WO2026010977A1 - Compositions et méthodes de traitement de maladies humaines avec des cellules souches mésenchymateuses modifiées par glycocalyx - Google Patents

Compositions et méthodes de traitement de maladies humaines avec des cellules souches mésenchymateuses modifiées par glycocalyx

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WO2026010977A1
WO2026010977A1 PCT/US2025/036118 US2025036118W WO2026010977A1 WO 2026010977 A1 WO2026010977 A1 WO 2026010977A1 US 2025036118 W US2025036118 W US 2025036118W WO 2026010977 A1 WO2026010977 A1 WO 2026010977A1
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mscs
glycocalyx
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disease
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Robert Sackstein
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)

Definitions

  • a method for treating bone diseases or skeletal conditions using glycocalyx-modified human mesenchymal stem cells is disclosed.
  • BACKGROUND OF THE INVENTION [0003] Abundant preclinical studies (encompassing both in vitro studies and animal studies) have provided compelling evidence for existence of multipotent mammalian adult stem cells defined by their capacity to (1) adhere to plastic, (2) expand in tissue culture (i.e., undergo culture-expansion), (3) express cell surface structures CD44, CD73, CD90, and CD105, without expression of CD45, CD11b, CD14, and CD19, and, most critically, (4) develop into various characteristic progeny cells including fibroblasts, osteoblasts, chondroblasts, and adipocytes under standard in vitro differentiation conditions.
  • This multipotent adult stem cell type is generally called a “mesenchymal stem cell” and is denoted by the acronym “MSC”, but is also referred to by various names including, but not limited to, “multipotent stromal cells” or “mesenchymal stromal cells” or “mesenchymal stem/stromal cells” or “medicinal signaling cells” or USA.617191053.7/45S “mesenchymal progenitor cells” or “mesenchymal precursor cells”. As used herein, the acronym “MSC” incorporates any cell type with the characteristics described above.
  • an allogeneic MSC as opposed to an autologous MSC (i.e., a non-self-sourced versus a self-sourced MSC, respectively)
  • an additional confounder is introduced due to the inherent variations of the immunobiology of alloreactivity/host defense between that of the animal and that of a human being, which can further limit the extrapolation of results of an MSC-based animal study to elucidate the clinical applicability of human MSCs in human diseases.
  • MSC biology varies as a function of the age of the donor from which the cells are obtained.
  • MSCs derived from younger mammals display much greater tissue reparative functions, have greater differentiation capacity, and higher proliferative ability than MSCs from older mammals.
  • Functional alterations and replicative exhaustion of human MSCs each rise precipitously as a function of age, with human MSCs become increasingly senescent after age 30.
  • all human MSC preparations that are in commercial development for therapeutic applications consist of cells harvested from healthy donors below age 30 years, indeed, most favorably from umbilical cord, umbilical cord blood, or placental sources.
  • MSCs While there can exist species-specific and age-specific variations in MSC biology, all mammalian MSCs share a fundamental biologic deficit: they all lack molecular effectors of cell migration that can guide the trafficking of intravascularly administered cells to sites of tissue injury. While there may be some localization of intravascularly administered MSCs within a given tissue due to vascular “entrapment” (a random process whereby these large cells (MSC diameter ranges from 20-40 microns) lodge within small bore ( ⁇ 10 micron diameter) microvessels), this process is random and does not result in efficient MSC colonization within an intended anatomic area.
  • vascular “entrapment” a random process whereby these large cells (MSC diameter ranges from 20-40 microns) lodge within small bore ( ⁇ 10 micron diameter) microvessels
  • direct injection ensures that MSCs are deposited within the intended tissue of interest (i.e., the inflamed/injured/diseased tissue itself)
  • direct injection is an invasive procedure that requires use of needles (or other administrative devices, e.g., catheters) with subsequent instillation of MSCs suspended in a crystalloid solution into the affected site as a bolus under hydrostatic pressure; altogether, this approach disrupts the native tissue microenvironment, and this technique can thus result in worsening the tissue injury within the affected site as well as collateral tissue damage in unaffected sites consequent to the invasive procedure itself.
  • direct tissue injection is only possible for tissues with strict anatomic boundaries, with tissue planes/histologic structure permitting placement of MSCs at defined affected sites, and with sufficient structural integrity/consistency capable to withstand the invasive injection (i.e., this approach is feasible for the heart, but not feasible for the vast majority of tissues (e.g., the lungs, GI tract, pancreas, or the central nervous system)).
  • tissue injection is not feasible for diseases and/or conditions that are multi-focal or generalized in nature (e.g., multiple sclerosis, muscular dystrophies, osteoporosis, etc.).
  • sLeX is the principal binding determinant (ligand) for E-selectin, an endothelial lectin: adhesive interactions between sLeX displayed on the surface of blood-borne cells and E-selectin expressed on endothelial cells promotes trafficking to the target tissue with subsequent colonization/engraftment of the sLeX- bearing circulating cell at the pertinent site.
  • E-selectin is constitutively displayed on microvessels of marrow and skin, and, notably, its expression is induced/upregulated at all endothelial beds at inflamed/diseased tissues.
  • E-selectin on marrow microvascular endothelial cells provides a “gateway” for highly efficient marrow colonization of sLeX-bearing blood-borne cells.
  • osteogenesis imperfecta Some of these conditions result from genetic deficits (e.g., osteogenesis imperfecta), some are due to trauma (e.g., fractures, and, in particular, delayed-healing or non-healing fractures), some are due to infections (e.g., osteomyelitis), some are due to metabolic derangements (e.g., renal osteodystrophy), some are due to drugs (e.g., steroids), some are due to endocrinopathies (e.g., hyperparathyroidism), some are due to malnutrition (e.g., inadequate calcium absorption or intake), and some are due to degenerative diseases (e.g., osteoporosis).
  • trauma e.g., fractures, and, in particular, delayed-healing or non-healing fractures
  • infections e.g., osteomyelitis
  • metabolic derangements e.g., renal osteodystrophy
  • drugs e.g., steroids
  • endocrinopathies e.
  • osteoporosis is the most prevalent and the most insidious, leading to fragility fractures and associated disability.
  • This generalized skeletal disease is most commonly caused by the loss of estrogen in menopause (“postmenopausal osteoporosis”) and it affects hundreds of millions of women world-wide.
  • postmenopausal osteoporosis the loss of estrogen in menopause
  • the incidence of osteoporosis is continuing to rise, especially among aging men.
  • osteoporosis mediated by osteoblasts, cells derived from mesenchymal stem cells (MSCs)
  • MSCs mesenchymal stem cells
  • osteoclasts cells derived from hematopoietic stem cells
  • Osteoporosis is “generalized” (i.e., a systemic disease) in that all skeletal sites are affected. As such, direct injection of reparative/regenerative cells – either the osteoblast (OB) itself or the mesenchymal stem cell (MSC) – into the marrow at all skeletal sites where osteoblast replenishment is needed is impossible. Both MSCs and OBs can be readily culture-expanded in numbers sufficient to treat a patient suffering from any skeletal condition.
  • the proximate hurdle is to develop the means to deliver the culture- expanded MSCs or the OBs to all sites that are affected so that they can exert their beneficial effect(s) where needed, an imperative that is best achieved by exploiting vascular delivery (i.e., introducing the osteorestorative/osteoreparative cells into the blood and then having the cells migrate to all the marrow located throughout the body).
  • the bone disease may not be generalized (e.g., at a given fracture site or at sites of several fractures, such as might occur with trauma) or in cases where there may be introduction of a scaffold to enable bone reconstruction (e.g., a bone graft or any pertinent scaffold/implant), it may be beneficial to seed the affected bone area with MSCs and/or with osteoblasts, using the vascular route of delivery.
  • a scaffold to enable bone reconstruction (e.g., a bone graft or any pertinent scaffold/implant)
  • osteotropism The migration of blood-borne cells to marrow is called “osteotropism”, a process occurring at specialized medullary microvascular endothelial cells that constitutively express E- selectin, a lectin binding a glycan determinant called “sialylated Lewis X” (sLeX; CD15s).
  • sLeX a lectin binding a glycan determinant
  • sLeX glylated Lewis X
  • Hematopoietic stem/progenitor cells HSPCs
  • E-selectin-sLeX interactions empower HSPC osteotropism requisite for successful hematopoietic stem cell transplantation.
  • sLeX is a tetrasaccharide, a “type 2” lactosamine (i.e., galactose (Gal) ⁇ (1,4)-linked to N- acetylglucosamine (GlcNAc)), bearing terminal sialic acid (NeuAc) and fucose (Fuc) substitutions: NeuAc- ⁇ (2,3)-Gal- ⁇ (1,4)-[Fuc- ⁇ (1,3)]-GlcNAc-R.
  • Gal galactose
  • GlcNAc N- acetylglucosamine
  • MSCs do not display sLeX yet uniformly express a CD44 glycovariant that bears terminal ⁇ (2,3)-sialylated type 2 lactosamines (i.e., NeuAc- ⁇ (2,3)-Gal- ⁇ (1,4)-GlcNAc-R) missing only Fuc ⁇ (1,3)-linked to GlcNAc to complete the sLeX motif.
