Multi-chimeric cells, and therapies for transplantation and treatment of immunodeficiency and genetic disorders

Abstract

Multichimeric cells created by ex vivo fusion of three or more types of hematopoietic stem cells, mesenchymal stem cells, myoblasts, pericytes, or satellite cells, or combinations thereof, from three or more different donors are provided for use in transplantation therapy and the treatment of immune deficiencies and genetic disorders.

Description

[Technical Field]

[0001] introduction This application claims priority to U.S. Provisional Patent Application No. 62 / 591,397, filed November 28, 2017 (the contents of which are incorporated herein by reference in their entirety).

[0002] This invention was made with government support under authorization number W81XWH-13-2-0053 granted by the Department of Defense. The government has certain rights in this invention. [Background technology]

[0003] background Stem cell transplantation is a promising new strategy for treating many human disorders. Hematopoietic stem cell (HSC) transplantation is the most promising example of the curative potential of stem cell-based therapies. While autologous HSCs are not a viable treatment option for many hematological diseases, the availability of allogeneic stem cell grafts is limited due to a lack of related compatible donors. In addition, unrelated compatible HSC transplantation is hampered by often severely high mortality and morbidity rates due to the toxic effects of conditioning regimens and graft-versus-host disease (GvHD). Overcoming the barrier of histocompatibility without the risk of GvHD and improving engraftment rates, efficacy, and transplant feasibility would greatly increase the application of HSC transplantation as a select new treatment modality for many, often incurable, diseases.

[0004] Chimeric cells have been developed using various ex vivo fusion methods (Siemionow, et al. (2012) Ann. Plast. Surg. 69(5):575-9; Cwykiel & Siemionow (2015) Plastic and Reconstructive Surgery: Experimental Models and Research Design, Siemionow (Ed.), Springer-Verlag London, Ltd., Chapters 71-72). Chimeric cell therapy applied to various experimental models, including vascularized composite allotransplantation (VCA), irradiation, and animal models of muscular dystrophy, is described (Arslan, et al. (2007) Microsurgery 27:190-9; Kulahci, et al. (2010) Transplantation 90(8):843-52; Hivelin, et al. (2016) Arch. Immunol. Ther. Exp. (Warsz). 64(4):299-310; Siemionow, et al. (2016) Microsurgery 36(8):676-683). [Overview of the project]

[0005] Summary of the present invention The present invention relates to a multi-chimeric cell composed of a fusion of three or more hematopoietic stem cells, mesenchymal stem cells, myoblasts, pericytes, satellite cells, or combinations thereof, from three or more different donors. In one embodiment, the hematopoietic stem cells are isolated from bone marrow, umbilical cord blood, peripheral blood, or combinations thereof. In another embodiment, the donors are related, unrelated, or a combination thereof. In some embodiments, the fusion is a combination of hematopoietic stem cells and two or more cells independently selected from hematopoietic stem cells and mesenchymal stem cells. In another embodiment, the fusion is a combination of hematopoietic stem cells and two or more cells independently selected from hematopoietic stem cells, mesenchymal stem cells, and pericytes. In yet another embodiment, the fusion is a combination of myoblasts and two cells independently selected from mesenchymal stem cells, myoblasts, and satellite cells. In specific embodiments, the fusion is a combination of hematopoietic stem cells, mesenchymal stem cells, and pericytes; three types of hematopoietic stem cells; myobacteria, mesenchymal stem cells, and satellite cells; or three types of myoblast cells. Kits are also provided that include a fusogenic agent and three or more donor cells selected from the group of hematopoietic stem cells, mesenchymal stem cells, myobacteria, pericytes, and satellite cells from three or more different donors.

[0006] The present invention also provides a method for treating immunodeficiency or hereditary disorders by administering an effective amount of the multi-chimeric cells of the present invention to a subject requiring treatment. In some embodiments, the multi-chimeric cells are administered by intraosseous, intravenous, or intramuscular injection. In other embodiments, the immunodeficiency or hereditary disorder is selected from bone marrow failure, adenosine deaminase (ADA) deficiency, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia telangiectasia, muscular dystrophy, type 1 diabetes mellitus, Gaucher disease, leukemia, aplastic anemia, sickle cell anemia, lymphoma, and multiple myeloma. In embodiments directed toward the treatment of muscular dystrophy, the multi-chimeric cells are preferably composed of a fusion of myofibril cells and two or more cells independently selected from mesenchymal stem cells, myofibril cells, and satellite cells. In embodiments directed towards the treatment of sickle cell anemia, the multi-chimeric cells are preferably fusions of hematopoietic stem cells and two or more cell types independently selected from hematopoietic stem cells and mesenchymal stem cells.

