Cell therapy and treatment methods for minor stroke

Abstract

Methods of treating a subject after the subject suffers from a small volume ischemic stroke and methods of treating a subject with stroke-induced movement disorders are disclosed. Compositions for treating small volume ischemic stroke are also disclosed. In one aspect, the method of treating a subject after a small volume ischemic stroke comprises administering a therapeutically effective amount of cells to the brain region surrounding the small volume ischemic core of the subject, the cells being derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding Notch intracellular domain.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 194,021, filed on 27 May 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure generally relates to the field of regenerative cell therapy, and more specifically to cell therapy and treatment methods for small volume stroke. [Background technology]

[0003] Globally, stroke is the second leading cause of death and the third leading cause of disability.[1] Post-ischemic stroke, the acute phase is generally defined as the period from several hours to several days after the stroke event or incident. Immediate post-stroke interventions for acute ischemic stroke focus on life-saving measures, including blood pressure, respiratory and cardiac control, monitoring of oxygen saturation and blood glucose levels, prevention of metabolic disorders, maintenance of organ function, and management of elevated intracranial pressure.

[0004] One of the only approved therapies for acute ischemic stroke in the United States is thrombolytics administered to patients within three hours of stroke onset. Some studies estimate that less than 5% of patients with acute ischemic stroke actually receive this therapy, possibly due to strict criteria for thrombolytic intervention, lack of appropriate facilities, and patients arriving beyond the three-hour timeframe.[2] Approximately 70% to 85% of patients who experience their first stroke develop hemiplegia or some form of paralysis on at least one side of their body. Within six months after a stroke event or incident, only 60% of patients who develop partial hemiplegia and require inpatient rehabilitation care achieve functional independence to perform simple daily activities.[3]

[0005] When stroke patients enter the chronic phase, generally defined as several months or more after a stroke event or incident, physiotherapy is often the only rehabilitation regimen prescribed for them. For these chronic stroke patients, there are no proven biological or pharmaceutical therapies that have been shown to significantly reverse damage and improve the patient's motor function. Various cell therapies have been proposed and studied, but clinical trials for these therapies have so far been limited to patients in whom such trials have shown only slight improvement [4-6]. Furthermore, there has been no investigation into whether a particular therapy is more effective in treating specific patient subpopulations, such as stroke patients with varying ischemic center sizes or volumes.

[0006] Therefore, there is a need for safe and effective therapies for treating patients with chronic stroke or chronic stroke-induced motor impairment. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Publication No. 2003 / 0003090 [Patent Document 2] U.S. Patent No. 5,486,359 [Patent Document 3] WO 2005 / 100552 [Patent Document 4] U.S. Patent No. 8,092,792 [Patent Document 5] U.S. Patent No. 10,245,286 [Patent Document 6] U.S. Patent No. 7,682,825 [Patent Document 7] U.S. Patent Application Publication No. 2010 / 0266554 [Patent Document 8] WO 2009 / 023251 [Patent Document 9] U.S. Patent Application Publication No. 2011 / 0229442 [Patent Document 10] U.S. Patent Publication No. 2019 / 0290846

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Non-Patent Literature

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[0009] Methods for treating a subject after a small volume ischemic stroke and methods for treating a subject with stroke-induced motor impairment are disclosed. Compositions for treating small volume ischemic stroke are also disclosed. [Means for solving the problem]

[0010] In one embodiment, a method for treating a subject after a small volume ischemic stroke includes the step of administering a therapeutically effective amount of cells to a brain region surrounding the small volume ischemic center of the subject. The cells may be derived from mesenchymal stem cells transiently transfected with polynucleotides encoding the Notch intracellular domain (NICD).

[0011] The cells may be prepared by a method comprising the steps of preparing a mesenchymal stem cell culture, contacting the mesenchymal stem cell culture with a polynucleotide encoding NICD, selecting cells containing the polynucleotide, and further culturing the selected cells in the absence of the polynucleotide selection. The mesenchymal stem cells may be human bone marrow-derived cells. Furthermore, the polynucleotide encoding NICD does not encode the full-length Notch protein.

[0012] A small-volume ischemic center may be an ischemic center having an ischemic center volume of less than 50 cubic centimeters (cc). For example, a small-volume ischemic center may be an ischemic center having an ischemic center volume of approximately 2cc to 50cc.

[0013] In some cases, treatment is particularly effective when the small volume of ischemic center is located in a region of the target brain other than the parietal region, such as the cortical frontal, cortical temporal, subcortical white matter, or subcortical gray matter of the target brain.

[0014] In some cases, treatment is effective even if the small volume ischemic stroke occurred more than six months before the cell administration process. For example, treatment may be effective even if the small volume ischemic stroke occurred between six and ninety months before the cell administration process.

[0015] The step of administering a therapeutically effective amount of cells may include the step of injecting a cell suspension containing the cells into the deposition site in the target brain. The cell suspension may contain cells suspended in a sterile isotonic crystalloid solution.

[0016] The step of administering a therapeutically effective amount of cells may further include injecting at least a portion of a cell suspension containing cells into one or more deposition sites on the periphery of an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area. For example, the step of administering a therapeutically effective amount of cells may include injecting at least a portion of a cell suspension containing cells into one or more deposition sites proximal to an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area.

[0017] The step of administering a therapeutically effective amount of cells may further include injecting at least a portion of a cell suspension containing cells into one or more deposition sites within an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area. In some examples, the step of administering a therapeutically effective amount of cells may include injecting at least a portion of a cell suspension containing cells into one or more deposition sites distal to the ischemic penumbra or within a small volume of ischemic central area.

[0018] The step of administering a therapeutically effective amount of cells may further include the step of stereotactically administering a therapeutically effective amount of cells by single burr hole craniotomy.

[0019] A therapeutically effective dose of cells may be approximately 25 million cells (i.e., 25 million cells ± 100,000 cells). The step of administering approximately 25 million cells may include injecting a cell suspension containing cells into five deposition sites along a first deposition or injection trajectory, five deposition sites along a second deposition or injection trajectory, and five deposition sites along a third deposition or injection trajectory. Approximately 20 μL of cell suspension may be injected into each deposition site. The cell suspension has a cell concentration of approximately 8.5*10 6 It may have a cell / mL content.

[0020] A therapeutically effective dose of cells may be approximately 5 million cells (i.e., 5 million cells ± 100,000 cells). The step of administering approximately 5 million cells may include injecting a cell suspension containing cells into five deposition sites along a first deposition or injection trajectory, five deposition sites along a second deposition or injection trajectory, and five deposition sites along a third deposition or injection trajectory. Approximately 20 μL of the cell suspension may be injected into each deposition site. The cell suspension has a cell concentration of approximately 17.0 * 10 6 It may have a cell / mL content.

[0021] The method may further include a step of subjecting a formulated dose of cells to a post-release test before administering the cells to the subject.

[0022] The method may further include the steps of determining the degree of disability of the subject by determining the subject's mRS score, and administering a therapeutically effective amount of cells only if the subject's mRS score is between 2 and 4.

[0023] A method for treating subjects with stroke-induced motor impairment is also disclosed. The method may include the steps of determining the volume of the ischemic center of the subject, and administering a therapeutically effective amount of cells to the brain region surrounding the ischemic center of the subject only if the volume of the ischemic center is determined to be less than 50 cubic centimeters (cc). For example, the method may include the step of administering a therapeutically effective amount of cells to the brain region surrounding the ischemic center of the subject only if the volume of the ischemic center is between approximately 2 cc and 50 cc.

[0024] Stroke-induced motor impairment may be a consequence of the ischemic stroke the subject suffered. In some cases, the ischemic stroke occurred more than 6 months before the cell administration process. For example, small volume ischemic stroke occurred between 6 and 90 months before the cell administration process.

[0025] In a particular case, the method includes administering a therapeutically effective dose of cells to the brain region surrounding the ischemic center of the subject, only if the ischemic stroke occurred more than six months prior (e.g., between six and ninety months prior).

[0026] The cells may be derived from mesenchymal stem cells transiently transfected with polynucleotides encoding the Notch intracellular domain (NICD).

[0027] The cells may be prepared by a method comprising the steps of preparing a mesenchymal stem cell culture, contacting the mesenchymal stem cell culture with a polynucleotide encoding NICD, selecting cells containing the polynucleotide, and further culturing the selected cells in the absence of the polynucleotide selection. The mesenchymal stem cells may be human bone marrow-derived cells. Furthermore, the polynucleotide encoding NICD does not encode the full-length Notch protein.

[0028] In certain cases, the method includes administering a therapeutically effective amount of cells to the brain region surrounding the ischemic center of a target brain, but only if the ischemic center is located in a region of the target brain other than the parietal region. For example, the method may include administering a therapeutically effective amount of cells to the brain region surrounding the ischemic center of a target brain, but only if a portion of the ischemic center is located in at least one of the cortical frontal, cortical temporal, subcortical white matter, and subcortical gray matter of the target brain.

[0029] The step of administering a therapeutically effective amount of cells may include the step of injecting a cell suspension containing the cells into the deposition site in the target brain. The cell suspension may contain cells suspended in a sterile isotonic crystalloid solution.