  • the missing Fuc can be placed stereospecifically using ⁇ (1,3)-fucosyltransferases, thereby engendering sLeX decorations on the MSC surface; when injected intravascularly, these “glycoengineered” MSCs are then capable of osteotropism.
  • MSCs can fuel osteorestoration via direct differentiation into osteoblasts and/or via MSC-mediated trophic effects to improve resident osteoblast biology and/or via MSC-mediated dampening of inflammation, thereby effectively treating bone conditions/diseases such as osteoporosis.
  • MSCs are located within many tissues of the body (e.g., bone marrow, fat, periodontal ligament, umbilical cord, amniotic membrane, etc.), and can also be found in blood (especially in umbilical cord blood).
  • tissues of the body e.g., bone marrow, fat, periodontal ligament, umbilical cord, amniotic membrane, etc.
  • osteoogenic capacity the greatest capacity to make new bone
  • a non-self (i.e., “allogeneic”) source of MSCs would engender “foreign” osteoblasts that would be immunologically rejected.
  • an allogeneic source of MSCs would engender “foreign” osteoblasts that would be immunologically rejected.
  • the platform glycoengineering approach described herein would be applicable to the requisite distribution of the osteoregenerative cells throughout the entire skeleton.
  • the osteoblast including gene-edited osteoblasts
  • intravascularly administering the cells thereby achieving increased osteoblast content within the affected skeletal sites.
  • Some embodiments provided herein are based, in part, on the surprising finding that a single intravascular infusion of human MSCs that have undergone cell surface glycoengineering (glycocalyx editing; glycosyltransferase-programmed stereosubstitution (GPS)) to enforce display of the cell surface glycan sLeX results in a profound beneficial reparative/restorative effect within a human tissue whose endothelial beds express E-selectin.
  • cell surface glycoengineering glycosyltransferase-programmed stereosubstitution
  • the embodiments provided herein are also based in part on the surprising finding that human MSCs obtained from “aged” donors (in particular, human beings older than age 30 years) are capable of exerting significant tissue reparative/restorative effects. Moreover, the embodiments provided herein are based in part on the surprising finding that culture-expanded human MSCs harvested from a tissue location that harbors a disease/inflammatory process retain the capacity to exert significant tissue reparative/restorative effects, i.e., contrary to numerous reports, MSCs sourced from a site of pathology are not compromised with regards to tissue regenerative properties.
  • human MSCs can be treated in vitro with glycosyltransferases and appropriate donor nucleotide donors under conditions sufficient to install display of sLeX on the MSC surface.
  • Glycosyltranferases can include a fucosyltransferase, a sialyltransferase, a galactosyltransferase, or an N-acetylglucosaminotransferase, either singly or in combination. These glycosyltransferases may be used following treatment of the MSCs with sialidases, with subsequent resialylation of the MSC surface followed by fucosylation.
  • the fucosyltransferase may be an ⁇ (1,3)-fucosyltransferase (for example, but not limited to, Fucosyltransferase VI (FTVI, FT6) or Fucosyltransferase VII (FTVII, FT7) or Fucosyltransferase III (FTIII, FT3) or Fucosyltransferase V (FTV or FT3)), and/or an ⁇ (2,3)-sialyltransferase (for example, but not limited to, ST3GalIII, ST3GalIV, or ST3GalVI).
  • FTVI Fucosyltransferase VI
  • FTVII Fucosyltransferase VII
  • FTV or FT3 Fucosyltransferase V
  • an ⁇ (2,3)-sialyltransferase for example, but not limited to, ST3GalIII, ST3GalIV, or ST3GalVI).
  • sLeX Confirmation of the enforced expression of sLeX on the cell surface can be verified by staining using antibodies directed to sLeX (e.g., the rat IgM mAb HECA-452 or the mouse IgG mAb CSLEX-1) and/or by staining E- selectin-Fc chimera using fluorochrome-tagged antibodies/reagents and standard flow cytometry techniques.
  • antibodies directed to sLeX e.g., the rat IgM mAb HECA-452 or the mouse IgG mAb CSLEX-1
  • E- selectin-Fc chimera using fluorochrome-tagged antibodies/reagents and standard flow cytometry techniques.
  • stem/progenitor cells obtained from older persons, and then culture-expanded could harbor age-associated mutations that could result in aberrant cell proliferation and/or differentiation leading to cancer; in the case of MSCs, the possibility of highly aggressive cancers derived from “older” MSCs called “sarcomas” (including osteosarcomas).
  • sarcomas highly aggressive cancers derived from “older” MSCs
  • culture-expanded MSCs obtained from the marrow of osteoporotic bone (marrow) of older persons when glycocalyx-modified and then intravascularly administered into a patient with osteoporosis, can improve osteogenic/osteoreparative capacity and thereby achieve osteorestoration sufficient to decrease fragility fractures.
  • the colonization of intravascularly administered culture- expanded human MSCs at all skeletal sites within the recipient was accomplished by modifying the composition of the cell “glycocalyx” (i.e., the sugar-coat displayed on the cell surface), via a process that is generically called “cell surface glycan engineering”, “glycoengineering”, “glycocalyx editing”, “glycosyltransferase-programmed stereosubstitution” (GPS), and/or “glycocalyx modification”.
  • GPS glycose-programmed stereosubstitution
  • This sugar (glycan) engineering of the human MSC surface CD44 involves the glycosyltransferase-mediated installation of fucose onto ⁇ (2,3)-sialylated type 2 lactosaminyl glycans of CD44 using an ⁇ (1,3)- fucosyltransferase together with GDP-fucose donor (“exofucosylation”), thereby enforcing expression of the sLeX-laden glycoform of CD44 called “Hematopoietic Cell E-/L-selectin Ligand” (HCELL), the most potent E-selectin ligand expressed on any human cell.
  • HCELL Hematopoietic Cell E-/L-selectin Ligand
  • HCELL is the “bone marrow homing receptor” – the cell surface molecule that mediates osteotropism.
  • the present disclosure provides a method of treating or ameliorating a skeletal/bone disease or condition in a subject in need thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the skeletal/bone disease or condition; modifying the glycocalyx of the MSCs, ex vivo, using one or more glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce “glycocalyx-modified MSCs”; introducing the glycocalyx- modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effective for bone regeneration.
  • MSCs culture-expanded mesenchymal stem cells
  • the present disclosure provides a method of treating or ameliorating a disease or condition (e.g., a bone disease) in a subject in need thereof.
  • a disease or condition e.g., a bone disease
  • the present disclosure provides the steps comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the disease or condition, and then differentiating the MSCs in vitro into osteoblasts, which may themselves be further culture-expanded; modifying the glycocalyx of the osteoblasts, ex vivo, using one or more glycosyltransferases to enforce cell surface expression of sLeX on the osteoblasts to produce “glycocalyx-modified osteoblasts”; introducing the glycocalyx-modified osteoblasts into the subject, wherein the glycocalyx-modified osteoblasts are effective for bone regeneration.
  • MSCs mesenchymal stem cells
  • the glycosyltransferase-mediated reactions enforce sLeX display on the intrinsic cell surface glycoprotein CD44, thereby engendering the expression of the distinct CD44 glycoform known as HCELL. on the surface of the glycocalyx-modified MSCs or glycocalyx-modified osteoblasts.
  • the population of culture-expanded MSCs is obtained from the bone marrow of the subject suffering from the skeletal/bone disease or condition.
  • the MSCs may be obtained from non-marrow sources, including, but not limited to, adipose tissue, dental pulp, periodontal ligament, umbilical cord, umbilical cord blood, menstrual blood, placenta, and amniotic membrane [0024]
  • the skeletal/bone disease or condition is caused by one or more of congenital/genetic diseases, trauma, degenerative diseases, metabolic diseases, endocrinopathies, neoplastic diseases, and iatrogenic (drug) effects.
  • the bone disease or condition is caused by one or more of dietary deficiencies/malnutrition, endocrinologic conditions, gastrointestinal conditions/malabsorption states, pregnancy/lactation- induced bone loss, or decreased bone density due to cancer or due to medications.
  • the bone disease or condition is osteoporosis caused by one or more of an autoimmune disorder, digestive/gastrointestinal disorder, medical procedure, cancer, hematologic/blood disorder, neurological/nervous system disorder, blood/bone marrow disorder, endocrine/hormonal disorder, and pregnancy/lactation.
  • the bone condition is one or more of fracture and trauma-related injuries (including crush injuries and bone destruction requiring repair with scaffolds/implants).
  • the MSCs or osteoblasts are glycocalyx-modified ex vivo by exofucosylation. In some embodiments, the MSCs or osteoblasts are glycocalyx-modified ex vivo by transfection with a nucleic acid encoding a fucosyltransferase. In some embodiments, the glycocalyx-modified MSCs or glycocalyx-modified osteoblasts are introduced into the subject one time per year. In some embodiments, the glycocalyx-modified MSCs or glycocalyx-modified osteoblasts are introduced into the subject multiple times per year (e.g., a plurality of times/year).
  • the subject suffers from the skeletal/bone disease osteoporosis.