[0007] The present invention further provides a method for preventing the development of GvHD or reducing the severity of GvHD in subjects undergoing allogeneic hematopoietic stem cell transplantation or organ transplantation by administering an effective amount of the multi-chimeric cells of the present invention to the subject. In this aspect of the present invention, the multi-chimeric cells are preferably fusions of hematopoietic stem cells and two or more cells independently selected from hematopoietic stem cells and mesenchymal stem cells. [Brief explanation of the drawing]

[0008] Simple description of the drawing [Figure 1]Figure 1 shows the ex vivo fusion procedure for creating human multi-chimeric cells (MCCs). Human hematopoietic stem cells (HSCs) are obtained from at least three related and / or unrelated donors. Prior to fusion, the cells are fluorescently labeled with PKH26 (red), PKH67 (green), or eFluor670 (magenta) dyes, respectively. Cell fusion of the fluorescently labeled cells is performed using polyethylene glycol (PEG). After fusion, the triple-stained (PKH26, PKH67, and eFluor670) cells are selected via fluorescence-activated cell sorting (FACS). Multi-chimeric (trimera) cells are delivered to transplant patients or immunocompromised patients via systemic intraosseous injection. [Modes for carrying out the invention]

[0009] Detailed description of the present invention Multi-chimeric cells for transplantation therapy and for treating patients with immunodeficiency and genetic disorders have now been developed. The multi-chimeric cells of this invention were created by the fusion of at least three (trimeras) or more (quadri-chimeras, multi-generation chimeras, etc.) human hematopoietic stem cells (autologous and / or allogeneic) derived from related and / or unrelated donors (Figure 1). Specifically, multiple-generation chimeric cells were created via PEG-mediated ex-vivo fusion of hematopoietic origin cells: hematopoietic stem cells (HSCs), umbilical cord blood (UCB) cells, or bone marrow (BM) cells, or combinations of HSCs and / or UCBs and / or BMs. The resulting multi-chimeric cells expressed HLA antigens specific to each of the multiple cell donors on their surface. The microenvironment established by multi-chimeric cells reduces the side effects of immunosuppression and pre-treatment regimens and enhances HSC engraftment without GvHD. This approach will enable the treatment of various immunological and genetic disorders.

[0010] Consequently, the present invention relates to multi-chimeric cells, which are the product of ex vivo fusion of three or more hematopoietic stem cells, mesenchymal stem cells, myobiocytes, pericytes, or satellite cells, or combinations thereof, from three or more different donors, wherein the fusion is carried out in the presence of an exogenous membrane fusion agent. For the purposes of the present invention, a “multi-chimeric cell” is a cell constructed from the cell fusion or hybridization (whole cell hybridization) of three or more living cells (parent cells). While the multi-chimeric cells of the present invention are referred to as “multi-chimeric cells,” the term “chimeric cell” is intended to mean a single cell or a group of cells.

[0011] "Hematopoietic stem cells" or "HSCs" refer to clonogenic, self-renewing pluripotent cells that are ultimately capable of differentiating into all cell types of the hematopoietic system, including B cells, T cells, NK cells, lymphoid dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes, and erythroid cells. Marker phenotypes useful for identifying HSCs are generally known in the art. For human HSCs, the cellular marker phenotype is preferably CD34 + CD38 - CD90 (Thy1) + Lin - . For mouse HSCs, exemplary cellular marker phenotypes include Sca-1 + CD90 + (see, for example, Spangrude, et al. (1988) Science 1:661-673) or c-kit + Thy lo Lin - Sca-1 + (see Uchida, et al. (1998) J. Clin. Invest. 101(5):961-966). Alternative HSC markers such as aldehyde dehydrogenase (see Storms, et al. (1999) Proc. Nat'l Acad. Sci. 96:9118-923), AC133 (see Yin, et al. (1997) Blood 90:5002-5012), and CD150 (SLAM) (see Kiel (2005) Cell 121(7):1109-1121) may also find advantageous use. In certain embodiments, the hematopoietic stem cells are isolated from bone marrow, umbilical cord blood, and / or peripheral blood.