[0030] The step of administering a therapeutically effective amount of cells may further include injecting at least a portion of a cell suspension containing cells into one or more deposition sites on the periphery of an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area. For example, the step of administering a therapeutically effective amount of cells may include injecting at least a portion of a cell suspension containing cells into one or more deposition sites proximal to an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area. The step of administering a therapeutically effective amount of cells may also include injecting at least a portion of a cell suspension containing cells into one or more deposition sites within an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area. In some cases, the step of administering a therapeutically effective amount of cells may include injecting at least a portion of a cell suspension containing cells into one or more deposition sites within a distal or small volume of ischemic center in an ischemic penumbra / chronic ischemic penumbra.

[0031] The step of administering a therapeutically effective amount of cells may further include the step of stereotactically administering a therapeutically effective amount of cells by single burr hole craniotomy.

[0032] A therapeutically effective dose of cells may be approximately 25 million cells (i.e., 25 million cells ± 100,000 cells). The step of administering approximately 25 million cells may include injecting a cell suspension containing cells into five deposition sites along a first deposition or injection trajectory, five deposition sites along a second deposition or injection trajectory, and five deposition sites along a third deposition or injection trajectory. Approximately 20 μL of cell suspension may be injected into each deposition site. The cell suspension has a cell concentration of approximately 8.5*10 6 It may have a cell / mL content.

[0033] A therapeutically effective dose of cells may be approximately 5 million cells (i.e., 5 million cells ± 100,000 cells). The step of administering approximately 5 million cells may include injecting a cell suspension containing cells into five deposition sites along a first deposition or injection trajectory, five deposition sites along a second deposition or injection trajectory, and five deposition sites along a third deposition or injection trajectory. Approximately 20 μL of the cell suspension may be injected into each deposition site. The cell suspension has a cell concentration of approximately 17.0 * 10 6 It may have a cell / mL content.

[0034] The method may further include a step of subjecting a formulated dose of cells to a post-release test before administering the cells to the subject.

[0035] The method may further include the steps of determining the degree of disability of the subject by determining the subject's mRS score, and administering a therapeutically effective amount of cells only if the subject's mRS score is between 2 and 4.

[0036] Compositions for treating small-volume ischemic stroke are also disclosed. The composition may comprise a therapeutically effective amount of cells and a pharmaceutically acceptable carrier or diluent. The cells may be derived from mesenchymal stem cells transiently transfected with polynucleotides encoding Notch intracellular domains (NICDs).

[0037] The cells may be prepared by a method comprising the steps of preparing a mesenchymal stem cell culture, contacting the mesenchymal stem cell culture with a polynucleotide encoding NICD, selecting cells containing the polynucleotide, and further culturing the selected cells in the absence of the polynucleotide selection. The mesenchymal stem cells may be transiently transfected with a plasmid containing a polynucleotide encoding NICD.

[0038] Mesenchymal stem cells may be derived from human bone marrow. Furthermore, the polynucleotide encoding NICD does not encode the full-length Notch protein.

[0039] A pharmaceutically acceptable carrier or diluent may include a sterile isotonic crystalloid solution (e.g., Plasma-Lyte A). The composition may be a cell suspension packaged in a sealed vial before administration.

[0040] A therapeutically effective dose of cells may be approximately 2.5 million cells. If the therapeutically effective dose of cells is approximately 2.5 million cells, the composition should have a cell concentration of approximately 8.5*10 6 A cell suspension having a cell / mL concentration of approximately 0.3 mL may be used.

[0041] A therapeutically effective dose of cells may be approximately 5 million cells. If the therapeutically effective dose of cells is approximately 5 million cells, the composition should have a cell concentration of approximately 17.0*10 6 A cell suspension having a cell / mL concentration of approximately 0.3 mL may be used.

[0042] In some cases, a therapeutically effective dose of cells may be approximately 2 million to 5 million cells.

[0043] The use of cells in the manufacture of a pharmaceutical product for treating a subject after a small volume ischemic stroke the subject has suffered is also disclosed, comprising the step of administering a therapeutically effective amount of cells to a brain region surrounding the small volume ischemic center of the subject, wherein the cells are derived from mesenchymal stem cells transiently transfected with polynucleotides encoding Notch intracellular domains (NICDs).

[0044] Further disclosures describe the use of cells in the manufacture of a pharmaceutical product for the treatment of a subject with stroke-induced motor impairment, comprising the steps of determining the volume of the ischemic center of the subject, and, only if the volume of the ischemic center is determined to be less than 50 cubic centimeters (cc), administering a therapeutically effective amount of cells to the brain region surrounding the ischemic center of the subject, wherein the cells are derived from mesenchymal stem cells transiently transfected with polynucleotides encoding Notch intracellular domains (NICDs).

[0045] Cells derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding a Notch intracellular domain (NICD) for use in a method of treating a subject after a small volume ischemic stroke in which the subject has suffered, wherein the method comprises the step of administering a therapeutically effective amount of cells to a brain region surrounding the small volume ischemic center of the subject.

[0046] Cells derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD) for use in a method for treating a subject with stroke-induced motor impairment, wherein the method comprises the steps of determining the volume of the ischemic center of the subject, and, only if the volume of the ischemic center is determined to be less than 50 cubic centimeters (cc), administering a therapeutically effective amount of the cells to the brain region surrounding the ischemic center of the subject, wherein the cells are derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). [Brief explanation of the drawing]

[0047] [Figure 1] This diagram shows methods for treating patients with stroke-induced motor impairment. [Figure 2] This diagram shows the ischemic center of a stroke patient surrounded by an ischemic penumbra. [Figure 3] Figure 3A is a graph showing the relationship between baseline FMMS and stroke volume for various treatments and control groups. Figure 3B is a graph showing the relationship between baseline mRS and baseline FMMS for various treatments and control groups. Figure 3C is a graph showing the relationship between baseline mRS and stroke volume for various treatments and control groups. [Figure 4] This graph shows the delta composite ratio relative to stroke volume for various medication dosage groups. [Figure 5] This diagram shows various regions or areas of the brain, particularly including the subcortical white matter, subcortical gray matter, cortical frontal lobe, cortical parietal lobe, and cortical temporal lobe. [Figure 6] This table contains information on stroke location, baseline characteristics, and delta response rate for research populations divided by population percentage. [Figure 7] This plot shows the delta composite response rate in Figure 6, plotted against the group percentage. [Modes for carrying out the invention]

[0048] The following terms are defined as follows for the purposes of this disclosure:

[0049] definition "Angiogenesis" or "angiogenic" refers to the formation of a new vascular system (e.g., blood vessels; e.g., veins, arteries, venules, arterioles, capillaries). Angiogenesis can occur through the budding of new blood vessels from existing ones and / or through the in situ adhesion of endothelial cells to form new blood vessels. Angiogenesis also includes incidental processes such as matrix remodeling and cell recruitment (e.g., recruitment of smooth muscle cells, monocytes, and / or pericytes). Angiogenesis further includes the proliferation and / or migration of endothelial cells.

[0050] The terms "DNTT-MSC" ("descendants of MSCs transiently transfected with NICD") and "SB623 cells" refer to a population of cells obtained after transient expression of exogenous Notch intracellular domains (NICDs) in MSCs. For example, a population of DNTT-MSCs can be obtained by transient transfection of MSCs with a vector containing a sequence that does not encode the full-length Notch protein but encodes NICD (e.g., derived from human Notch 1 protein), followed by selection (e.g., by G418). The selected cells may be further cultured in a standard culture medium optionally supplemented with serum, in the absence of any added growth factors or differentiation factors (other than factors that may be present in serum if serum is present in the culture medium). DNTT-MSCs may originate from human bone marrow MSCs obtained by transient transfection of human bone marrow MSCs with NICD (e.g., human Notch 1 intracellular domain (NICD1)), subsequent selection, and subsequent proliferation. This process generates a cell population that exhibits superior angiogenic and neurogenic properties in vitro compared to the parent MSCs [7-9]. The neurogenic effect of DNTT-MSCs is due to increased expression of FGF1, FGF2, and BMP, as well as increased secretion of these proteins [7, 9].

[0051] The Fugl-Meyer assessment, or FMA, is an index or scale used to assess sensorimotor impairment in individuals who have had a stroke. The FMA index or scale is clinically applied to determine the severity of impairment, assess motor recovery, and plan treatment. The FMA index or scale was first proposed in 1975 by Axel Fugl-Meyer and his colleagues as a standardized assessment of post-stroke recovery.

[10] The assessment consists of five areas: (1) motor function (both arms and legs), (2) sensory function, (3) balance, (4) range of motion, and (5) joint pain. A total of 155 items are assessed across all five areas, with a maximum score of 226 points.

[0052] The Fugl-Meyer Motor Scale or Score, or FMMS, is a subscale used to assess motor function or impairment in individuals with stroke, including range of motion in both the upper and lower limbs. The FMMS ranges from 0 points (hemiplegia) to 100 points (normal motor function). The maximum FMMS score of 100 points is approximately half of the maximum total FMA score (226 points) across all five ranges. The maximum FMMS score is divided into 66 points for both arms (based on 33 assessment items) and 34 points for both legs (based on 17 assessment items).

[0053] "FMMS bilateral arms" or "FMMS UE" are more specific subscales used to assess motor function in both arms or upper limbs of individuals who have had a stroke.