  • the subject suffers from the bone disease postmenopausal osteoporosis (also called “age-related osteoporosis” because this entity also occurs in elderly men).
  • the subject is greater than 1 month old.
  • the subject suffers from a congenital/genetic condition whereby production of bone is compromised, such as occurs in osteogenesis imperfecta, hypophosphatasia, and idiopathic juvenile osteoporosis.
  • the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has evidence of bone loss by imaging studies (e.g., dual- energy X-ray absorptiometry scan (DXA scan) that can identify reduced bone density.
  • DXA scan dual- energy X-ray absorptiometry scan
  • the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject suffers from one or more bone fractures.
  • the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has suffered a traumatic fracture of a bone.
  • the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has suffered an atraumatic fracture of a bone.
  • the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has suffered an atraumatic fracture of one or more of vertebra, hip, distal forearm/carpal joint, and pelvis.
  • the MSC is obtained from the bone marrow (i.e., bone marrow-derived MSCs (“BM-MSCs”) of the subject (i.e., “autologous” BM-MSCs (“autoBM-MSCs”) suffering from the bone disease and the subject is greater than 1 month old.
  • BM-MSCs bone marrow-derived MSCs
  • autoBM-MSCs autologous BM-MSCs
  • the MSC is obtained from osteoporotic bone marrow of the subject. In some embodiments, the MSC is obtained from osteoporotic bone marrow of the subject and the subject is greater than 30 years old.
  • the MSC surface is glycocalyx-modified ex vivo by exofucosylation with a fucosyltransferase VI or fucosyltransferase VII (“exofucosylated” autoBM (“Fuc-autoBM-MSCs”)). In some embodiments, the MSC is glycocalyx-modified ex vivo by transfection or transduction with a nucleic acid encoding a fucosyltransferase VI or a fucosyltransferase VII.
  • the subject is undergoing one or more osteoporosis treatments.
  • the subject is undergoing one or more osteoporosis treatments including, but not limited to, calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents/analogs.
  • the glycocalyx-modified MSCs are effective to increase plasma levels of bone neoformation marker N-terminal propeptide of type I procollagen (P1NP).
  • P1NP type I procollagen
  • 0.5x106 to 10x106 glycocalyx-modified MCSs/kg of recipient body weight is introduced to the subject.
  • the MSC is introduced to the subject via intravascular administration.
  • the present disclosure provides: a method of treating a disease in a subject in need thereof, wherein the endothelial beds of the diseased tissue express E- selectin, comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the diseased tissue of the subject; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject.
  • MSCs culture-expanded mesenchymal stem cells
  • the disease is a skeletal/bone disease or condition.
  • the skeletal/bone disease or condition is osteoporosis.
  • modifying the glycocalyx of the MSCs comprises exofucosylation using one or more fucosyltransferases.
  • the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E-/L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.
  • introducing the glycocalyx-modified MSCs comprises intravascular administration.
  • 0.5x10 6 to 50x10 6 glycocalyx-modified MSCs/kg of recipient body weight are introduced to the subject.
  • the subject is undergoing one or more treatments for the disease in addition to the introduction of glycocalyx- modified MSCs.
  • the present disclosure provides a method of treating a skeletal/bone disease in a subject in need thereof comprising: obtaining a population of culture- expanded mesenchymal stem cells (MSCs) from diseased tissue of the subject, wherein the subject suffers from the skeletal/bone disease; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effective for skeletal regeneration.
  • MSCs mesenchymal stem cells
  • the skeletal/bone disease is osteoporosis.
  • the glycocalyx of the MSCs comprises exofucosylation using one or more of fucosyltransferases.
  • the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E-/L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.
  • introducing the glycocalyx-modified MSCs comprises intravascular administration.
  • 0.5x106 to 50x106 glycocalyx-modified MSCs/kg of recipient body weight are introduced to the subject.
  • the subject is undergoing one or more treatments for the skeletal/bone disease in addition to the introduction of glycocalyx-modified MSCs.
  • the one or more treatments include at least one of calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents.
  • the present disclosure provides a method of treating a disease in a subject in need thereof comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from a subject older than 30 years; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject.
  • the subject is older than 50 years.
  • modifying the glycocalyx of the MSCs comprises exofucosylation using one or more fucosyltransferases.
  • the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E-/L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.
  • introducing the glycocalyx-modified MSCs comprises intravascular administration.
  • 0.5x10 6 to 50x10 6 glycocalyx-modified MSCs/kg of recipient body weight are introduced to the subject.
  • the disease is a skeletal disease or condition.
  • the skeletal disease or condition is osteoporosis.
  • the present disclosure provides a method of treating osteoporosis in a subject in need thereof comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from a subject older than 30 years; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on CD44 molecules of the MSCs to produce glycocalyx-modified MSCs expressing HCELL; and introducing the glycocalyx-modified MSCs into the subject via intravascular administration.
  • the glycosyltransferases comprise one or more fucosyltransferases.
  • modifying the glycocalyx of the MSCs comprises exofucosylation using the one or more of fucosyltransferase VI and fucosyltransferase VII.
  • 0.5x106 to 50x106 glycocalyx-modified MSCs/kg of recipient body weight are introduced to the subject.
  • the subject is undergoing one or more additional treatments for osteoporosis.
  • the one or more additional treatments include at least one of calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents.
  • the present disclosure provides a method of making immunomodulatory/restorative/reparative mesenchymal stem cells (MSCs) comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and/or wherein the subject is greater than 30 years old.
  • MSCs immunomodulatory/restorative/reparative mesenchymal stem cells
  • the present disclosure provides a method of preparing a secretome comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and/or wherein the subject is greater than 30 years old; and harvesting the secretome (e.g. extracellular vesicles) from the MSCs.
  • the secretome e.g. extracellular vesicles
  • Fig. 1A Temporal distribution of patient fragility (i.e., spontaneous) fractures before and after Fuc-autoBM-MSC infusion. Timeline of years/months are shown at bottom of figure, and each patient is presented as a separate row, in sequence by date of accrual to study.
  • the date of Fuc-autoBM-MSC administration is represented by solid purple circle, and date of completion of the protocol-mandated 2-year evaluation period is shown by blue vertical bar; the date and number of fragility fractures (if >1, so listed as “x#”) are shown in red cross- marks on horizontal bars depicting the patient evaluation interval before (pink bars) and after (blue bars) Fuc-autoBM-MSC infusion. Horizontal bars colored orange indicate the treatment period for those patients who received PTH analog (teriparatide), in each case administered as per discretion of primary physician.
  • Fig 2A-B Changes in plasma levels of bone neoformation markers after Fuc- autoBM-MSC infusion.
  • Fig. 2A shows levels of each biomarker at designated point of measurement.
  • Red solid line patient #4 indicates initiation of teriparatide (PTH analog) treatment; red dashed line (patients #9 and #10) indicates discontinuation of teriparatide.
  • Fig.2B top table shows mean of aggregated delta percent change from baseline levels for each biomarker per patient for the first 24 months post-infusion.
  • Bottom left table shows Mean ⁇ SEM of aggregated delta percent change from baseline levels of each biomarker for all patients;
  • Bottom right table shows Mean ⁇ SEM of aggregated delta percent change from baseline levels for each biomarker excluding values from teriparatide-treated patients (#4, #9, and #10).
  • P1NP N-terminal propeptide of type I procollagen
  • BGLAP osteocalcin
  • Bone ALP bone alkaline phosphatase.
  • Red solid line indicates initiation of teriparatide (PTH analog) treatment; red dashed line (patients #9 and #10) indicates discontinuation of teriparatide.
  • Top table shows mean of aggregated delta percent change from baseline levels of each biomarker per patient for the first 24 months post-infusion.
  • Bottom left table shows Mean ⁇ SEM of aggregated delta percent change from baseline levels of each biomarker for all patients;
  • Bottom right table shows Mean ⁇ SEM of aggregated delta percent change from baseline levels of each biomarker excluding values from teriparatide-treated patients (#4, #9, and #10).
  • Fig. 4A Histomorphometrical analysis of bone tissue area in subjects before (day 0) and 120 days after systemic administration of fucosylated autologous bone marrow-derived MSC. Bone Tissue Area (BTA) was measured within bone biopsies (obtained at the same anatomic area for each subject) before (basal, day +0) and 120 days following MSC infusion (day +120). Each high-resolution analysis per histological slice is shown as a circle.
  • Panel at left shows data for each tissue plane (mm2) for each subject ⁇ Mean + SD for BTA values are shown, and differences between basal and day 120 values are as noted: significantly increased in 7 patients (*p ⁇ 0 ⁇ 05, ***p ⁇ 0 ⁇ 001), significantly decreased in patient #6 (3 ⁇ 5 mm2 to 2 ⁇ 5 mm2) and patient #7 (2 ⁇ 2 mm2 to 0 ⁇ 8 mm2)( ⁇ p ⁇ 0 ⁇ 001), or unchanged in patient #9 (2 ⁇ 4 mm2 to 2 ⁇ 3 mm2; ns) (Welch’s t-test).
  • Fig. 4B Bone Tissue Area (BTA, mm2) and Volumetric Bone Mineral Density (QTS Score).