[0012] Mesenchymal stem cells, or MSCs, are cells that can give rise to connective tissue, bone, cartilage, and other cells in the circulatory and lymphatic systems. Mesenchymal stem cells are found in a portion of the embryonic mesoderm, consisting of loosely packed, spindle-shaped or star-shaped unspecialized cells. Mesenchymal stem cells can be obtained by conventional methods and marked with the following markers: CD29, CD31. - CD34 - CD44 CD45 - CD51, CD73, CD90 / Thy-1, CD105, CD166, Integrin α1, PDGF Rα, Nestin, Sca-1 + They can be identified by one or more of the following: SCF R / c-Kit, STRO-1, and VCAM-1. In some embodiments, mesenchymal stem cells are derived from or obtained from bone marrow (BM) or adipose tissue (ASC). In specific embodiments, mesenchymal stem cells are derived from or obtained from human bone marrow.

[0013] In the art, the term "myoblast" conventionally refers to primordial muscle cells that have the potential to develop into muscle fibers. Myoblasts are characterized by the expression of desmin and CD56 and can be obtained from fetal or adult tissue using methods known in the art. For example, see WO 93 / 03768, which discloses the isolation of myoblasts from a crude cell population by flow cytometry (e.g., FACs). Alternatively, myoblasts can be obtained by propagating myoblasts in culture from muscle biopsy cells. For example, see Springer, et al. (1997) In: Current Human Genetics. Unit 13.4, Boyle Ed. John Wiley & Sons, NY.

[0014] As used herein, a "pericyte" is a pluripotent cell associated with the wall of microvessels. Previous studies have shown that pericytes can differentiate into various cell types including adipocytes, chondrocytes, fibroblasts, and macrophages. Pericytes are characterized by the expression of one of the following markers: vimentin, neuro-glial 2 (NG2), platelet-derived growth factor receptor beta (PDGFR-β), and α-smooth muscle actin (α-SMA).

[0015] "Satellite cells" are mononuclear myoprogenitor cells of mature muscle fibers that are responsible for postnatal muscle growth and regeneration in vivo. Markers used to characterize satellite cells include, but are not limited to, M-cadherin, CD34, and c-met. Satellite cells may also be isolated from muscle tissue by known methods (see, for example, US 2007 / 0224168).

[0016] As used herein, a donor is a subject that provides cells used for preparing the multichimeric cells of the present invention. The donor is preferably a healthy donor, that is, an individual not suffering from any hereditary disorder or hereditary disease. Further, the donor may be any mammal including human, mouse, rat, dog, cat, horse, and the like. In specific embodiments, the donor is a human.

[0017] The donor may be a genetic relative (e.g., a parent or sibling) of the subject or a cell bank donor. Accordingly, in some embodiments, one or more cells used to generate the multichimeric cell are autologous. In other embodiments, all of the cells used to generate the multichimeric cell are autologous. In further embodiments, one or more cells used to generate the multichimeric cell are allogeneic. In still other embodiments, all of the cells used to generate the multichimeric cell are allogeneic.

[0018] As used herein, when referring to the isolation and transplantation of cells, "autologous" refers to cells in which the donor and recipient are the same individual. Thus, autologous cells are recovered from a subject and then returned to the same subject. In contrast, "allogeneic" cells are cells from genetically non-identical individuals of the same species as the donor and recipient. Comparatively, "xenogeneic" cells are cells from different species as the donor and recipient.

[0019] As noted, the multi-chimeric stem cell of the present invention is a fusion of three or more types of cells selected from hematopoietic stem cells, mesenchymal stem cells, myogenic cells, pericytes, or satellite cells, or a combination thereof. In some embodiments, the multi-chimeric stem cell is a fusion comprising or consisting of three types of cells selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, myogenic cells, pericytes, and satellite cells. In other embodiments, the multi-chimeric stem cell is a fusion comprising or consisting of 4, 5, 6, 7, 8, 9, or 10 types of cells selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, myogenic cells, pericytes, and satellite cells. In embodiments directed to a fusion of three types of cells, specific combinations of cells are provided in Table 1. [Table 1]

[0020] In embodiments directed to a fusion of four types of cells, combinations of four types of cells are provided in Table 2. [Table 2]

[0021] In some embodiments, multi-chimeric stem cells are ex vivo fusions of hematopoietic stem cells and two cell types independently selected from hematopoietic stem cells, mesenchymal stem cells, and pericytes. In specific embodiments, multi-chimeric stem cells are ex vivo fusions of hematopoietic stem cells and two cell types independently selected from hematopoietic stem cells and mesenchymal stem cells. In another embodiment, multi-chimeric stem cells are ex vivo fusions of hematopoietic stem cells, mesenchymal stem cells, and pericytes. In yet another specific embodiment, multi-chimeric stem cells are ex vivo fusions of three types of hematopoietic stem cells.