[0054] "FMMS Bilateral Leg" or "FMMS LE" is a more specific subscale used to assess motor function in both legs or lower limbs of individuals with a stroke.

[0055] The terms "introduction" and "transplantation" are used to refer to the introduction of exogenous cells into a subject or patient. Exogenous cells may be self (i.e., obtained from the subject) or allogeneic (i.e., obtained from an individual other than the subject).

[0056] "Mesenchymal cells" refers to mesenchymal tissues (e.g., chondrocytes, chondrocytes, osteoblasts, osteocytes, adipocytes) and their precursor cells, including, for example, fibroblasts (e.g., human precipitous fibroblasts), MSCs (as defined herein), and cells derived from MSCs such as DNTT-MSCs as defined herein.

[0057] The "minimum clinically significant change" (MCID) is the smallest change in treatment outcome that a patient or individual perceives or identifies as meaningful or important. For example, since the MCID is related to the FMA, it may be the smallest change in the score used to assess a stroke patient in one or more of the five FMA domains. As a more specific example, an MCID threshold for assessing stroke treatment might require that a stroke patient exhibits an improvement of at least 6 points in their FMMS UE score from a baseline measurement.

[0058] The Modified Rankin Scale, or mRS, is a clinician-reported ordinal scale used to measure the degree or level of disability in patients with stroke. The scale ranges from grade or score 0 (no symptoms) to 6 (death). Patients with grade or score 1 have no significant disability and can perform all normal movements and activities. Patients with grade or score 2 have slight disability and cannot perform all previous activities but can care for themselves without assistance. Patients with grade or score 3 have moderate disability and require some assistance but can walk without assistance. Patients with grade or score 4 have moderately severe disability, cannot walk, and cannot care for their own physical needs without assistance. Patients with grade or score 5 have severe disability, are bedridden, incontinent, and require constant nursing care and attention.

[0059] "MSCs" ("mesenchymal stem cells") refer to adherent, non-hematopoietic pluripotent cells obtained from bone marrow. These cells are variously known as mesenchymal stem cells, mesenchymal stromal cells, bone marrow adherent stromal cells, bone marrow adherent stem cells, and bone marrow stromal cells. MSCs may be obtained, for example, from umbilical cord blood, adipose tissue, dental pulp, Wharton's jelly, and various connective tissues. MSCs may be obtained by selecting adherent cells (i.e., cells that adhere to tissue culture plastics) from bone marrow (for example, by growth in a culture medium). To obtain an MSC population with a sufficient number of cells for therapeutic use, a population of adherent cells is grown in a culture medium after selection for adherence. Growth in a culture medium also enriches the MSCs because contaminating cells (such as monocytes) do not grow under the culture conditions.

[0060] Exemplary disclosures of MSCs are provided in U.S. Patent Publication 2003 / 0003090; Prockop (1997) Science 276: pp. 71-74 and Jiang (2002) Nature 418: pp. 41-49. Methods for isolating and purifying MSCs can be found, for example, in U.S. Patent No. 5,486,359; Pittenger et al. (1999) Science 284: pp. 143-147 and Dezawa et al. (2001) Eur. J. Neurosci. 14: pp. 1771-1776. Human MSCs are commercially available (e.g., BioWhittaker, Walkersville, Md.) or may be obtained from a donor by bone marrow aspiration, then cultured and adherent bone marrow cells selected. See, for example, WO 2005 / 100552.

[0061] MSCs may be isolated from umbilical cord blood. See, for example, Campagnoli et al. (2001) Blood 98:2396-2402; Erices et al. (2000) Br. J. Haematol. 109:235-242 and Hou et al. (2003) Int. J. Hematol. 78:256-261. Further sources of MSCs include, for example, adipose tissue, dental pulp, and Wharton's jelly.

[0062] "Neurogenesis" or "neurogenic" refers to or relates to the growth and / or differentiation of neural progenitor cells (NPCs) into neurons and / or glial cells (e.g., astrocytes, oligodendrocytes). Examples of neurogenesis include, but are not limited to, NPC proliferation, neurogenesis (e.g., formation of new neurons), and glialogenesis (e.g., formation of astrocytes and / or oligodendrocytes). Other processes relate to neuronal development, including, for example, neurite elongation, axon elongation, and dendritic elongation.

[0063] The Notch protein (e.g., Notch-1 protein) is a transmembrane receptor found in all metazoans that influences cell differentiation through intracellular signaling. When the Notch extracellular domain (e.g., the extracellular domain of Notch-1 protein) is brought into contact with a Notch ligand (e.g., delta, serate, jagged), two proteolytic cleavages of the Notch protein occur. The second cleavage is catalyzed by γ-secretase, releasing the Notch intracellular domain (NICD) into the cytoplasm. In the mouse Notch protein, this cleavage occurs between amino acids gly1743 and val1744. The NICD travels to the nucleus, where it acts as a transcription factor, recruiting further transcriptional regulatory proteins (e.g., MAM, histone acetylase) to mitigate transcriptional repression of various target genes (e.g., Hes1). Further details and information regarding Notch signaling can be found, for example, in Artavanis-Tsakonas et al. (1995) Science 268:225-232; Mumm and Kopan (2000) Develop. Biol. 228:151-165; and Ehebauer et al. (2006) Sci. STKE 2006 (364), cm7. [DOI: 10.1126 / stke.3642006cm7].

[0064] "Stroke" is the name given to a condition resulting from impaired blood flow in the brain. Such cerebrovascular disorders can result, for example, from intracranial hemorrhage or from reduced or blocked blood flow in the brain (i.e., cerebral ischemia). Ischemic occlusion can result from thrombosis (i.e., in situ formation of a blood clot in the cranial blood vessels or blood vessels supplying the brain) or cerebral embolism (migration of a blood clot to a part of the brain). Damage resulting from ischemic or hemorrhagic stroke usually results in impairment of specific neurological and physiological functions. Further information on the various types of stroke and their characteristics can be found in the shared U.S. Patents 8,092,792 and 10,245,286, whose disclosures are incorporated herein by reference throughout for the purpose of describing the various types of stroke and their characteristics.

[0065] treatment Figure 1 shows a method 100 for treating a subject with stroke-induced motor impairment. Stroke-induced motor impairment may be a result of an ischemic stroke suffered by the subject. Method 100 may be considered a method for treating a subject after an ischemic stroke or a method for treating a subject after a small-volume ischemic stroke.

[0066] Method 100 may include in task 102 determining the volume of the ischemic center of a subject with stroke-induced motor impairment. In some cases, task 102 may include receiving data or information regarding the volume of the ischemic center of the subject.

[0067] For the purposes of this disclosure, the ischemic central area or infarct central area may refer to brain tissue that has already formed an infarct (i.e., has undergone necrosis or tissue death) or is irreversibly destined to form an infarct as a result of the ischemic stroke suffered by the subject.

[0068] The volume of the ischemic center can be determined using computed tomography (CT) perfusion (CTP), diffusion-weighted magnetic resonance imaging (DWI), or, in some cases, baseline non-contrast CT (NCCT). When measured using CTP, the ischemic center can be defined as brain tissue with a relative cerebral blood flow (CBF) level of less than 30% of normal cerebral blood flow. When measured using DWI, the ischemic center is 620 μm². 2 It can be defined as brain tissue having an apparent diffusion coefficient of less than / s

[11] .

[0069] In some cases, method 100 may include proceeding with cell therapy or treatment only if the ischemic center is determined to be less than 50 cubic centimeters (cc). For example, if the ischemic center is determined to be between approximately 2cc and 50cc, method 100 may include only proceeding with cell therapy or treatment.

[0070] Method 100 may further include determining the degree of disability of the subject by determining the subject's mRS score in task 104. In some examples, task 104 may include receiving data or information regarding the subject's mRS score.

[0071] Figure 1 shows that operation 104 follows operation 102, but it should be understood by those skilled in the art that the order of operations may be reversed, and that operations may be performed in parallel.

[0072] In some cases, method 100 may include proceeding with cell therapy or treatment only if the subject's mRS score is between 2 and 4. For example, subjects with an mRS score of less than 2 are considered not to have a sufficient disability to warrant the cell therapy disclosed herein, while subjects with an mRS score greater than 4 are considered to have an overly severe disability to warrant treatment.

[0073] Method 100 may further include, in operation 106, administering a therapeutically effective amount of cells to the brain region surrounding the ischemic center of the subject. In some cases, the brain region surrounding the ischemic center may be the ischemic penumbra of the subject. Method 100 may include administering a therapeutically effective amount of cells to the ischemic penumbra without direct injection into the ischemic center.

[0074] For the purposes of this disclosure, ischemic penumbra may refer to brain tissue with reduced perfusion or at risk of irreversible damage, but still salvageable. Some studies have defined ischemic penumbra as an area with reduced CBF, where CBF levels are reduced to approximately 10 and 15 ml / 100g / min to approximately 25 ml / 100g / min

[12] .

[0075] Method 100 may include administering a therapeutically effective dose of cells to the ischemic penumbra in the subject in operation 106. In some cases, Method 100 may include administering a therapeutically effective dose of cells to the ischemic penumbra in the subject only if it is determined that the volume of the ischemic center is less than 50 cc. For example, Method 100 may include administering a therapeutically effective dose of cells to the ischemic penumbra in the subject only if it is determined that the volume of the ischemic center is between approximately 2 cc and 50 cc.