  • BTA and QTS scores are shown, with dashed and solid lines in each panel representing respective Mean + SEM.
  • Left Panel BTA was measured within iliac crest bone biopsies (obtained at the same anatomic area for each subject) at baseline (day 0) and 120 days after Fuc-autoBM-MSC infusion (day +120). BTA increased significantly post-infusion from Mean + SEM of 1 ⁇ 42 ⁇ 0 ⁇ 03 mm 2 to 2 ⁇ 41 ⁇ 0 ⁇ 05 mm 2 (***p ⁇ 0 ⁇ 001, Welch’s t-test).
  • Right Panel QTS scores for all patients at baseline (day 0) and month 24 after Fuc-autoBM-MSC infusion.
  • the present disclosure provides a method of treating or ameliorating a disease or condition in a subject in need thereof using glycocalyx-modified MSCs, wherein the glycocalyx-modified MSCs are obtained from a diseased/affected tissue.
  • the present disclosure provides a method of treating or ameliorating a disease or condition in a subject in need thereof using glycocalyx-modified MSCs, wherein the MSCs are obtained from the subject and the subject is of advanced age (e.g. greater than: 30 years old, 40 years old, 50 years old, 60 years old, 70 years old).
  • the present disclosure provides a method of treating or ameliorating a bone disease or condition in a subject in need thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the bone disease or condition; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; introducing the glycocalyx-modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effective for bone regeneration.
  • MSCs culture-expanded mesenchymal stem cells
  • osteorestoration can be achieved using the population of culture-expanded MSCs from the bone tissue (marrow) of a subject suffering from a bone disease, such as osteoporosis.
  • the population of culture-expanded MSCs are obtained from elderly subjects.
  • the culture-expanded MSCs can be used alone or in a composition to treat bone disease.
  • a single infusion of exofucosylated MSCs would be sufficient to drive forward a beneficial clinical effect.
  • the terms "treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient.
  • the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development.
  • treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population.
  • a given subject or subject population may fail to respond or respond inadequately to treatment.
  • the terms “ameliorate”, “ameliorating” and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.
  • the term “substantially free of divalent metal cation co-factors” or “substantially free of stabilizer compounds” means that the pertinent solution/buffer may have a concentration of a pertinent divalent cation and/or of a cryoprotective agent/cell stabilizer, respectively, at a molar level that does not cause cell death or cytotoxicity.
  • an "effective amount" of a therapeutic is an amount of such therapeutic that is sufficient to effect beneficial or desired results as described herein when administered to a subject.
  • Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine.
  • a suitable dose of a therapeutic according to the disclosure will be that amount of the agent, which is the lowest dose effective to produce the desired effect with no or minimal side effects.
  • compositions, pharmaceutical compositions, including cell populations disclosed herein for therapeutic indications can be achieved in a variety of ways, in each case as clinically warranted, using a variety of anatomic access devices, a variety of administration devices, and a variety of anatomic approaches, with or without support of anatomic imaging modalities (e.g., radiologic, MRI, ultrasound, etc.) or mapping technologies (e.g., epiphysiologic mapping procedures, electromyographic procedures, electrodiagnostic procedures, etc.).
  • anatomic imaging modalities e.g., radiologic, MRI, ultrasound, etc.
  • mapping technologies e.g., epiphysiologic mapping procedures, electromyographic procedures, electrodiagnostic procedures, etc.
  • compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered systemically, via either peripheral vascular access (e.g., intravenous placement, peripheral venous access devices, etc.) or central vascular access (e.g., central venous catheter/devices, arterial access devices/approaches, etc.).
  • peripheral vascular access e.g., intravenous placement, peripheral venous access devices, etc.
  • central vascular access e.g., central venous catheter/devices, arterial access devices/approaches, etc.
  • the compositions, pharmaceutical compositions and cell populations of the present disclosure can be delivered intravascularly into anatomic feeder vessels of an intended tissue site using catheter-based approaches or other vascular access devices (e.g., arterial catheterization, etc.) that will deliver a vascular bolus of cells to the intended site.
  • compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered directly into body cavities or anatomic compartments by either catheter-based approaches or direct injection into a pertinent anatomic site (e.g., intrabone/intramedullary (i.e., within the marrow itself) or within scaffolds/implants.
  • a pertinent anatomic site e.g., intrabone/intramedullary (i.e., within the marrow itself) or within scaffolds/implants.
  • the compositions, pharmaceutical compositions and cell populations of the present disclosure can be introduced by direct local tissue injection, using either intravascular approaches, or percutaneous approaches, or via surgical exposure/approaches to the tissue, or via arthroscopic approaches, or directly into the accessible tissue sites and/or guided by imaging techniques.
  • compositions, pharmaceutical compositions and cell populations of the present disclosure can also administered into tissue or structural support devices (e.g., tissue scaffold devices and/or embedded within scaffolds placed into tissues, etc.), and/or administered in gels, and/or administered together with enhancing agents (e.g., admixed with supportive cells, cytokines, growth factors, resolvins, anti- inflammatory agents, etc.).
  • tissue or structural support devices e.g., tissue scaffold devices and/or embedded within scaffolds placed into tissues, etc.
  • enhancing agents e.g., admixed with supportive cells, cytokines, growth factors, resolvins, anti- inflammatory agents, etc.
  • the compositions, pharmaceutical compositions and cell populations of the present disclosure are administered to the subject with an enforced expression of glycosylation.
  • the enforced glycosylation on the surface of administered cells will aid in tissue repair/regeneration.
  • the enforced expression of sLeX on administered cells promotes lodgment of cells within the affected tissue milieu, in apposition to cells bearing E-selectin (i.e., endothelial cells) and/or L-selectin (i.e., leukocytes), respectively, within the target site.
  • E-selectin i.e., endothelial cells
  • L-selectin i.e., leukocytes
  • cells of the present disclosure are contacted with a glycosyltransferase to enforce a glycan on the cell surface.
  • the glycosylstransferase is a human glycosyltransferase.
  • the glycosyltransferase is a non-human glycosyltransferase.
  • fucosylated lactosaminyl glycans are enforced by a member of the ⁇ (1,3)-fucosyltransferase family.
  • the human ⁇ (1,3)-fucosyltransferase family includes Fucosyltransferase III (also called FTIII, FT3, FUTIII, or FUT3), Fucosyltransferase IV (also called FTIV, FT4, FUTIV, or FUT4), Fucosyltransferase V (also called FTV, FT5, FUTV, or FUT5), Fucosyltransferase VI (also called FTVI, FT6, FUTVI, or FUT6), Fucosyltransferase VII (also called FTVII, FT7, FUTVII, or FUT7), Fucosyltransferase IX (also called FTIX, FT9, FUTIX, or FUT9), and variants thereof.
  • Fucosyltransferase III also called FTIII, FT3, FUTIII, or FUT3
  • Fucosyltransferase IV also called FTIV, FT4, FUTIV, or FUT4
  • the cDNA/protein sequences for the ⁇ (1,3)-fucosyltransferase family are as follows Name GenBank Acc. No. F lt f III [0061]
  • the notation for a fucosyltransferase should not be construed as limiting to the nucleotide sequence or the amino acid sequence.
  • the notation of Fucosyltransferase VII, FTVII, FT7, FUTVII or FUT7 are used interchangeably as meaning the nucleotide, amino acid sequence, or both, of Fucosyltransferase VII.
  • cells are contacted by one or more of the ⁇ (1,3)-fucosyltransferase family members to enforce fucosylated lactosaminyl glycans.
  • fragments of ⁇ (1,3)-fucosyltransferase family members are contacted with a cell.
  • a peptide/nucleotide having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an ⁇ (1,3)-fucosyltransferase family member is contacted with a cell.
  • the term “identity” and grammatical versions thereof means the extent to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. Percent (%) identity is calculated by multiplying the number of matches in a sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps. [0063]
  • the cells may be contacted with the desired fucosyltransferase via exofucosyltation using, for example, the methods disclosed herein.
  • Nos.7,875,585 and 8,084,236, (which disclosures are expressly incorporated by reference as if recited in full herein) provide non-limiting examples of compositions and methods for ex vivo modification of cell surface glycans on a viable cell, which may be used to enforce expression of fucosylated lactosaminyl glycans on a cell according to the present disclosure.
  • the cells may be contacted with a purified glycosyltransferase polypeptide and a physiologically acceptable solution, for use together with appropriate donor nucleotide sugars in reaction buffers and reaction conditions specifically formulated to retain cell viability.
  • the physiologically acceptable solution may be free or substantially free of divalent metal co-factors, to such extent that cell viability is not compromised.
  • the cells may be contacted with a solution that is also free or substantially free of stabilizer compounds such as for example, glycerol, again, to such extent that cell viability is not compromised.
  • Glycosyltransferases of the present disclosure include for example, one or more fucosyltransferase.
  • the fucosyltransferase is an ⁇ (1,3)-fucosyltransferase such as an ⁇ (1,3)-fucosyltransferase III, ⁇ (1,3)-fucosyltransferase IV, an ⁇ (1,3)-fucosyltransferase V, an ⁇ (1,3)-fucosyltransferase VI, an ⁇ (1,3)-fucosyltransferase VII, or an ⁇ (1,3)- fucosyltransferase IX.