[0022] In another embodiment, a multi-chimeric stem cell is an ex vivo fusion of a myofibril and two cell types, or a combination thereof, independently selected from mesenchymal stem cells, myofibrils, and satellite cells. In a specific embodiment, a multi-chimeric stem cell is an ex vivo fusion of a myofibril, a mesenchymal stem, and a satellite cell. In yet another specific embodiment, a multi-chimeric stem cell is an ex vivo fusion of three myofibrils.

[0023] The cells used in the preparation of multi-chimeric stem cells of the present invention may be isolated and optionally purified. As used herein, the term “isolated” is intended to describe cells of interest that are in an environment different from the environment in which their elements naturally occur. As used herein, “purified” means cells that have been removed from the environment in which they were produced and that are at least 60% free, preferably 75% free, and most preferably 90% free of other components (those naturally associated with the cells, or otherwise associated with the cells during production).

[0024] The purification and / or identification of target cells can be achieved by any means known in the art, such as immunological means. Histochemical staining, flow cytometry, fluorescence-activated cell sorting (FACS), Western blot analysis, enzyme-linked immunosorbent assay (ELISA), density gradient separation (e.g., Ficol®, polysucrose 400), immunomagnetic bead separation, or a combination thereof may be used. Flow immunocytochemistry may be used to detect cell surface markers, and immunohistochemistry (e.g., of fixed cells) may be used for intracellular or cell surface markers. Western blot analysis may be performed on cell extracts. Enzyme-linked immunosorbent assay may be used on cell extracts or products secreted into culture medium. Antibodies for the identification of stem cell markers may be obtained from commercial sources, such as Chemicon International (Temecula, CA).

[0025] The multi-chimeric stem cells of the present invention are prepared by ex vivo contact of three or more cells from three or more different donors with an external membrane fusion agent, thereby promoting ex vivo fusion of the three or more different donor cells. “Ex vivo” means that the cells are manipulated outside of a living organism. Cell fusion is the process of combining three or more cells into one by fusing their plasma membranes. Multi-chimeric stem cells may be prepared using methods known in the art, including but not limited to exposure of cells to membrane fusion agents and fusion-promoting chemicals such as polyethylene glycol (PEG); the use of inactivated viruses such as Sendai virus; and the use of electrical stimulation. For a review of commonly used methods based on Sendai virus-induced cell fusion or polyethylene glycol (PEG)-induced cell fusion, see, for example, Kennett (1979) Methods Enzymol. 58:345–359. In short, the cells to be fused are incubated with a membrane fusion agent such as Sendai virus or PEG. Centrifugation or stirring may be used to promote the close juxtaposition of aggregates and cell membranes. Variables such as time, temperature, cell concentration, and membrane fusion agent concentration may be optimized for each cell combination. With regard to electrofusion, short electrical pulses pass through the cell mixture to stimulate fusion. See, for example, Neil & Zimmermann (1993) Methods Enzymol. 220:174-196. In one embodiment, multi-chimeric stem cells are prepared by polyethylene glycol cell fusion.

[0026] Prior to fusion, donor cells may or may not be cultured to increase their number. Furthermore, donor cells may or may not be labeled (e.g., with membrane dyes) to monitor donor cell fusion. For example, HSCs from a first donor are labeled with PKH26 (emitting at 567 nm), MSCs from a second donor are labeled with eFluor670 (emitting at 670 nm), and pericytes from a third donor are labeled with PKH67 (emitting at 502 nm) so that the fused cells can fluoresce at 502 nm, 567 nm, and 670 nm at fusion (Figure 1). To identify the multi-chimeric cells of the present invention, fused cells are selected via cell sorting, e.g., fluorescence-activated cell sorting (FACS). Once identified, the multi-chimeric cells may be cryopreserved, stored, and banked as cell lines. Alternatively, successful fusion of three or more cell types can be assessed by morphological, phenotypic, or genotypic characterization.