[0076] In these and other examples, Method 100 may include administering a therapeutically effective dose of cells to the ischemic penumbra of the subject only if the subject's mRS score is between 2 and 4. Method 100 may also include administering a therapeutically effective dose of cells to the ischemic penumbra of the subject only if the volume of the ischemic center is determined to be less than 50 cc and the subject's mRS score is between 2 and 4.

[0077] As will be discussed in more detail in the following sections, the applicant found that in subjects with small volume stroke and an mRS score of 2–4 (e.g., ischemic central volume less than 50 cc), there was a statistically significant difference (p-value = 0.02) in the proportion of such subjects in the treatment group (i.e., subjects administered cells) compared to the same subjects in the sham / control group, in which subjects showed clinically significant improvement in motor function.

[0078] In certain specific cases, Method 100 may be considered a method for treating a subject with chronic stroke-induced motor impairment, or a method for treating chronic stroke. For example, Method 100 may include administering a therapeutically effective dose of cells to the brain region surrounding the ischemic center of the subject, only if the ischemic stroke occurred more than six months prior to the administration of the cells (e.g., between six and ninety months prior).

[0079] Method 100 can also be considered a method for treating small-volume chronic stroke (as opposed to acute stroke). For example, Method 100 may include administering a therapeutically effective dose of cells to the brain region surrounding the ischemic center of the subject only if the ischemic stroke occurred more than six months prior to cell administration (e.g., between six and ninety months prior) and only if the volume of the ischemic center is determined to be less than 50 cc.

[0080] If the stroke incident occurred more than six months prior to treatment (e.g., cell administration), the penumbra surrounding the chronic ischemic center of the subject is considered a chronic penumbra (as opposed to an acute penumbra). The chronic penumbra may exhibit even lower CBF levels than the acute penumbra of a subject who has recently suffered a stroke. In these cases, method 100 for treating such patients may include administering a therapeutically effective dose of cells to the chronic penumbra surrounding the chronic ischemic center of the subject. In some cases, method 100 may include administering a therapeutically effective dose of cells to the chronic penumbra surrounding the chronic ischemic center of the subject only if the volume of the chronic ischemic center is determined to be less than 50 cc.

[0081] DNTT-MSC The cells to be administered may be allogeneic cells derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). The cells may be prepared by a method comprising: preparing a culture of mesenchymal stem cells; contacting the mesenchymal stem cell culture with a polynucleotide encoding the NICD (the polynucleotide does not encode the full-length Notch protein); selecting cells containing the polynucleotide; and further culturing the selected cells in the absence of polynucleotide selection. The mesenchymal stem cells may be human bone marrow-derived cells. For the purposes of this disclosure, the cells to be administered may be referred to as DNTT-MSCs.

[0082] As previously discussed, DNTT-MSCs are obtained from bone marrow adherent stromal cells, also known as MSCs, by transiently expressing the intracellular domain of the Notch protein in the MSCs. Transient expression of the Notch intracellular domain (e.g., NICD derived from human Notch 1 protein) in MSCs may be sufficient to convert a population of MSCs into a population of DNTT-MSCs. Further treatment with growth and / or differentiation factors is not required. Thus, a population of MSCs is converted into a population of DNTT-MSCs by transient transfection of MSCs with a vector containing a sequence encoding NICD (but not the full-length Notch protein), and then cells containing the vector may be selected and further cultured in serum-containing medium in the absence of further exposure to growth and / or differentiation factors. For example, see U.S. Patent No. 7,682,825 (March 23, 2010); U.S. Patent Application Publication No. 2010 / 0266554 (October 21, 2010); and WO 2009 / 023251 (February 19, 2009), whose disclosures are incorporated herein by reference throughout, for the purpose of describing the isolation of mesenchymal stem cells and the conversion of mesenchymal stem cells to DNTT-MSCs (referred to hereafter as “neural progenitor cells” and “neural regenerating cells”).

[0083] In these methods, any polynucleotide encoding the Notch intracellular domain (e.g., a vector) can be used, and any method for selecting and enriching transfected cells can be used. For example, MSCs may be transfected with a vector containing a sequence encoding the Notch intracellular domain (e.g., human Notch 1 intracellular domain) and a sequence encoding a selection marker (e.g., drug resistance, e.g., resistance to G418). In some examples, two vectors, one containing a sequence encoding the Notch intracellular domain and the other containing a sequence encoding a drug resistance marker, can be used for transfecting MSCs. In these examples, selection is achieved by adding a sufficient amount of the selection agent (e.g., G418) to the cell culture after transfection with a single or multiple vectors, so as to kill the vector-free cells but allow the vector-containing cells to survive. Absence of selection involves removing the selection agent or reducing the concentration of the selection agent to a level that does not kill the vector-free cells. After selection (for example, for 7 days), the selected drug may be removed, and the cells may be further cultured in serum-containing culture medium (for example, for two passages).

[0084] Depending on the properties of the selection marker used and / or the concentration of the selection agent, it is not necessarily required to kill all cells lacking the vector encoding the selection marker during the selection process. For example, the selection agent may inhibit the growth of cells that do not contain the selection marker, and after the removal of the selection agent, these cells may recover and resume growth.

[0085] Thus, the preparation of DNTT-MSCs involves transient expression of exogenous Notch intracellular domains in MSCs. For this purpose, MSCs may be transfected with a vector containing a sequence encoding a Notch intracellular domain (e.g., human Notch 1 intracellular domain), the sequence not encoding the full-length Notch protein. All such sequences are known and readily available to those skilled in the art. For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Biol. 228:151-165 provide the amino acid sequence surrounding the so-called S3 cleavage site that releases the intracellular domain, derived from the mouse Notch protein. In summary, these references provide to those skilled in the art any peptide containing a Notch intracellular domain that is not the full-length Notch protein, and therefore any polynucleotide containing a sequence encoding a Notch intracellular domain that is not encoding the full-length Notch protein. The aforementioned references (Del Amo and Mumm) are incorporated by reference throughout to disclose the amino acid sequences of the full-length Notch protein and the Notch intracellular domain, respectively.

[0086] Similar information is available for Notch protein and nucleic acids from further species, including rats, clawed frogs, fruit flies, and humans. See, for example, Weinmaster et al. (1991) Development 113:pp. 199-205; Schroeter et al. (1998) Nature 393:pp. 382-386; NCBI reference sequence NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are incorporated by reference throughout for the purpose of disclosing the amino acid sequences of the full-length Notch protein and the Notch intracellular domain in multiple different species.

[0087] In some cases, DNTT-MSCs may be prepared by introducing nucleic acids containing a sequence encoding the intracellular Notch domain into MSCs, such that the MSCs do not express the exogenous Notch extracellular domain. This can be achieved, for example, by transfecting MSCs with a vector containing a sequence encoding the intracellular Notch domain, the sequence not encoding the full-length Notch protein.

[0088] Further details regarding the preparation of DNTT-MSCs and methods for producing cells having properties similar to those of DNTT-MSCs, which can be used in the methods disclosed herein, are found in U.S. Patent No. 7,682,825 (March 23, 2010); and U.S. Patent Publications 2010 / 0266554 (October 21, 2010) and 2011 / 0229442 (September 22, 2011), the disclosures of which are incorporated herein by reference in their entirety for the purpose of describing alternative methods for the preparation of DNTT-MSCs and providing methods for producing cells having properties similar to those of DNTT-MSCs. See also Dezawa et al. (2004) J. Clin. Invest. 113:1701–1710.

[0089] cell culture Standard methods for cell culture are known in the art. See, for example, R.I. Freshney, "Culture of Animal Cells: A Manual of Basic Technique," 5th edition, Wiley, New York, 2005.

[0090] Transfection Methods for introducing exogenous DNA into cells (i.e., transfection) and the selection of transfected cells are also known in the art; see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, New York, 1987 and its periodic updates.

[0091] Figure 2 illustrates the ischemic center 200 in a subject who has suffered an ischemic stroke. As shown in Figure 2, the ischemic center 200 may be surrounded by an ischemic penumbra 202. If the ischemic stroke occurred more than 6 months before the start of treatment, the ischemic center 200 is considered a chronic ischemic center, and the ischemic penumbra 202 is considered a chronic ischemic penumbra.

[0092] As discussed earlier, the ischemic central 200 may have a central volume or ischemic central volume. The central volume can be determined using other imaging techniques, such as computed tomography perfusion or diffusion-weighted magnetic resonance imaging. If the central volume is determined to be less than 50 cc (e.g., approximately 2 cc to 50 cc), the ischemic stroke is considered a small-volume ischemic stroke.

[0093] Furthermore, as previously discussed, a method for treating small-volume ischemic stroke may include administering a therapeutically effective dose of allogeneic cells to the ischemic penumbra surrounding the small-volume ischemic central 200 in a subject suffering from small-volume ischemic stroke. The cells may be derived from mesenchymal stem cells transiently transfected with polynucleotides encoding NICD. For the purposes of this disclosure, the cells may be called DNTT-MSCs. Moreover, if the ischemic stroke occurred more than six months before the initiation of treatment, the method may be considered a method for treating small-volume chronic ischemic stroke.