  • fucosyltransferases other than these, for example the ⁇ (1,3)-fucosyltransferase from H.
  • glycans are glycocalyx-modified on the surface of a cell by contacting a population of cells with one or more glycosyltransferase compositions described above.
  • the cells are contacted with the glycosyltransferase composition together with an appropriate nucleotide sugar donor (e.g., GDP-fucose) under conditions in which the glycosyltransferase has enzymatic activity.
  • an appropriate nucleotide sugar donor e.g., GDP-fucose
  • cells may be incubated for 60 min at 37oC in fucosyltransferase reaction buffer composed of Hank’s Balanced Salt Solution (HBSS) (without Ca 2+ and Mg 2+ ) (Lonza) containing 20 mM HEPES (Lonza), 0.1% human serum albumin (HSA) (Grifols, Barcelona, Spain), 30 ⁇ g/ml fucosyltransferase, and 1 mM GDP-fucose.
  • HBSS Hank’s Balanced Salt Solution
  • HSA human serum albumin
  • Glycan modification results in cells according to the present disclosure that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more viability at 24 hours or more after treatment.
  • the cells of the present disclosure have at least 70% viability at 48 hours after treatment.
  • the cells of the present disclosure have at least 75% viability at 48 hours after treatment.
  • the cells of the present disclosure have at least 80% viability at 48 hours after treatment.
  • the phenotype of the cells of the present disclosure is preferably preserved after treatment.
  • glycosyltransferases are contacted with cells of the present disclosure in reaction buffer that is free of or substantially free of) divalent metal co- factors (e.g. divalent cations such as manganese, magnesium, calcium, zinc, cobalt or nickel) and stabilizers such as glycerol.
  • divalent metal co- factors e.g. divalent cations such as manganese, magnesium, calcium, zinc, cobalt or nickel
  • stabilizers such as glycerol.
  • a purified glycosyltransferase polypeptide and a physiologically acceptable solution free of or substantially free of divalent metal co-factors is used to enforce a desired glycosylation pattern.
  • a composition is free of or substantially free of cryoprotective agents/stabilizer compounds (such as for example, glycerol), in any case such that the composition contains such agents/stabilizers at levels that do not affect cell viability.
  • glycosyltransferases used with solutions that are free or substantially free of divalent metal cofactors, and free of or substantially free of cryoprotective agents/stabilizer compounds include for example, ⁇ (1,3)-fucosyltransferases such as an ⁇ (1,3)-fucosyltransferase III, ⁇ (1,3)- fucosyltransferase IV, an ⁇ (1,3)- fucosyltransferase VI, an ⁇ (1,3)- fucosyltransferase VII, or an ⁇ (1,3)-fucosyltransferase IX.
  • the glycosyltransferase is biologically active.
  • biologically active means that the glycosyltransferase is capable of transferring a sugar molecule from a donor to acceptor.
  • a glycosyltransferase according to the present disclosure is capable of transferring 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 5, 10 or more ⁇ moles of sugar per minute at pH 6.5 at 37° C.
  • the contacting of a glycosyltransferase with a cell occurs in a physiologically acceptable solution, which is any solution that does not cause cell damage, e.g. death.
  • the viability of the cell is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more after treatment with the compositions of the invention.
  • suitable physiologically acceptable solutions include, for example, Hank's Balanced Salt Solution (HBSS), Dulbecco's Modified Eagle Medium (DMEM), a Good's buffer such as a HEPES buffer, a 2-Morpholinoethanesulfonic acid (MES) buffer, or phosphate buffered saline (PBS).
  • HBSS Hank's Balanced Salt Solution
  • DMEM Dulbecco's Modified Eagle Medium
  • MES 2-Morpholinoethanesulfonic acid
  • PBS phosphate buffered saline
  • the human or mammalian cells of the present disclosure may be contacted with a desired fucosyltransferase by transfecting a DNA or RNA nucleotide sequence encoding the desired fucosyltransferase into the cell.
  • modified RNA (modRNA) encoding the relevant ⁇ (1,3)-FT transcripts is used to enforce the desired pattern of fucosylated lactosaminyl glycans.
  • the transfected nucleotide sequence encodes a full length or partial peptide sequence of the desired fucosyltransferase.
  • the nucleotide sequence encodes a naturally existing isoform of a fucosyltransferase.
  • a fucosyltransferase See, e.g., Mondal N. et al. Distinct human ⁇ (1,3) – fucosyltransferases drive Lewis-X/sialyl Lewis-X assembly in human cells. J Biol Chem. 2018; 293(19):7300-7314.)
  • the cells may be contacted with the desired fucosyltransferase by transfecting the cells with a recombinant DNA or RNA molecule.
  • the term “recombinant DNA or RNA” means a DNA or RNA molecule formed through recombination methods to splice fragments of DNA or RNA from a different source or from different parts of the same source.
  • the recombinant DNA may comprise a plasmid vector, which controls expression of the DNA in the cell. Proteins, such as the enzymes disclosed herein, which are encoded by recombinant DNA or RNA are recombinant proteins.
  • the nucleic acid may be in the form of modified RNA (“modRNA”).
  • the MSC or the osteoblast may have undergone in vitro pre- treatment with agents which stimulate osteogenic activity (agents having “osteoinductive effects”) prior to intravascular administration of the cells.
  • agents include, but are not limited to, growth factors (e.g., bone morphogenetic proteins (BMPs, in particular BMP-2), TGF- ⁇ among others), and chemicals (e.g., fucoidan, dexamethasone, ascorbic acid, ⁇ -glycerophosphate, among others).
  • growth factors e.g., bone morphogenetic proteins (BMPs, in particular BMP-2), TGF- ⁇ among others
  • chemicals e.g., fucoidan, dexamethasone, ascorbic acid, ⁇ -glycerophosphate, among others.
  • the patient will receive agents that stimulate new bone formation (osteoanabolic agents) prior to and/or coincident with and/or following the intravascular administration of glycocalyx-modified MSCs or glycocalyx-modified osteoblasts; such osteoanabolic agents include, but are not limited to, hormones (e.g., parathyroid hormone, analogs of parathyroid hormone (e.g., teriparatide, abaloparatide), among others) and romosozumab.
  • hormones e.g., parathyroid hormone, analogs of parathyroid hormone (e.g., teriparatide, abaloparatide), among others
  • romosozumab e.g., romosozumab.
  • the patient will receive agents that inhibit bone resorption (antiresorptive agents) prior to and/or coincident with and/or following the intravascular administration of glycocalyx-modified MSCs or glycocalyx-modified osteoblasts; such agents include, but are not limited to, bisphosphonates, denosumab, and estrogens/estrogen analogs.
  • the culture-expanded MSCs or culture-expanded osteoblasts may have undergone transfection or transduction to introduce nucleic acid sequences encoding the production of proteins that promote osteogenesis (e.g., transcription factors such as osterix, and RUNX2, BMPs and other growth factors, among others).
  • Administration of cells can be achieved in a variety of ways, in each case as clinically warranted, using a variety of anatomic access devices, a variety of administration devices, and a variety of anatomic approaches, with or without support of anatomic imaging modalities (e.g., radiologic, MRI, ultrasound, etc.) or mapping technologies (e.g., epiphysiologic mapping procedures, electromyographic procedures, electrodiagnostic procedures, etc.).
  • anatomic imaging modalities e.g., radiologic, MRI, ultrasound, etc.
  • mapping technologies e.g., epiphysiologic mapping procedures, electromyographic procedures, electrodiagnostic procedures, etc.
  • compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered systemically, via syringe-based injection, or either peripheral vascular access (e.g., intravenous placement, peripheral venous access devices, etc.) or central vascular access (e.g., central venous catheter/devices, arterial access devices/approaches, etc.).
  • peripheral vascular access e.g., intravenous placement, peripheral venous access devices, etc.
  • central vascular access e.g., central venous catheter/devices, arterial access devices/approaches, etc.
  • the compositions, pharmaceutical compositions and cell populations of the present disclosure can be delivered intravascularly into anatomic feeder vessels of an intended tissue site using catheter- based approaches or other vascular access devices (e.g., cardiac catheterization, etc.) that will deliver a vascular bolus of cells to the intended site.
  • compositions, pharmaceutical compositions and cell populations of the present disclosure can be introduced into the spinal canal and/or intraventricularly intrathecally, into the subarachnoid space to distribute within cerebrospinal fluid and/or within the ventricles).
  • the compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered directly into body cavities or anatomic compartments by either catheter-based approaches or direct injection (e.g., intraperitoneal, intrapleural, intrapericardial, intravesicularly (e.g., into bladder, into gall bladder, into bone marrow, into biliary system (including biliary duct and pancreatic duct network), intraurethrally, via renal pelvis/intraureteral approaches, intravaginally, etc.)).