[0027] The multi-chimeric stem cells of the present invention are specifically used in treating immunodeficiency and hereditary disorders. Consequently, the present invention also provides a method for treating immunodeficiency or hereditary disorders in subjects in need, by administering to a subject a multi-chimeric stem cell or composition of the present invention containing multi-chimeric stem cells in an amount effective for treating the immunodeficiency or hereditary disorder. "Treating" a subject with a disease or disorder means performing one or more of the following: (a) reducing the severity of the disease; (b) delaying the progression of the disease or disorder; (e) inhibiting the worsening of the disease or disorder; (d) limiting or preventing the recurrence of the disease or disorder in a patient who has previously had the disease or disorder; (e) inducing regression of the disease or disorder; (f) improving or eliminating the symptoms of the disease or disorder; and (g) improving survival. In some embodiments, the methods of the present invention use a fusion of three or more hematopoietic stem cells, mesenchymal stem cells, or myofibrillar cells from three or more different donors.

[0028] Immunodeficiencies and hereditary disorders that may be treated according to the present invention include bone marrow failure, adenosine deaminase (ADA) deficiency, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia telangiectasia, muscular dystrophy, type 1 diabetes mellitus, Gaucher disease, leukemia, aplastic anemia, lymphoma, and multiple myeloma. In one embodiment, the immunodeficiency and hereditary disorder is selected from bone marrow failure, muscular dystrophy, sickle cell anemia, leukemia, and type 1 diabetes mellitus. In another embodiment, the hereditary disorder is muscular dystrophy.

[0029] Muscular dystrophy is a group of genetic disorders characterized by the progressive weakness and degeneration of the skeletal muscles that control movement. Examples of muscular dystrophy include Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreyfus muscular dystrophy, Fukuyama congenital muscular dystrophy, Miyoshi myopathy, Ulrich congenital muscular dystrophy, and Steinert muscular dystrophy. In one embodiment, the muscular dystrophy is Duchenne muscular dystrophy (DMD). In a specific embodiment, the muscular dystrophy is treated with myofibril cells and multi-chimeric cells, which are fusions of myofibril cells and two or more cell types independently selected from mesenchymal stem cells, myofibril cells, and satellite cells.

[0030] In another embodiment, an immunodeficiency or hereditary disorder is sickle cell anemia. According to this embodiment, sickle cell anemia is treated with hematopoietic stem cells and multi-chimeric cells, which are fusions of hematopoietic stem cells and two or more cell types selected from hematopoietic stem cells and mesenchymal stem cells.

[0031] To the extent that the multi-chimeric cells of the present invention provide tolerogenic and immunomodulatory effects in the context of organ and HSC transplantation, the present invention also encompasses a method for preventing the development of GvHD or reducing the severity of GvHD in subjects undergoing allogeneic hematopoietic stem cell transplantation or organ transplantation. As is known in the art, "graft-versus-host response" or "GVH" or "GVHD" refers to a cellular response that occurs when lymphocytes of different MHC classes are introduced into a host, resulting in a lymphocyte response to the host. According to this method, subjects undergoing allogeneic hematopoietic stem cell transplantation or organ transplantation are administered an amount of multi-chimeric cells effective in preventing the development of GvHD or reducing the severity of GvHD.

[0032] Multi-chimeric cells may be administered individually to a subject and / or incorporated as part of a hematopoietic stem cell transplant or organ transplant. Treatment with the multi-chimeric cells of the present invention may significantly reduce the incidence and severity of GvHD, prevent allogeneic transplant rejection, improve the immune response, and / or reduce / eliminate the need for lifelong immunosuppression in transplant patients. In this regard, the multi-chimeric cells of the present invention can be used to prevent the development of GvHD-related symptoms and signs in various organs and systems, including skin, nails, mouth, eyes, female genitalia, gastrointestinal tract, liver, lungs, muscles, fascia, joints, etc. In a specific embodiment, GvHD is treated with multi-chimeric cells, which are fusions of hematopoietic stem cells and two or more cells selected independently from hematopoietic stem cells and mesenchymal stem cells.

[0033] According to the treatment methods described herein, multi-chimeric cells or compositions containing such cells are administered to subjects requiring treatment. In some embodiments, combinations of multi-chimeric cells of the present invention may be administered. Multi-chimeric cells or combinations of cells may be administered by engraftment, where the cells are injected into the subject, for example, intravenously, intramuscularly, intraarterially, intraosseously, etc. In specific embodiments, multi-chimeric stem cells are administered by intraosseous, intravenous, or intramuscular injection. In some embodiments, administration is about 10 2 , 10 4 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 12 This involves engrafting one or more cells. The number of cells to be engrafted may be selected based on the administration route for cell engraftment and / or the severity of the disease (condition). Advantageously, the multi-chimeric stem cells of the present invention successfully engraft and complement genetic / immunogenic defects, as well as providing immune tolerance-inducing and immunomodulatory effects in the context of organ and HSC transplantation.