[0094] As shown in Figure 2, the method may include administering a therapeutically effective amount of cells by injecting a cell suspension containing cells into one or more deposition sites 204 in the target brain. The step of administering a therapeutically effective amount of cells may further include injecting at least a portion of the cell suspension containing cells into one or more deposition sites 204 on the periphery of an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area. For example, the step of administering a therapeutically effective amount of cells may include injecting at least a portion of the cell suspension containing cells into one or more deposition sites 204 proximal to an ischemic penumbra or chronic ischemic penumbra surrounding a small volume of ischemic central area.

[0095] The step of administering a therapeutically effective amount of cells may include injecting at least a portion of a cell suspension containing cells into one or more deposition sites 204 within an ischemic penumbra surrounding a small volume of ischemic central area. In some examples, the step of administering a therapeutically effective amount of cells may include injecting at least a portion of a cell suspension containing cells into one or more deposition sites 204 distal to an ischemic penumbra or ischemic chronic penumbra. In these and other examples, the step of administering a therapeutically effective amount of cells may include injecting at least a portion of a cell suspension containing cells into one or more deposition sites 204 within a small volume of ischemic central area.

[0096] The cell suspension may contain cells suspended in a pharmaceutically acceptable carrier or diluent. In some cases, the pharmaceutically acceptable carrier or diluent may be a sterile isotonic crystalloid solution. For example, the cell suspension may contain cells suspended in Plasma-Lyte® A (Baxter Healthcare Corporation). The cells may be suspended in another physiologically suitable carrier, such as phosphate-buffered saline.

[0097] The cells may be stereotactically administered via single burr hole craniotomy. Further details regarding stereotactic administration of cells can be found in U.S. Patent Publication No. 2019 / 0290846 (Sep. 26, 2019), the contents of which are incorporated herein by reference in their entireties for the purpose of describing stereotactic administration of DNTT-MSCs and the equipment used for such purposes.

[0098] The method may further comprise subjecting the formulated dose of cells to post-release testing prior to administering the cells to the subject.

[0099] A therapeutically effective amount of cells (or DNTT-MSCs) may be approximately 2.5 million cells (i.e., 2.5 million cells ± 100,000 cells). Where the amount of cells administered is approximately 2.5 million cells, the method of treatment may comprise injecting a cell suspension comprising the cells into 5 deposition sites 204 along a first deposition trajectory 206A or injection trajectory (see Figure 2), 5 deposition sites 204 along a second deposition trajectory 206B or injection trajectory, and 5 deposition sites along a third deposition trajectory 206C or injection trajectory. Approximately 20 μL of the cell suspension may be injected at each deposition site. Further, the cell suspension may have a cell concentration of approximately 8.5 * 10 6 cells / mL.

[0100] A therapeutically effective amount of cells (or DNTT-MSCs) may be approximately 5 million cells (i.e., 5 million cells ± 100,000 cells). Where the amount of cells administered is approximately 5 million cells, the method of treatment may comprise injecting a cell suspension comprising the cells into 5 deposition sites 204 along a first deposition trajectory 206A or injection trajectory (see Figure 2), 5 deposition sites 204 along a second deposition trajectory 206B or injection trajectory, and 5 deposition sites along a third deposition trajectory 206C or injection trajectory. Approximately 20 μL of the cell suspension may be injected at each deposition site. Further, the cell suspension may have a cell concentration of approximately 17.0 * 10 6 cells / mL.

[0101] In some cases, a therapeutically effective dose of cells (or DNTT-MSCs) may be approximately 2.5 million to 5 million cells. For example, a therapeutically effective dose of cells (or DNTT-MSCs) may be approximately 3 million cells (i.e., 3 million cells ± 100,000 cells), 3.5 million cells (i.e., 3.5 million cells ± 100,000 cells), 4 million cells (i.e., 4 million cells ± 100,000 cells), or 4.5 million cells (i.e., 4.5 million cells ± 100,000 cells).

[0102] As shown in Figure 2, at least a portion of the deposition sites 204 along each of the deposition trajectories or injection trajectories may be within the ischemic penumbra or chronic ischemic penumbra. In some cases, at least one or more of the deposition sites 204 along each of the deposition trajectories or injection trajectories may be proximal to the ischemic penumbra or chronic ischemic penumbra, or on the periphery of the ischemic penumbra or chronic ischemic penumbra. In other cases, the entire deposition trajectory or injection trajectory may be within the ischemic penumbra or chronic ischemic penumbra. In further cases, at least one or more of the deposition sites 204 along each of the deposition trajectories or injection trajectories may be within the ischemic center or chronic ischemic center, or on the periphery of the ischemic center or chronic ischemic center.

[0103] In certain cases, the treatment is particularly effective when the ischemic center 200 is located in a region, lobe, or area of ​​the target brain other than the parietal or lobe. For example, a method for treating a small volume ischemic stroke may include administering a therapeutically effective dose of cells to the brain region surrounding the ischemic center 200 (e.g., ischemic penumbra 202) only if a portion of the ischemic center 200 is located in at least one of the cortical frontal, cortical temporal, subcortical white matter, and subcortical gray matter of the target brain (see, for example, Figure 5). In some cases, the method may include administering a therapeutically effective dose of cells to the brain region surrounding the ischemic center 200 (e.g., ischemic penumbra 202) only if the ischemic center 200 is located in a region of the target brain other than the parietal.

[0104] Compositions, formulations, and kits Compositions, formulations, and kits for treating small volume ischemic stroke or small volume chronic ischemic stroke are also disclosed. Compositions may comprise a therapeutically effective amount of cells and a pharmaceutically acceptable carrier or diluent. As previously discussed, the cells, also known as DNTT-MSCs, may be derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). The cells (DNTT-MSCs) may be prepared by a method comprising preparing a culture of mesenchymal stem cells (e.g., human bone marrow-derived cells) and contacting the mesenchymal stem cell culture with the polynucleotide encoding the NICD. For example, the mesenchymal stem cells may be transiently transfected with a plasmid containing the polynucleotide encoding the NICD. The method may further comprise selecting cells containing the polynucleotide and, in the absence of the polynucleotide selection, further culturing the selected cells. In certain specific examples, the polynucleotide does not encode the full-length Notch protein. The compositions disclosed herein may, in particular, be useful for stimulating the proliferation and differentiation of neural progenitor cells and / or endothelial cells.

[0105] The "therapeutic amount" of a composition may contain a quantity of cells suitable for treating small volume ischemic stroke or small volume chronic ischemic stroke, in particular, by stimulating the proliferation and differentiation of neural progenitor cells and / or endothelial cells. In some examples, the therapeutic amount of a composition may contain approximately 2.5 million cells (i.e., 2.5 million cells ± 100,000 cells). The therapeutic amount of a composition may contain approximately 5 million cells (i.e., 5 million cells ± 100,000 cells). The therapeutic amount of a composition may contain approximately 2 million to approximately 5 million cells.

[0106] In other examples, the therapeutically effective dose of the composition may vary based on the nature and severity of the injury, the patient's weight and overall health, and other criteria known to those skilled in the art. For example, the dosage may vary between approximately 100, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 50,000, 100,000, 500,000, 1,000,000, 2,500,000, and 5,000,000 to 10,000,000 cells or more (or any integer value in between), and the frequency of administration, for example, single dose, once daily, twice weekly, once weekly, twice monthly, or once monthly, may depend on, for example, body weight, route of administration, severity of the disease, etc.

[0107] The cells described herein may be suspended in a pharmaceutically acceptable carrier or diluent to form a cell suspension. A pharmaceutically acceptable carrier may be a physiologically compatible carrier for transfer. As used herein, the term “physiologically compatible carrier” may mean a carrier that is compatible with the other components of the formulation and is not harmful to the recipient of the formulation. Examples of suitable carriers or diluents include cell culture media (e.g., Eagle’s Minimal Essential Medium), phosphate-buffered saline, Hanks’ equilibrium salt solution + / - glucose (HBSS), and polyelectrolyte solutions. A pharmaceutically acceptable carrier or diluent may be, or may contain, a sterile isotonic crystalloid solution such as Plasma-Lyte® A (Baxter Healthcare Corporation).

[0108] Various pharmaceutical compositions, as well as techniques for their preparation and use, are known to those skilled in the art from the perspective of this disclosure. For a detailed enumeration of suitable pharmacological compositions and techniques for their administration, refer to textbooks such as Remington's Pharmaceutical Sciences, 17th edition, 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005; University of the Sciences in Philadelphia (ed.), "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (ed.), "Remington: The Principles of Pharmacy Practice," Lippincott Williams & Wilkins, 2008.

[0109] The composition may include a cell suspension packaged in a sealed vial. In some examples, the sealed vial contains a cell concentration of approximately 8.5*10 6 It may contain 0.3 mL of cell suspension with a cell / mL concentration. Alternatively, a sealed vial may contain a cell concentration of approximately 17.0*10 6 It may contain 0.3 mL of a cell suspension with a cell / mL concentration.

[0110] Other examples of substances that can function as pharmaceutically acceptable carriers are also disclosed, including sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; bacum; gelatin; talc; excipients, e.g., cocoa butter and suppository waxes; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings, preservatives, and antioxidants may also be present in the composition.