  • catheter-based approaches or direct injection e.g., intraperitoneal, intrapleural, intrapericardial, intravesicularly (e.g., into bladder, into gall bladder, into bone marrow, into biliary system (including biliary duct
  • compositions, pharmaceutical compositions and cell populations of the present disclosure can be introduced by direct local tissue injection, using either intravascular approaches (e.g., endomyocardial injection), or percutaneous approaches, or via surgical exposure/approaches to the tissue, or via laparoscopic/thoracoscopic/endoscopic/colonoscopic approaches, or directly into anatomically accessible tissue sites and/or guided by imaging techniques (e.g., intra-articular, intra-ocular, into spinal discs and other cartilage, into bones, into muscles, into skin, into connective tissues, and into relevant tissues/organs such as central nervous system, peripheral nervous system, heart, liver, kidneys, spleen, joints, eye, etc.).
  • intravascular approaches e.g., endomyocardial injection
  • percutaneous approaches e.g., percutaneous approaches, or via surgical exposure/approaches to the tissue, or via laparoscopic/thoracoscopic/endoscopic/colonoscopic approaches, or directly into anatomically accessible tissue sites and/or guided by imaging techniques (e.g., intra-
  • compositions, pharmaceutical compositions and cell populations of the present disclosure can also be placed directly onto relevant tissue surfaces/sites (e.g., placement onto tissue directly, onto ulcers, onto burn surfaces, onto serosal or mucosal surfaces, onto epicardium, etc.).
  • the compositions, pharmaceutical compositions and cell populations of the present disclosure can also administered into tissue or structural support devices (e.g., tissue scaffold devices and/or embedded within scaffolds placed into tissues, etc.), and/or administered in gels, and/or administered together with enhancing agents (e.g., admixed with supportive cells, cytokines, growth factors, resolvins, anti-inflammatory agents, etc.).
  • compositions, pharmaceutical compositions and cell populations of the present disclosure are administered to the subject following enforced glycosylation of the cell surface (i.e., following glycocalyx modification).
  • the enforced glycosylation on the surface of administered cells will aid in revascularization, in host defense (e.g., against infection or cancer) and/or in tissue repair/regeneration and/or mediate immunomodulatory processes that will dampen inflammation and/or prevent inflammation.
  • the enforced glycosylation pattern guides delivery of intravascularly administered cells to the marrow, to the skin, or to sites of inflammation/tissue injury by mediating binding of blood-borne cells to vascular E-selectin expressed on endothelial cells at sites of inflammation.
  • the enforced expression of ligands for E-selectin and/or L-selectin on administered cells promotes lodgment of cells within the affected tissue milieu, in apposition to cells bearing E-selectin (i.e., endothelial cells) and/or L-selectin (i.e., leukocytes), respectively, within the target site.
  • E-selectin i.e., endothelial cells
  • L-selectin i.e., leukocytes
  • the present methods augment efficiency in the delivery of relevant cells at or to a site of inflammation, tissue injury, or cancer, including, for example, the capacity to deliver immunomodulatory cells (e.g., mesenchymal stem cells).
  • immunomodulatory cells e.g., mesenchymal stem cells.
  • the disease, disorder, or medical condition having associated inflammation can be treated using the instant methods even in the absence of differentiation of the cell population in the subject. That is, there are trophic effects of administered cells at the site of inflammation without persistent engraftment and/or repopulation of the administered cells, irrespective of the type of tissue involved.
  • trophic effects include release of cytokines/growth factors that promote revascularization (e.g., VEGF), that promote tissue repair (e.g., TGF- ⁇ ), that are immunomodulatory (e.g., IL-10), that stimulate growth/proliferation of tissue-resident progenitors (e.g., SCF, LIF, etc) and many other tissue-reparative processes (e.g., mitochondria delivery to cells).
  • cytokines/growth factors that promote revascularization
  • tissue repair e.g., TGF- ⁇
  • immunomodulatory e.g., IL-10
  • stimulate growth/proliferation of tissue-resident progenitors e.g., SCF, LIF, etc
  • tissue-reparative processes e.g., mitochondria delivery to cells.
  • administered cells e.g., MSCs
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising conditioned media obtained from a population of CD44 + cells that have been (1) glycocalyx-modified ex vivo via exofucosylation to enforce hematopoietic cell E-Selectin/L-Selectin Ligand (HCELL) expression and treated with E- selectin or L-selectin for a period of time sufficient to prime the cells to produce interleukin-10 (IL-10) and at least one additional anti-inflammatory cytokine; or (2) modified ex vivo via treatment with hyaluronic acid (HA) for a period of time sufficient to prime the cells to produce interleukin-10 (IL-10) and at least one additional anti-inflammatory cytokine, wherein the modified cells of (1) or (2) produce elevated levels of IL-10 and at least one additional anti- inflammatory cytokine relative to a native population of CD44 + cells.
  • HCELL hematopoietic cell E-Selectin/L-Selectin Ligand
  • the conditioned media may be administered to a subject by any of the methods and routes of administration disclosed herein. In some embodiments, the conditioned media may be administered to a subject in aerosolized form. Indications [0075] The present disclosure is directed to the treatment of a disease, disorder, or medical condition wherein E-selectin is expressed in endothelial beds of the affected tissue(s) and/or L- selectin-expressing leukocytes have infiltrated/accumulated in the affected tissue(s).
  • E-selectin and L-selectin each bind to sialylated, fucosylated carbohydrates, and enforced expression of these sialofucosylated glycan structures on the cell surface serves to program binding to these selectins.
  • Pertinent salutary clinical effects may be engendered by the biologic activity of the infiltrating cell itself (e.g., immunologic effects mediated by a leukocyte, a CAR-T/NK cell, a regulatory T cell or B cell, etc.), and/or by the release of tissue-reparative/anti-inflammatory (trophic) factors, and/or by release of (desired) cytotoxic agents by the infiltrating cell, and/or or by differentiation of the infiltrating cell into a given cell type within the tissue milieu.
  • the methods described herein have utility in improving the outcome of any cell-based therapeutic approach, be it in host defense (e.g., infusion of any type of leukocyte), immunotherapy applications (e.g.
  • stem and/or progenitor cells or other tissue- reparative cells for tissue regeneration/restoration use of stem and/or progenitor cells or other tissue- reparative cells for tissue regeneration/restoration; use of culture-expanded stem cells and/or culture-expanded progenitor cells for tissue regeneration/restoration).
  • administered cells may themselves contribute to regenerate the target tissue by way of long-term engraftment (with attendant proliferation/differentiation) yielding tissue-specific cells (e.g. , such as in transplantation of hematopoietic stem cells for blood cell production) and/or may deliver a tissue restorative/reparative effect without long-term engraftment or differentiation into tissue-resident cells (e.g.
  • tissue injury/damage or neoplastic conditions may be treated in accordance with the methods described herein, including, but not limited to those initiated by direct tissue injury (e.g., burns, trauma, bone fracture, bone deformities, decubitus ulcers, etc.), ischemic/vascular events (e.g., myocardial infarct, stroke, shock, hemorrhage, coagulopathy, etc.), infections (e.g. , cellulitis, pneumonia, meningitis, cystitis, sepsis, SIRS, etc.), neoplasia (e.g.
  • direct tissue injury e.g., burns, trauma, bone fracture, bone deformities, decubitus ulcers, etc.
  • ischemic/vascular events e.g., myocardial infarct, stroke, shock, hemorrhage, coagulopathy, etc.
  • infections e.g. , cellulitis, pneumonia, meningitis, cystitis, sepsis, SIRS
  • immunologic/autoimmune conditions e.g. , acute or chronic GVHD, multiple sclerosis, diabetes, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), rheumatoid arthritis, psoriasis, etc.), degenerative diseases (e.g., osteoporosis, osteoarthritis, spinal disc degeneration, Alzheimer's disease, atherosclerosis, etc.), congenital/genetic diseases (e.g.
  • Acute Leukemias e.g., Acute Biphenotypic Leukemia, Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), and Acute Undifferentiated Leukemia;
  • Myelodysplastic Syndromes Amyloidosis (of any type), Chronic Myelomonocytic Leukemia (CMML), Refractory Anemia (RA), Refractory Anemia with Excess Blasts (RAEB), Refractory Anemia with Excess Blasts in Transformation (RAEB-T), and Refractory Anemia with Ringed Sideroblasts
  • Adrenoleukodystrophy Alpha Mannosidosis, Gaucher's Disease, Hunter's Syndrome (MPS-II), Hurler's Syndrome (MPS-IH), Krabbe Disease, Maroteaux-Lamy Syndrome (MPS-VI), Metachromatic Leukodystrophy, Morquio Syndrome (MPS-IV), Mucolipidosis II (l-cell Disease), Mucopolysaccharidoses (MPS), Niemann-Pick Disease, Sanfilippo Syndrome (MPS-III), Scheie Syndrome (MPS-IS), Sly Syndrome, Beta- Glucuronidase Deficiency (MPS-VII), and Wolman Disease; [0083] Inherited Erythrocyte Abnormalities, e.g.,_Beta Thalassemia, Blackfan-Diamond Anemia, Pure Red Cell Aplasia, and Sickle Cell Disease; [0084] Inherited Platelet Abnormalities, e.g., Amegakaryocytosis/Congenital Thrombocyto
  • Articular and skeletal diseases/conditions e.g., disc degeneration, synovial disease, cartilage degeneration, cartilage trauma, cartilage tears, arthritis, bone fractures, bone deformities, bone reconstruction, osteogenesis imperfecta, congenital bone diseases/conditions, genetic bone diseases/conditions, osteoporosis, osteopetrosis, hypophosphatasia, metabolic bone disease, etc.