[0034] Compositions containing multi-chimeric cells or combinations of multi-chimeric cells may be prepared by combining the cells or combinations of cells with a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically acceptable” means molecular entities and compositions that, when administered to animals or humans, do not produce adverse, allergic, or other untoward reactions. As used herein, “pharmaceutically acceptable carrier” includes all kinds of solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the cells of this disclosure, its use in therapeutic compositions is intended. Pharmaceutical compositions may be administered by those skilled in the art, for example, depending on the intended route of administration, form of delivery, and desired dosage. For example, Remington, The Science and Practice of Pharmacy, 22 nd See Edition, 2012.

[0035] The compositions of the present invention can be incorporated into injectable formulations. The formulations may also include necessary, physiologically acceptable carrier materials, excipients, lubricants, buffers, surfactants, antibacterial agents, fillers (such as mannitol), antioxidants (such as ascorbic acid or sodium bisulfite), etc.

[0036] Acceptable formulation materials are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition may contain formulation materials to modify, maintain, or preserve, for example, the pH, molar osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release, adsorption or penetration rate of the composition.Suitable formulation materials are not limited to these, but include: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (such as borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, Flavoring agents and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, TRITON® (surfactant), trimamine, lecithin, cholesterol, or tyloxapal); stability enhancers This may include enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, or sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, for example, Remington, The Science and Practice of Pharmacy, Id.

[0037] The main vehicle or carrier in the pharmaceutical composition may be either aqueous or non-aqueous. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, perhaps supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. The pharmaceutical composition may contain Tris buffer at approximately pH 7.0–8.5 or acetate buffer at approximately pH 4.0–5.5, which may further include sorbitol or a suitable substitute. The pharmaceutical compositions of the present invention may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing a selected composition having a desired purity with any formulation agents (Remington, The Science and Practice of Pharmacy, Id.).

[0038] Cells or compositions may be provided by a sustained-release system, by encapsulation, or by an implantable device. Compositions may be administered by bolus injection, by continuous infusion, or by an implantable device. Compositions may also be administered locally via implantation of a membrane, sponge, or other suitable material (on which cells (single) or cells (plural) are absorbed or encapsulated). When an implantable device is used, the device may be implanted in any suitable tissue or organ. The injection is given as a single treatment and may be repeated to achieve the desired therapeutic effect (daily, weekly, monthly, yearly, etc.).

[0039] Methodologies for cell encapsulation that enable the transplantation of encapsulated cells in the treatment of Parkinson's disease (Tresco, et al. (1992) ASAIO J. 38:17-23) or amyotrophic lateral sclerosis (Aebischer, et al. (1996) Hum. Gene Ther. 7:851-860) have been described. In this embodiment, cells are encapsulated by a compound that forms a microporous membrane. The capsule containing the target cells (e.g., approximately 1 cm in length) may be prepared using a hollow microporous membrane made from polyethersulfone (PES) (Akzo Nobel Faser AG, Wuppertal, Germany; Deglon, et al. (1996) Hum. Gene Ther. 7:2135-2146).

[0040] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial activity can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. The inclusion of isotonic agents, such as sugars and sodium chloride, may also be desired.

[0041] Supplementary active ingredients may also be incorporated into the composition. The active compositions of this disclosure may include classic pharmaceutical preparations. Administration of these compositions would be via any common route, insofar as the target tissue is available through that route, in accordance with this disclosure.

[0042] As used herein, the terms “effective amount,” “effective dose,” or “therapeutically effective amount” refer to the amount of cells or composition sufficient to achieve the desired result. The amount of cells or composition constituting an “effective amount” or “therapeutically effective amount” may vary depending on the severity of the disease, the condition of the patient being treated, weight, or age, the frequency of administration, or the route of administration, but can be determined conventionally by those skilled in the art. A clinician may also titer the dosage or route of administration to obtain the optimal therapeutic effect.