[0111] Exemplary formulations include, but are not limited to, formulations encapsulated in micelles, liposomes, or drug-release capsules (with active agents incorporated within a biocompatible coating designed for slow release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments, and gels; and other formulations, such as inhalants, aerosols, and sprays, which are suitable for parenteral administration, for example, intrapulmonary, intravenous, intra-arterial, intraocular, intracranial, submeningeal, or subcutaneous administration. The dosage of the compositions of this disclosure may vary according to the degree and severity of the need for treatment, the activity of the composition being administered, the overall health of the subject, and other considerations well known to those skilled in the art.

[0112] In further embodiments, the compositions described herein may be delivered topically. Topical delivery allows for non-systemic delivery of the composition and therefore reduces the internal load of the composition compared to systemic delivery. Such topical delivery may be achieved by the use of various medically implantable devices, including but not limited to stents and catheters, and may be achieved by inhalation, injection, or surgery. Methods for coating, implanting, incorporating, and otherwise attaching desired agents to medical devices such as stents and catheters are established in the Art and intended herein.

[0113] Another aspect of this disclosure relates to a kit for administering cells to a subject. For example, the kit may include, as one or more distinct pharmaceutical preparations, a composition of cells formulated as necessary (e.g., in a pharmaceutical carrier).

[0114] Compositions containing DNTT-MSC can be used in combination with other compositions containing substances that stimulate angiogenesis ("angiogenic agents"). The compositions may be administered sequentially or in parallel in any order. Accordingly, the therapeutic compositions disclosed herein may contain both DNTT-MSC and an angiogenic agent. In further embodiments, separate therapeutic compositions, one containing DNTT-MSC and the other containing an angiogenic agent, may be administered to a subject separately or together.

[0115] In some cases, the angiogenic agent may be a protein (e.g., fibroblast growth factor, platelet-derived growth factor, transforming growth factor alpha, hepatocyte growth factor, vascular endothelial growth factor, Sonic Hedgehog, MAGP-2, HIF-1, PR-39, RTEF-1, c-Myc, TFII, Egr-1, ETS-1) or a nucleic acid encoding such a protein. See, for example, Vincent et al. (2007) Gene Therapy 14: pp. 781-789. In other cases, the angiogenic agent may be a small RNA molecule (e.g., siRNA, shRNA, microRNA) or ribozyme that targets nucleic acids encoding angiogenesis inhibitors. Furthermore, the angiogenic agent may be a triple-stranded nucleic acid that binds to a DNA sequence that regulates the expression of an angiogenesis inhibitor, for example, to block the transcription of the gene encoding that protein.

[0116] Angiogenic agents may be transcription factors that activate the expression of angiogenic molecules (e.g., proteins). Naturally occurring transcription factors (e.g., HIF-1 alpha) that regulate the expression of angiogenic proteins are known. Furthermore, synthetic transcription regulatory proteins may be constructed by genetic engineering. For example, methods for designing zinc finger DNA-binding domains that bind to a target sequence, and methods for fusing such zinc finger DNA-binding domains to transcriptional activation and repression domains are described. See, for example, U.S. Patents 6,534,261; 6,607,882; 6,785,613; 6,794,136; 6,824,978; 6,933,16,979,539; 7,013,219; 7,177,766; 7,220,719; and 7,788,044. These methods may be used to synthesize non-naturally occurring proteins that activate the transcription of any gene encoding a pro-angiogenic protein. Furthermore, synthetic zinc finger transcription activators of the vascular endothelial growth factor (VEGF) gene have been described. See, for example, U.S. Patents 7,026,462; 7,067,317; 7,560,440; 7,605,140; and 8,071,564. Thus, non-naturally occurring (i.e., synthetic) zinc finger proteins that activate the transcription of the VEGF gene can be used in combination with SB623 cells to enhance angiogenesis, for example, in the treatment of stroke. Furthermore, natural or synthetic transcription regulatory proteins (e.g., synthetic zinc finger transcription regulatory proteins) that inhibit the transcription of anti-angiogenic molecules may be used as pro-angiogenic agents.

[0117] Clinical trials A randomized, double-blind, sham surgery-controlled clinical trial was conducted to evaluate the safety and efficacy of stereotactic intracranial injection of DNTT-MSCs (also known as SB623 cells) in subjects with chronic motor impairment resulting from ischemic stroke. Subjects were randomized as part of the trial in an approximately 1:1:1 ratio to receive either (1) approximately 2.5 million DNTT-MSCs, (2) approximately 5 million DNTT-MSCs, or (3) sham surgery (control group). A total of 158 subjects were randomized into 51 receiving sham surgery, 52 receiving approximately 2.5 million DNTT-MSCs, and 55 receiving approximately 5 million DNTT-MSCs.

[0118] Study population and administration procedure The study population included adults with chronic motor impairment caused by an ischemic stroke event that occurred 6 to 90 months (7.5 years) prior to the stroke. The 6-90 month post-stroke interval was selected based on studies showing that over 90% of ischemic stroke subjects remained stable up to 90 days post-stroke. All subjects had an mRS score of 2-4 at the time of screening. Subjects with an mRS score of less than 2 were not considered to have sufficient disability to justify the risk of such a procedure, while subjects with an mRS score greater than 4 were considered to have an increased risk due to severe disability.

[0119] As previously discussed, a total of 158 subjects were randomized as part of the trial into two groups: 51 subjects to receive sham surgery and 107 subjects to receive treatment (either approximately 2.5 million or approximately 5 million DNTT-MSCs). Of the 158 subjects, 77 had previously suffered a small volume ischemic stroke or a stroke with an ischemic central volume of less than 50 cc (based on CT or MRI scans included as part of the subjects' medical history).

[0120] Participants received either a sham surgery or intracranial administration of approximately 250 million DNTT-MSCs or approximately 5 million DNTT-MSCs. DNTT-MSCs were administered stereotactically via a single burr-hole craniotomy.

[0121] A single burr-hole craniotomy (approximately 1 cm to 1.5 cm) was performed under local anesthesia and sedation. The dura mater of the subject was opened and an implantable cannula was inserted. Five 20 μL volumes of cell suspension containing DNTT-MSCs were slowly injected into five transplantation sites selected by stereotactic targeting (thus, a total of 100 μL of cell suspension was injected into five transplantation sites along a single deposition trajectory). This procedure was repeated for two other deposition trajectories with different trajectories inserted via the same burr-hole craniotomy (see, for example, Figure 2). Subjects receiving approximately 2.5 million DNTT-MSCs had a target concentration of approximately 8.5*10 6 For subjects receiving approximately 5 million DNTT-MSCs by injection of a cell suspension containing cells / mL, the target concentration is approximately 17*10 6 A cell suspension containing cells / mL was injected.

[0122] The sham surgery group underwent the same stereotactic planning procedure, partial thickness cranial plate burr hole, and cranial suture, but without dura mater penetration or cell transplantation, under local anesthesia and sedation. The sham surgery procedure was designed to mimic the procedure performed on the treatment group as closely as possible.

[0123] DNTT-MSC or SB623 cells are divided into 8 x 10 units per 1 mL unit volume. 6 A sterile cell suspension containing cells / mL or more was prepared and cryopreserved in CryoStore® freezing medium in a 2 mL vial. The cryopreserved cells were thawed, washed, centrifuged, and resuspended in Plasma-Lyte A to achieve the target concentration described above. Before administration, a post-release test was performed on the injection-formulated dose, and the injection-formulated dose was administered to the subjects within 3 hours of the post-release test.

[0124] Clinical trial results Regarding safety, DNTT-MSC administered intracranially to subjects at doses of approximately 2.5 million cells or approximately 5 million cells was well-tolerated in all subjects with stroke-induced chronic motor impairment.

[0125] Regarding efficacy, this study did not meet the primary efficacy endpoint of a statistically significant difference in the proportion of subjects in the "combined treatment group" (subjects administered either approximately 2.5 million cells or approximately 5 million cells) who showed at least a 10-point improvement in total FMMS compared to the control / sham group at week 24 (approximately 6 months). Response rates were similar in the sham surgery group (approximately 15.6%) and the combined treatment group (approximately 15.0%), and based on statistical analysis using generalized linear mixed model (GLMM) analysis or logistic regression analysis, there was no statistically significant difference between the combined treatment group response rate and the sham surgery response rate.

[0126] Retrospective analysis of clinical trial data A retrospective analysis was conducted on the data collected from the aforementioned clinical trials.

[0127] Regarding efficacy, one unexpected result discovered by the applicant from a retrospective analysis was the statistically significant difference (p-value = 0.02) in the proportion of subjects in the combination treatment group (i.e., subjects administered either approximately 2.5 million cells or approximately 5 million cells) that showed clinically significant improvement in motor function compared to the sham / control group in subjects with small volume stroke (e.g., ischemic central volume less than 50 cc). Considering these unexpected findings, administration of DNTT-MSC appears to be an effective treatment for subjects suffering from chronic motor impairment caused by small volume ischemic stroke.

[0128] Furthermore, another unexpected result discovered by the applicant was the statistically significant difference (p-value = 0.05) in the proportion of subjects in the combined treatment group that showed clinically significant improvement in motor function compared to the sham / control group, in subjects who suffered stroke in a brain region / lobe other than the parietal lobe (i.e., the ischemic portion of the stroke was located in a brain region / lobe other than the parietal lobe).

