In vitro studies showed that QPSC has not only immunomodulatory and anti-ROS capabilities (fig. 10), but also the ability to express multiple neurotrophic factors and growth factors (fig. 12). In vivo studies showed that following QPSC treatment, SCA mice exhibited improved spinodal performance under oxidative stress (figure 11). In addition, QPSC can also prevent neuronal loss both in vitro (fig. 13) and in vivo (fig. 14). Although questions have been raised about the potential for cells to migrate across the Blood Brain Barrier (BBB), the ability of QPSC to be intracranially localized via intravenous infusion has been shown (fig. 8 and 9).

Therefore, it is reasonable to conclude that: via intravenous infusion, QPSC can reach the cerebellum through the BBB and protect neuronal cells of SCA individuals from ROS and hyper-immune reactions. QPSC also secrete multiple neurotrophic and growth factors to maintain neuronal cell numbers, delaying the progression of poly-Q diseases such as polyglutamine spinocerebellar ataxia, machado-joseph disease, huntington's disease, DRPLA and SMAX 1/SBMA.

Human clinical trials

The human clinical trial was intended to be studied by a randomized, double-blind, placebo-controlled study design

Infusion for therapeutic efficacy and safety of the treatment of polyglutamine-mediated diseases such as polyglutamine spinocerebellar ataxia, macchardol-joseph disease, huntington's disease, DRPLA and SMAX 1/SBMA. Eligible individuals will receive infusion via intravenous infusion

In one example for polyglutamine spinocerebellar ataxia, the individual undergoing the test has genotypically identified spinocerebellar ataxia type 2 or spinocerebellar ataxia type 3. The individual's baseline SARA score ranged from 5 to 15 points.

Mixing 2.5X 10⁷Per cell/kg body weight

Thawed, prepared and loaded into a syringe. Cells were injected slowly within one hour of thawing. Will be provided with

Each individual was given intravenous infusion and 3 cell administrations were performed at biweekly intervals. After one or more treatment cycles, the individual's SARA score decreases and the SCA2 or SCA3 condition improves.