[0043] The present invention is also directed toward kits for treating immunodeficiency or hereditary disorders and / or for preventing or mitigating GvHD. The kit is useful for practicing the inventive methods described herein. The kit is an assembly of materials or components comprising at least one of the inventive compositions. Thus, in some embodiments, the kit comprises a membrane fusion agent for performing ex vivo cell fusion, and three or more donor cells selected from the group of hematopoietic stem cells, mesenchymal stem cells, myobiocytes, pericytes, and satellite cells from different donors (e.g., donor cells from a cell bank), and optionally, materials for obtaining the donor cells as described above.

[0044] The exact nature of the components configured in an original kit depends on its intended purpose. For example, several embodiments are configured for the purpose of treating muscular dystrophy. In one embodiment, the kit is configured specifically for the purpose of treating human subjects. In another embodiment, the kit is configured specifically for the purpose of treating adult human subjects. In another embodiment, the kit is configured specifically for the purpose of treating children. In another embodiment, the kit is configured specifically for the purpose of treating DMD and may include myofibrillators and, independently, two or more cell types selected from mesenchymal stem cells, myofibrillators, and satellite cells. In another embodiment, the kit is configured specifically for the purpose of treating GvHD and may include hematopoietic stem cells and, independently, two or more cell types selected from hematopoietic stem cells and mesenchymal stem cells. In another embodiment, the kit is configured specifically for the purpose of treating sickle cell anemia and may include hematopoietic stem cells and, independently, two or more cell types selected from hematopoietic stem cells and mesenchymal stem cells. In another embodiment, the kit is configured specifically for the purpose of providing a dosage for continuous daily use. In another embodiment, the kit is configured for the purpose of providing specific, on-demand dosages. In a further embodiment, the kit is configured for veterinary applications treating subjects such as farm animals, domestic animals, and laboratory animals, but is not limited to these.

[0045] Instructions for use may be included in the kit. These instructions typically include tangible representations describing techniques employed in the use of the kit components that affect the desired outcome, such as treating muscular dystrophy, sickle cell anemia, or GvHD. Optionally, the kit may also include other useful components such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandage materials, or other useful tools readily recognizable to those skilled in the art.

[0046] The materials or components collected in the kit may be stored and provided to the practitioner in any convenient and suitable form that maintains their operability and practicality. For example, components may be in fused, dehydrated, or freeze-dried form; they may be provided at room temperature, refrigerated, or frozen temperature. Components are typically contained in suitable packaging material(s). As used herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit. The packaging material is preferably constructed in a well-known manner to provide a sterile environment free of contaminants. The packaging material used in the kit is one that is conventionally used in therapeutic procedures. As used herein, the term “package” refers to a suitable solid matrix or material, such as glass, plastic, paper, or foil, capable of holding individual kit components. The packaging material generally has external markings indicating the contents and / or the purpose of the kit and / or its components.

[0047] The following non-limiting examples are provided to further illustrate the present invention.

[0048] Example 1: Ex vivo generation of human multichimeric cells Multi-chimeric cells (MCCs) are generated by ex vivo cell fusion of human hematopoietic cells from three or more related and / or unrelated donors, as well as from one or more hematopoietic stem cell sources, namely bone marrow ("BM"), peripheral blood ("PB"), and / or umbilical cord blood ("UCB"), for example, from commercially available sources.

[0049] Mononuclear cells were isolated and purified from three unrelated donors. Subsequently, mononuclear cells from each of the three donors were pre-stained with different fluorescent cell membrane dyes (i.e., PKH 26, PKH 67, and CELLVUE® Claret (near-infrared fluorescent dye); Sigma Aldrich). The fluorescently labeled cells were mixed in a 1:1:1 ratio and fused using polyethylene glycol. Based on triple staining, the MCCs were sorted (purity 80-90%).

[0050] To confirm fusion, triple-labeled (PKH26, PKH67, and CELLVUE® Claret) chimeric cells were evaluated using confocal microscopy and flow cytometry. The viability, genotypic, and phenotypic stability of the multi-chimeric cells were assessed using standard assays. In particular, short tandem repeat PCR was used for genotypic characterization. These analytical results are presented in Table 3. [Table 3]

[0051] Currently, there are no available treatments to support organ and HSC transplantation without the side effects of immunosuppression and the development of GvHD. Multi-chimeric cell therapy would have a significant impact on eliminating the side effects of immunomodulatory protocols required to prevent organ and HSC transplant rejection. This treatment would significantly increase the lifespan and quality of life of transplant patients. The risk of GvHD, the most limiting and dangerous complication of HSC transplantation, would be significantly reduced in patients treated with multi-chimeric cells. Treatment with immuno-privileged chimeric cells would provide more effective HSC engraftment and bone marrow (BM) compartment rearrangement, thus leading to the induction of immune tolerance and better outcomes for transplant recipients and immunocompromised patients.