[0129] Furthermore, another unexpected result discovered by the applicant was the observation of a larger statistically significant difference (p<0.01) in the proportion of subjects in the combined treatment group that showed clinically significant improvement in motor function compared to the sham / control group, in subjects with small-volume strokes in brain regions other than the parietal / lobar region (e.g., ischemic central volume less than 50 cc).

[0130] As part of a retrospective analysis, participants were considered “composite responders” if they demonstrated a clinically significant improvement in motor function by achieving at least one of the following MCID thresholds ("MCID thresholds"): 1. Improvement of at least 9 points in the change from baseline in total FMMS ("CFB") at week 24 ("W24") (W24 CFB total FMMS improvement of 9 points or more). 2. At least 6 points of total FMMS UE improvement in CFB at week 24 (W24 CFB FMMS UE improvement of 6 points or more) 3. At least 4 points of total FMMS LE improvement in CFB at week 24 (W24 CFB FMMS LE improvement of 4 points or more)

[0131] Tables 1 and 2 below show the composite response rate, mean baseline score, and mean stroke volume for the entire population and the small-volume stroke subpopulation, respectively.

[0132] [Table 1]

[0133] [Table 2]

[0134] Table 3 below shows the delta composite response rates and their associated p-values ​​for the entire population and for the small stroke subgroup. For the purposes of this disclosure, "delta composite response rate" refers to the difference in response rates between the treatment group and the control group.

[0135] [Table 3]

[0136] As shown in Tables 1-3 above, the subgroup of the population with small-volume stroke (e.g., patients with ischemic stroke with a central ischemic volume of less than 50 cc) had a significantly higher response rate (approximately 49%) in the treatment group compared to the control group (approximately 19%). The delta composite response rate of approximately 30% for this subgroup was determined to be statistically significant (p-value = 0.02) based on GLMM and logistic regression analysis. This is in stark contrast to the delta composite response rate of 8% for the entire population, which was determined to be not statistically significant (p-value = 0.42).

[0137] Retrospective analysis also determined that this statistically significant delta composite response rate was driven by the volume of the stroke in the subjects, rather than by the baseline mRS of the subjects. More specifically, the volume of the stroke in the subjects had no significant effect (and virtually no effect) on the baseline mRS of the subjects.

[0138] Figures 3A-3C are plots showing the relationship between baseline FMMS, baseline mRS, and stroke volume (e.g., ischemic center volume in stroke patients) for the 2.5 million (2.5M) dose group, the 5 million (5.0M) dose group, and the control group. Figure 3A shows the relationship between baseline FMMS and stroke volume for the 2.5M dose group, the 5.0M dose group, and the control group. Spearman correlation was calculated between these two factors, yielding a correlation of -0.23 with a p-value of 0.002. This indicates that the baseline FMMS of the subjects was significantly correlated with the stroke volume of the subjects, meaning that subjects with smaller stroke volumes generally exhibited better motor function than subjects with larger stroke volumes.

[0139] Figure 3B shows the relationship between baseline mRS and baseline FMMS for the 2.5M dosage group, the 5.0M dosage group, and the control group. The Spearman correlation between these two factors was calculated, yielding a correlation of -0.34 with a p-value of less than 0.001. This indicates that the baseline mRS of the subjects was significantly correlated with their baseline FMMS, meaning that subjects with lower baseline motor function generally experienced greater disability than those with higher baseline motor function.

[0140] Figure 3C shows the relationship between baseline mRS and stroke volume for the 2.5M dosage group, the 5.0M dosage group, and the control group. The Spearman correlation between these two factors was calculated, yielding a correlation of 0.06 with a p-value of 0.44. This indicates that the baseline mRS of the subjects did not significantly correlate with the stroke volume of the subjects.

[0141] Medication Dosage Comparison Table 4 below shows the composite response rate, mean baseline score, and mean stroke volume for all subjects, segmented by administered dose, control group, and overall population.

[0142] [Table 4]

[0143] Table 5 below shows the composite response rate, mean baseline score, and mean stroke volume for subjects with small-volume stroke (e.g., patients with ischemic stroke with an ischemic central volume of less than 50 cc), segmented by administered dose, control group, and overall population.

[0144] [Table 5]

[0145] Table 6 below shows the composite response rate, mean baseline score, and mean stroke volume for subjects with a stroke volume of 50 cc or more (e.g., patients with ischemic stroke with an ischemic central volume of 50 cc or more), divided by administered dose, control group, and overall population.

[0146] [Table 6]

[0147] Table 7 below shows the composite response rate, mean baseline score, and mean stroke volume for subjects with a stroke volume of 100cc or more (e.g., patients with ischemic stroke with an ischemic central volume of 100cc or more), divided by administered dose, control group, and overall population.

[0148] [Table 7]

[0149] Tables 4-7 above show that when the treatment population was subdivided by dosage (e.g., 2.5M cell dosage or 5.0M cell dosage) and compared to the control population, subjects administered approximately 2.5 million DNTT-MSCs responded better than subjects administered approximately 5 million DNTT-MSCs. This was maintained across all stroke volume subpopulations, as shown in Tables 5-7.

[0150] Figure 4 shows that across almost all target stroke volumes, the delta composite response rate (i.e., the difference in the composite response rate between the treatment group and the control group) for the 2.5M dosage group was higher than that for the 5.0M dosage group. As can be seen from Figure 4, the difference in delta composite response rates is particularly pronounced in subjects with small volume strokes (e.g., patients with ischemic stroke with an ischemic central volume of less than 50 cc). These results are unexpected and suggest that a dosage of 2.5 million DNTT-MSCs may be a more therapeutically effective dose in the treatment of chronic ischemic stroke than a dosage of 5 million DNTT-MSCs, even though the latter contains more cells. This may be particularly true in subjects with small volume strokes (e.g., patients with ischemic stroke with an ischemic central volume of less than 50 cc).

[0151] The graph in Figure 4 excludes results for subjects with a stroke volume of less than 2cc (e.g., patients with ischemic stroke with an ischemic central volume of less than 2cc) because, from a relative perspective, the number of subjects with a stroke volume of less than 2cc in the control group significantly exceeded the number of such subjects in the 5.0M dosage group.

[0152] Tables 5-7 show that, with the exception of the small-volume stroke subgroup (e.g., patients with ischemic stroke with a central ischemic volume of less than 50 cc) (see Table 5), the control group actually responded better than either the dosage group for subjects with a stroke volume of 50 cc or more (see Table 6) or 100 cc or more (see Table 7). These results suggest that DNTT-MSC may not be very effective in treating chronic stroke patients with a central ischemic volume greater than 50 cc.

[0153] Location of the stroke Retrospective analysis also investigated the effectiveness of treatment based on stroke location. In the study population, stroke (or, more appropriately, the ischemic center of such stroke) was recorded in the following regions / regions of the brain: (1) subcortical white matter, (2) subcortical gray matter, (3) cortical frontal lobe, (4) cortical parietal lobe, and (5) cortical temporal lobe. Figure 5 shows these regions / regions of the brain along with other regions / regions of the brain.

[0154] As previously mentioned, one unexpected result discovered by the applicant was the statistically significant difference (p-value = 0.05) in the proportion of subjects in the combined treatment group that showed clinically significant improvement in motor function compared to the sham / control group, among subjects who suffered stroke in brain regions other than the corticoparietal / lobar region.

[0155] Furthermore, another unexpected result discovered by the applicant was the observation of a larger statistically significant difference (p-value <0.01) in the proportion of subjects in the combined treatment group that showed clinically significant improvement in motor function compared to the sham / control group, in subjects with small-volume strokes in brain regions other than the corticoparietal / lobar region (e.g., ischemic central volume less than 50 cc).

[0156] Table 8 below shows the delta composite response rate and its associated p-value for the entire population and for the control group that did not suffer a stroke in the corticoparietal region.

[0157] [Table 8]

[0158] Table 9 below shows the various delta response rates and scores at 24 weeks for a small-volume stroke group (ischemic central volume less than 50 cc) that either suffered or did not suffer a stroke in the corticoparietal region.

[0159] [Table 9]

[0160] As shown in Table 8 above, the study group that did not experience a stroke in the cortical parietal region had a significantly higher response rate (approximately 42%) in the treatment group compared to the control group (approximately 19%). The delta composite response rate of approximately 23% for this subgroup was determined to be statistically significant (p-value = 0.05) based on GLMM or logistic regression analysis.

[0161] Table 9 above shows that the highest delta composite response rate (45.8%) was observed in the group with small-volume stroke (ischemic central volume less than 50 cc) that did not suffer from a stroke in the corticoparietal region.

[0162] Considering these unexpected findings, DNTT-MSC administration appears to be an effective treatment for patients suffering from chronic motor impairment caused by ischemic stroke in which the central ischemic area was not located in the corticoparietal region of the patient. Furthermore, DNTT-MSC administration may be an even more effective treatment for patients suffering from chronic motor impairment caused by small-volume (central volume less than 50cc) ischemic stroke in which the central ischemic area was not located in the corticoparietal region of the patient.

[0163] Figure 6 is a reference table created as part of a retrospective analysis, containing information on stroke location, baseline characteristics, and delta response rate for study populations segmented by population percentage.