  • Skin/soft tissue diseases and conditions such as bullous diseases, psoriasis, eczema, epidermolysis bullosa, ulcerative skin conditions, soft tissue deformities (including post-surgical skin and soft tissue deformities), plastic surgery/reconstructive surgery indications, etc.
  • associated inflammation symptoms that include, without limitation, fever, pain, edema, hyperemia, erythema, bruising, tenderness, stiffness, swollenness, chills, respiratory distress, hypotension, hypertension, stuffy nose, stuffy head, breathing problems, fluid retention, blood clots, loss of appetite, weight loss, polyuria, nocturia, anuria, dyspnea, dyspnea on exertion, muscle weakness, sensory changes, increased heart rate, decreased heart rate, arrythmias, polydipsia, formation of granulomas, fibrinous, pus, non-viscous serous fluid, or ulcers.
  • the actual symptoms associated with an acute and/or chronic inflammation are well known and can be determined by a person of ordinary skill in the art by taking into account factors, including, without limitation, the location of the inflammation, the cause of the inflammation, the severity of the inflammation, the tissue or organ affected, and the associated disorder.
  • Specific patterns of acute and/or chronic inflammation are seen during particular situations that arise in the body, such as when inflammation occurs on an epithelial surface, or pyogenic bacteria are involved.
  • granulomatous inflammation is an inflammation resulting from the formation of granulomas arising from a limited but diverse number of diseases, include, without limitation, tuberculosis, leprosy, sarcoidosis, and syphilis.
  • Purulent inflammation is an inflammation resulting in large amount of pus, which consists of neutrophils, dead cells, and fluid. Infection by pyogenic bacteria such as staphylococci is characteristic of this kind of inflammation.
  • Serous inflammation is an inflammation resulting from copious effusion of non- viscous serous fluid, commonly produced by mesothelial cells of serous membranes, but may be derived from blood plasma. Skin blisters exemplify this pattern of inflammation.
  • Ulcerative inflammation is an inflammation resulting from the necrotic loss of tissue from the epithelial surface, exposing lower layers and forming an ulcer.
  • An acute and/or chronic inflammation symptom can be associated with a large, unrelated group of disorders which underlay a variety of diseases and disorders.
  • Non-immune diseases with etiological origins in acute and/or chronic inflammatory processes include amyloidosis, cancer, atherosclerosis, and ischaemic heart disease.
  • an acute and/or chronic inflammation comprises a tissue inflammation.
  • tissue inflammation is an acute and/or chronic inflammation that is confined to a particular tissue or organ.
  • a tissue inflammation may comprise a skin inflammation, a muscle inflammation, a tendon inflammation, a ligament inflammation, a bone inflammation, a cartilage/joint inflammation, a lung inflammation, a heart inflammation, a liver inflammation, a gall bladder inflammation, a pancreatic inflammation, a kidney inflammation, a bladder inflammation, an gum inflammation, an esophageal inflammation, a stomach inflammation, an intestinal inflammation, an anal inflammation, a rectal inflammation, a vessel inflammation, a vaginal inflammation, a uterine inflammation, a testicular inflammation, a penile inflammation, a vulvar inflammation, a neuron inflammation, an oral inflammation, an ocular inflammation, an aural inflammation, a brain inflammation, a ventricular/meningial inflammation and/or inflammation involving central or peripheral nervous system cells/elements.
  • an acute and/or chronic inflammation comprises a systemic inflammation.
  • systemic inflammation is not confined to a particular tissue but rather involves multiple sites within the body, involving the epithelium, endothelium, nervous tissues, serosal surfaces and organ systems.
  • sepsis can be used, with bacteremia being applied specifically for bacterial sepsis and viremia specifically to viral sepsis.
  • Vasodilation and organ dysfunction are serious problems associated with widespread infection that may lead to septic shock and death.
  • an acute and/or chronic inflammation is induced by an arthritis.
  • Arthritis includes a group of conditions involving damage to the joints of the body due to the inflammation of the synovium including, for example, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthropathies like ankylosing spondylitis, reactive arthritis (Reiter's syndrome), psoriatic arthritis, enteropathic arthritis associated with inflammatory bowel disease, Whipple disease and Behcet disease, septic arthritis, gout (also commonly referred to as gouty arthritis, crystal synovitis, metabolic arthritis), pseudogout (calcium pyrophosphate deposition disease), and Still's disease.
  • osteoarthritis rheumatoid arthritis
  • juvenile idiopathic arthritis juvenile idiopathic arthritis
  • spondyloarthropathies like ankylosing spondylitis reactive arthritis (Reiter's syndrome)
  • psoriatic arthritis enteropathic arthritis associated with inflammatory bowel disease
  • Arthritis can affect a single joint (monoarthritis), two to four joints (oligoarthritis) or five or more joints (polyarthritis) and can be either an autoimmune disease or a non-autoimmune disease.
  • an acute and/or chronic inflammation is induced by an autoimmune disorder.
  • Autoimmune diseases can be broadly divided into systemic and organ- specific autoimmune disorders, depending on the principal clinico-pathologic features of each disease.
  • Systemic autoimmune diseases include, for example, systemic lupus erythematosus (SLE), Sjogren's syndrome, Scleroderma, rheumatoid arthritis and polymyositis.
  • Local autoimmune diseases may be endocrinologic (Diabetes Mellitus Type 1 , Hashimoto's thyroiditis, Addison's disease, etc.), dermatologic (pemphigus vulgaris), hematologic (autoimmune haemolytic anemia), neural (multiple sclerosis) or can involve virtually any circumscribed mass of body tissue.
  • endocrinologic Diabetes Mellitus Type 1 , Hashimoto's thyroiditis, Addison's disease, etc.
  • dermatologic pemphigus vulgaris
  • hematologic autoimmune haemolytic anemia
  • neural multiple sclerosis
  • Types of autoimmune disorders include, without limitation, acute disseminated encephalomyelitis (ADEM), Addison's disease, an allergy or sensitivity, amyotrophic lateral sclerosis (ALS), anti-phospholipid antibody syndrome (APS), arthritis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune pancreatitis, bullous pemphigoid, celiac disease, Chagas disease, chronic obstructive pulmonary disease (COPD) (including acute exacerbations thereof), diabetes mellitus type 1 (IDDM), endometriosis, fibromyalgia, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD), interstitial cystitis, lupus (including discoid lup
  • the acute and/or chronic inflammation results from or is otherwise caused by diabetes in the subject. In another particular embodiment, the acute and/or chronic inflammation results from or is otherwise caused by multiple sclerosis in the subject.
  • an acute and/or chronic inflammation is induced by a myopathy.
  • myopathies are caused when the immune system inappropriately attacks components of the muscle, leading to inflammation in the muscle.
  • a myopathy includes, for example, an inflammatory myopathy and an autoimmune myopathy.
  • Myopathies include, for example, dermatomyositis, inclusion body myositis, and polymyositis.
  • an acute and/or chronic inflammation is induced by a vasculitis.
  • Vasculitis is a varied group of disorders featuring inflammation of a vessel wall including lymphatic vessels and blood vessels like veins (phlebitis), arteries (arteritis) and capillaries due to leukocyte migration and resultant damage.
  • the inflammation may affect any size blood vessel, anywhere in the body. It may affect either arteries and/or veins.
  • the inflammation may be focal, meaning that it affects a single location within a vessel, or it may be widespread, with areas of inflammation scattered throughout a particular organ or tissue, or even affecting more than one organ system in the body.
  • Vasculitis include, without limitation, Buerger's disease (thromboangiitis obliterans), cerebral vasculitis (central nervous system vasculitis), ANCA- associated vasculitis, Churg-Strauss arteritis, cryoglobulinemia, essential cryoglobulinemic vasculitis, giant cell (temporal) arteritis, Golfer's vasculitis, Henoch-Schonlein purpura, hypersensitivity vasculitis (allergic vasculitis), Kawasaki disease, microscopic polyarteritis/polyangiitis, polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatoid vasculitis, Takayasu arteritis, Wegener's granulomatosis, and vasculitis secondary to connective tissue disorders like systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), relaps
  • an acute and/or chronic inflammation is induced by a skin disorder.
  • Skin disorders include, for example, an acne, including acne vulgaris, a bullous phemigoid, a dermatitis, including atopic dermatitis and acute and/or chronic actinic dermatitis, an eczema-like atopic eczema, contact eczema, xerotic eczema, seborrhoeic dermatitis, dyshidrosis, discoid eczema, venous eczema, dermatitis, dermatitis herpetiformis, neurodermatitis, and autoeczematization, and stasis dermatitis, diabetic skin complications, hidradenitis suppurativa, lichen planus, psoriasis including plaqure psoriasis, nail psoriasis, guttate psoriasis, scalp
  • an acute and/or chronic inflammation is induced by a gastrointestinal disorder.