[0052] The unique characteristics of MCCs include their ability to express hematopoietic markers and / or other markers of the parent cell (e.g., myobiocytes) as well as immunotolerogenic cytokines. In addition, chimeric cells can be cryopreserved, stored, and deposited in banks, thereby making them readily available for both acute and boosting long-term treatments. Due to these unique characteristics, MCC therapy has significant advantages over other stem cell-based strategies.

[0053] Example 2: Fusion and phenotypic characterization of human umbilical cord blood cells Individual fusions of 30 human UCB cells from three unrelated donors were performed as shown in Figure 1. Ex vivo fusion was confirmed by the presence of triple-fluorescently labeled (PKH26 / PKH67 / eFluor 670) MCCs using confocal microscopy and flow cytometry. Additional phenotypic features of the MCCs were also evaluated. In particular, the ex vivo fusion procedure was found not to alter the expression of CD4, CD90, CD45, and CD19 by the MCCs compared to the control (i.e., eFluor670 growth dye-labeled UCB cells). Furthermore, 90–95% of the MCCs were viable, as determined by trypan blue staining. Proliferative characteristics were determined by in vivo colony-forming unit assays, including multicentric, multipotential granulocyte, erythroid, macrophage, megakaryocyte colony-forming units (CFU-GEMM), granulocyte colony-forming unit (CFU-G), granulocyte-macrophage colony-forming unit (CFU-GM), macrophage colony-forming unit (CFU-M), and erythroid burst-forming unit (BFU-E) assays. Genotyping of MCCs using serological HLA typing confirmed the presence of alleles specific to each of the three unrelated donors in the genetic material of the MCCs (Table 4). [Table 4]

[0054] Genotyping analysis of MCCs using STR PCR further confirmed the presence of markers specific to each of the three unrelated donors in the genetic material of the MCCs (Table 5). [Table 5]

[0055] The oncogenicity of MCCs in the NSG mouse model was also evaluated. As outlined in Table 6, MCCs were administered to mice via intraosseous (IO) or intravenous (IV) routes. [Table 6]

[0056] Clinical findings of NSG mice 90 days post-injection indicated that the animals were active, possessed normal weight gain, normal fur, and showed no tumorous growth (including tumorous growth at the injection site as determined by palpation). Furthermore, magnetic resonance imaging (MRI) of NSG mice 90 days post-cell delivery indicated the absence of tumorous structures in mice receiving a mixture of non-fusion human umbilical cord blood cells from three unrelated donors or mice receiving fusion MCCs.

Claims

1. A composition for treating human immunodeficiency or hereditary disorders, comprising multi-chimeric cells consisting of ex vivo fusions of three or more hematopoietic stem cells that have been previously isolated and purified from three or more different, unrelated healthy donors.

2. The composition according to claim 1, wherein the hematopoietic stem cells are isolated from bone marrow, umbilical cord blood, peripheral blood, or a combination thereof.

3. The composition according to claim 1, comprising ex vivo fusions of three types of hematopoietic stem cells that have been previously isolated and purified from three different donors.

4. The composition according to claim 1, wherein the composition is administered by intraosseous, intravenous, or intramuscular injection.

5. The composition according to claim 1, wherein the immunodeficiency or hereditary disorder is selected from bone marrow failure, adenosine deaminase (ADA) deficiency, severe combined immunodeficiency (SCID), DiGeorge syndrome, type 1 diabetes mellitus, Gaucher disease, leukemia, aplastic anemia, sickle cell anemia, lymphoma, and multiple myeloma.

6. The composition according to claim 6, wherein the immunodeficiency or hereditary disorder is sickle cell anemia.

7. A composition for preventing the onset of GvHD or reducing the severity of GvHD in subjects undergoing allogeneic hematopoietic stem cell transplantation or organ transplantation, comprising multi-chimeric cells consisting of ex vivo fusions of three or more hematopoietic stem cells that have been previously isolated and purified from three or more different healthy, unrelated donors.

8. (i) membrane fusion agent, and (ii) Donor cells consisting of three or more hematopoietic stem cells that have been previously isolated and purified from three or more different healthy, unrelated donors. A kit for treating human immunodeficiency or genetic disorders, including [mention specific product / method].

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