[0164] Figure 7 is a plot showing the delta composite response rate from Figure 6, plotted against the population percentage. As shown in Figure 6, point P1 on the graph corresponds to approximately 9% of the population and includes subjects with cerebellar stroke volume (ischemic central volume less than 50 cc) where the stroke did not occur in the subcortical gray matter or cortical frontal region; point P2 on the graph corresponds to approximately 11% of the population and includes subjects with cerebellar stroke volume where the stroke did not occur in the cortical parietal region; point P3 on the graph corresponds to approximately 25% of the population and includes subjects with cerebellar stroke volume where the stroke did not occur in the cortical temporal region; and point P4 on the graph corresponds to approximately 31% of the population and includes subjects with cerebellar stroke volume where the stroke did not occur in either the cortical parietal or cortical temporal region. Point P5 on the graph represents approximately 47% of the population and includes all study subjects with cerebellar stroke volume (regardless of stroke location); point P6 on the graph represents approximately 51% of the population and includes all study subjects in which the stroke did not occur in the cortical parietal region; point P7 on the graph represents approximately 63% of the population and includes all study subjects in which the stroke occurred at least partially in the subcortical white matter region; point P8 on the graph represents approximately 79% of the population and includes all study subjects who were administered cells processed in less than 180 minutes (considered a short cell processing time); and point P9 on the graph represents approximately 97% of the population (essentially the entire study population).

[0165] Figures 6 and 7 show that the group divisions represented by points P5 (cerebellar stroke volume) and P6 (no stroke in the corticoparietal region) were the only two group divisions in which response rates, including the delta composite response rate, were calculated from nearly 50% of the entire study population, and both delta composite response rates were statistically significant.

[0166] Of the 83 subjects without a stroke in the corticoparietal region, 55 also had a small-volume stroke, so it should be noted that there is a significant overlap between the P5 (cerebellar stroke volume) and P6 (no stroke in the corticoparietal region) groups.

[0167] Progress in composite responders at 48 weeks To determine whether subjects who were W24 composite responders showed improvement in their disability scores at 24 weeks (i.e., at 48 weeks or W48), clinical trial data for mRS CFB at W48 were also analyzed for the entire population and for subpopulations with cerebellar stroke volume (ischemic central volume < 50 cc).

[0168] Tables 10 and 11 below show the changes in W48 mRS for the entire population and for subpopulations of cerebellar stroke, respectively. It is important to note that a decrease in mRS in a subject indicates an improvement in the degree or level of disability in that subject.

[0169] [Table 10]

[0170] [Table 11]

[0171] As shown in Tables 10 and 11 above, composite responders in the cerebellar stroke volume (ischemic central volume less than 50 cc) subgroup exhibited better delta mRS CFB at 48 weeks than the overall population. Since improvement in the subject's motor response is essential for improvement in the subject's disability (known as the "read-lag effect"), the results indicate that the read-lag effect was present in composite responders in both the overall population and the cerebellar stroke volume subgroup at 48 weeks, and that the effect was more pronounced in composite responders in the cerebellar stroke volume subgroup.

[0172] Multiple embodiments are described. Nevertheless, it will be understood by those skilled in the art that various changes and modifications may be made to this disclosure without departing from the spirit and scope of the embodiments. Elements of mechanisms, devices, apparatus and methods shown in any embodiment are illustrative to a particular embodiment and may be used in combination with or differently to other embodiments in this disclosure. For example, the steps for any method illustrated in the drawings or described in this disclosure do not require a specific sequence or set of steps shown or described in order to achieve the desired result. Furthermore, other steps may be given, and steps or operations may be deleted or omitted from the described methods or steps to achieve the desired result. Furthermore, any component or part of any apparatus or mechanism described in this disclosure or illustrated in the drawings may be removed, deleted or omitted to achieve the desired result. Furthermore, certain components or parts of mechanisms, devices or apparatus shown or described herein are omitted for brevity and clarity.

[0173] Therefore, other embodiments are within the scope of the following claims, and this specification and / or the figures may be considered illustrative rather than restrictive.

[0174] Each of the individual variations or embodiments described and shown herein has individual components and characteristics that can be readily separated from or combined with any of the characteristics of other variations or embodiments. Modifications may be made to adapt specific situations, substances, compositions, methods, procedures, or steps to the object, spirit, or scope of the present invention.

[0175] The methods enumerated herein may be carried out not only in the order in which the events are enumerated, but also in any logically possible order of the enumerated events. Furthermore, additional steps or tasks may be given, or steps or tasks may be omitted, in order to achieve the desired result.

[0176] Furthermore, if a range of values ​​is given, each value that lies between the upper and lower limits of that range, as well as any other values ​​that are specified or lies between the specified range, are included within the present invention. Also, any optional feature of the variations of the present invention described may be shown independently and claimed, or may be combined with any one or more of the features described herein. For example, a description of the range 1 to 5 should be considered to have disclosed sub-ranges such as 1 to 3, 1 to 4, 2 to 4, 2 to 5, 3 to 5, and individual numbers within those ranges, such as 1.5, 2.5, etc., and any whole or partial increments between them.

[0177] All existing subject matter (e.g., publications, patents, patent applications) referenced herein are incorporated by reference in their entirety, except where such subject matter may conflict with the subject matter of the present invention (i.e., where the subject matter present herein takes precedence). The referenced items are provided solely for disclosure prior to the filing date of this application. Nothing herein should be construed as acknowledging that the present invention does not have prior rights to such prior inventions.

[0178] A reference to a single item includes the possibility of multiple references to the same item. More specifically, as used herein and in the appended claims, the singular forms “a,” “an,” “the,” and “the” include multiple references unless the context otherwise explicitly specifies. It should be further noted that claims may be drafted to exclude optional elements. Thus, this statement is intended to function as an antecedent for the use of exclusive technical terms such as “alone,” “only,” and similar terms, or for the use of “negative” limitation, in relation to the enumeration of claim elements. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains.

[0179] A reference to the phrase “at least one of” means any combination of one or more of the items or components (or an enumerated list of items or components) that such a phrase modifies. For example, the phrase “at least one of A, B, and C” means (i) A, (ii) B, (iii) C, (iv) A, B, and C, (v) A and B, (vi) B and C, or (vii) A and C.

[0180] For understanding the scope of this disclosure, the term “including” and its derivatives are intended to be open-ended terms that, when used herein, specify the presence of specified features, elements, components, groups, integers, and / or processes, but do not exclude the presence of other unspecified features, elements, components, groups, integers, and / or processes. The foregoing also applies to terms having similar meanings to “including,” “having,” and their derivatives. Furthermore, the terms “part,” “section,” “part,” “component,” “element,” or “component” may have a dual meaning when used in the singular, referring to a singular part or a plural part. When used herein, the following directional terms “forward, backward, upward, downward, vertical, horizontal, below, lateral, lateral, and vertical” and any other similar directional terms refer to the location of a part of the equipment or facility, or the direction of a part of the equipment or facility being moved or relocated.

[0181] Finally, when used herein, degree terms such as “substantially,” “about,” and “approximately” mean the specified value, or a reasonable deviation from the specified value (e.g., a deviation of up to ±0.1%, ±1%, ±5%, or ±10%, where such variation is appropriate), and as a result, the final result does not change significantly or substantially. For example, “about 1.0 cm” may be interpreted as “1.0 cm” or “0.9 cm to 1.1 cm.” When degree terms such as “about” or “approximately” are used to refer to a number or value that is part of a range, the term may be used to modify both the minimum and maximum number or value.

[0182] This disclosure is not intended to be limited to the specific forms shown, but is intended to cover alternatives, modifications, and equivalents to the variations or embodiments described herein. Furthermore, the scope of this disclosure fully encompasses other variations or embodiments that may become apparent to those skilled in the art in consideration of this disclosure. (References) TIFF0007914933000012.tif191170 [Explanation of symbols]

[0183] 100 ways 102 Task 104 Task 106 Task 200 central ischemia 202 Ischemic Penumbra 204 Deposition site 206A First deposition trajectory 206B Second deposition trajectory 206C Third deposition trajectory

Claims

1. A composition for treating small volume ischemic stroke, A therapeutically effective amount of cells, which are mesenchymal stem cells transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD), or cultures of mesenchymal stem cells transiently transfected with a polynucleotide encoding the NICD, and Pharmacologically acceptable carrier or diluent Includes, A small volume ischemic center is an ischemic center having an ischemic center volume ranging from 2 cubic centimeters (cc) to 50 cc. A composition comprising administering a therapeutically effective amount of cells intracranially only when the ischemic central volume is between 2cc and 50cc, for the treatment of small-volume ischemic stroke.

2. The composition according to claim 1, wherein the polynucleotide does not encode a full-length notch protein.

3. The composition according to claim 1, wherein mesenchymal stem cells are transiently transfected with a plasmid containing a polynucleotide encoding NICD.

4. The composition according to claim 1, wherein the therapeutically effective amount of cells is approximately 2.5 million to 4 million cells.

5. The cell suspension is packaged in a sealed vial, with a volume of approximately 0.3 mL, and approximately 8.5*10 6 The composition according to claim 4, having a cell concentration of cells / mL.

6. The composition according to claim 1, wherein the therapeutically effective amount of cells is approximately 5 million cells.

7. The cell suspension is packaged in a sealed vial, with a volume of approximately 0.3 mL, and approximately 17.0 * 10 6 The composition according to claim 6, having a cell concentration of cells / mL.

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