  • a gastrointestinal disorder includes, for example, irritable bowel disease (IBD), an inflammatory bowel disease including Crohn's disease and an ulcerative colitis like ulcerative proctitis, left-sided colitis, pancolitis, and fulminant colitis.
  • IBD irritable bowel disease
  • an acute and/or chronic inflammation is induced by a cardiovascular disease. When LDL cholesterol becomes embedded in arterial walls, it can invoke an immune response. Acute and/or chronic inflammation eventually can damage the arteries, which can cause them to burst.
  • cardiovascular disease is any of a number of specific diseases that affect the heart itself and/or the blood vessel system, especially the veins and arteries leading to and from the heart.
  • cardiovascular disorders including, for example, a hypertension, endocarditis, myocarditis, heart valve dysfunction, congestive heart failure, myocardial infarction, a diabetic cardiac conditions, blood vessel inflammation like arteritis, phlebitis, vasculitis; arterial occlusive disease like arteriosclerosis and stenosis, inflammatory cardiomegaly, a peripheral arterial disease; an aneurysm; an embolism; a dissection; a pseudoaneurysm; a vascular malformation; a vascular nevus; a thrombosis; a thrombophlebitis; a varicose veins; a stroke.
  • Symptoms of a cardiovascular disorder affecting the heart include, without limitation, chest pain or chest discomfort (angina), pain in one or both arms, the left shoulder, neck, jaw, or back, shortness of breath, dizziness, faster heartbeats, nausea, abnormal heartbeats, feeling fatigued.
  • Symptoms of a cardiovascular disorder affecting the brain include, without limitation, sudden numbness or weakness of the face, arm, or leg, especially on one side of the body, sudden confusion or trouble speaking or understanding speech, sudden trouble seeing in one or both eyes, sudden dizziness, difficulty walking, or loss of balance or coordination, sudden severe headache with no known cause.
  • Symptoms of a cardiovascular disorder affecting the legs, pelvis and/or arm include, without limitation, claudication, which is a pain, ache, or cramp in the muscles, and cold or numb feeling in the feet or toes, especially at night.
  • claudication which is a pain, ache, or cramp in the muscles, and cold or numb feeling in the feet or toes, especially at night.
  • an acute and/or chronic inflammation is induced by a cancer.
  • inflammation orchestrates the microenvironment around tumors, contributing to proliferation, survival and migration.
  • fibrinous inflammation results from a large increase in vascular permeability which allows fibrin to pass through the blood vessels. If an appropriate procoagulative stimulus is present, such as cancer cells, a fibrinous exudate is deposited.
  • an acute and/or chronic inflammation is a pharmacologically induced inflammation.
  • Certain drugs or exogenic chemical compounds including deficiencies in key vitamins and minerals, are known to effect inflammation.
  • Vitamin A deficiency causes an increase in an inflammatory response
  • Vitamin C deficiency causes connective tissue disease
  • Vitamin D deficiency leads to osteoporosis.
  • Certain pharmacologic agents can induce inflammatory complications, e.g., drug-induced hepatitis.
  • an acute and/or chronic inflammation is induced by an infection.
  • An infectious organism can escape the confines of the immediate tissue via the circulatory system or lymphatic system, where it may spread to other parts of the body. If an organism is not contained by the actions of acute inflammation it may gain access to the lymphatic system via nearby lymph vessels.
  • An infection of the lymph vessels is known as lymphangitis, and infection of a lymph node is known as lymphadenitis.
  • a pathogen can gain access to the bloodstream through lymphatic drainage into the circulatory system.
  • Infections include, without limitation, bacterial cystitis, bacterial encephalitis, pandemic influenza, viral encephalitis, and viral hepatitis (A, B and C).
  • an acute and/or chronic inflammation is induced by a tissue or organ injury. Tissue or organ injuries include, without limitation, a burn, a laceration, a wound, a puncture, or a trauma.
  • an acute and/or chronic inflammation is induced by a transplant rejection. Transplant rejection occurs when a transplanted organ or tissue is not accepted by the body of the transplant recipient because the immune system of the recipient attacks the transplanted organ or tissue.
  • transplant rejection is mediated through both T-cell-mediated and humoral immune (antibodies) mechanisms.
  • a transplant rejection can be classified as a hyperacute rejection, an acute rejection, or a chronic rejection.
  • Acute and/or chronic rejection of a transplanted organ or tissue is where the rejection is due to a poorly understood acute and/or chronic inflammatory and immune response against the transplanted tissue.
  • graft-versus-host disease GVHD
  • GVHD graft-versus-host disease
  • GVHD is a common complication of allogeneic bone marrow transplantation in which functional immune cells in the transplanted marrow recognize the recipient as "foreign" and mount an immunologic attack. It can also take place in a blood transfusion under certain circumstances.
  • GVHD is divided into acute and chronic forms. Acute and chronic GVHD appear to involve different immune cell subsets, different cytokine profiles, somewhat different host targets, and respond differently to treatment.
  • an acute and/or chronic inflammation is induced by a Th1 -mediated inflammatory disease.
  • Th1 -mediated inflammatory disease In a well- functioning immune system, an immune response should result in a well-balanced pro- inflammatory Th1 response and anti-inflammatory Th2 response that is suited to address the immune challenge.
  • Th1 response In a well- functioning immune system, an immune response should result in a well-balanced pro- inflammatory Th1 response and anti-inflammatory Th2 response that is suited to address the immune challenge.
  • Th1 response the body relies on the anti-inflammatory response invoked by a Th2 response to counteract this Th1 response.
  • Th2 type cytokines such as, e.g., IL- 4, IL-5, and IL-13 which are associated with the promotion of IgE and eosinophilic responses in atopy, and also IL-10, which has an anti-inflammatory response.
  • Th1 -mediated inflammatory disease involves an excessive proinflammatory response produced by Th1 cells that leads to acute and/or chronic inflammation.
  • the Th1 -mediated disease may be virally, bacterially or chemically (e.g., environmentally) induced.
  • a virus causing the Th1 -mediated disease may cause a chronic or acute infection, which may cause a respiratory disorder or influenza.
  • an acute and/or chronic inflammation comprises an acute and/or chronic neurogenic inflammation.
  • Acute and/or chronic neurogenic inflammation refers to an inflammatory response initiated and/or maintained through the release of inflammatory molecules like Substance P (SP) or calcitonin gene-related peptide (CGRP) which released from peripheral sensory nerve terminals (i.e., an efferent function, in contrast to the normal afferent signaling to the spinal cord in these nerves).
  • SP Substance P
  • CGRP calcitonin gene-related peptide
  • Acute and/or chronic neurogenic inflammation includes both primary inflammation and secondary neurogenic inflammation.
  • Primary neurogenic inflammation refers to tissue inflammation (inflammatory symptoms) that is initiated by, or results from, the release of substances from primary sensory nerve terminals (such as C and A-delta fibers).
  • Secondary neurogenic inflammation refers to tissue inflammation initiated by non- neuronal sources (e.g., extravasation from vascular bed or tissue interstitium-derived, such as from mast cells or immune cells) of inflammatory mediators, such as peptides or cytokines, stimulating sensory nerve terminals and causing a release of inflammatory mediators from the nerves.
  • inflammatory mediators such as peptides or cytokines
  • the net effect of both forms (primary and secondary) of acute and/or chronic neurogenic inflammation is to have an inflammatory state that is maintained by the sensitization of the peripheral sensory nerve fibers.
  • the retained biological activity makes it possible to achieve a clinical benefit from administering MSCs obtained from diseased tissue and/or tissue of subjects of advanced age in a single administration and/or low cell dose.
  • the glycocalyx-modified cells disclosed herein it is possible to treat subjects with autologous cells in a single administration and/or low cell dose, even when MSCs are obtained from diseased tissue and/or tissue of subjects of advanced age.
  • the low dose compositions including glycocalyx-modified cell populations, pharmaceutical compositions and use of such compositions in the methods disclosed herein comprises at least about 50,000 cells/kg (based on the weight of the subject), at least about 200,000 cells/kg (based on the weight of the subject), at least about 400,000 cells/kg (based on the weight of the subject), at least about 500,000 cells/kg (based on the weight of the subject), at least about 600,000 cells/kg (based on the weight of the subject), at least about 700,000 cells/kg (based on the weight of the subject), at least about 800,000 cells/kg (based on the weight of the subject), or at least about 900,000 cells/kg (based on the weight of the subject).
  • the low dose compositions, including cell populations, pharmaceutical compositions and use of such compositions in the methods disclosed herein comprises about 50,000 glycocalyx-modified cells, about 200,000 glycocalyx-modified cells, about 400,000 glycocalyx-modified cells, about 500,000 glycocalyx-modified cells, about 600,000 glycocalyx- modified cells, about 700,000 glycocalyx-modified cells, about 800,000 glycocalyx-modified cells, or about 900,000 glycocalyx-modified cells.
  • the low dose compositions, including cell populations, pharmaceutical compositions and use of such compositions in the methods disclosed herein comprise less than 200,000 glycocalyx-modified cells.
  • the MSCs disclosed herein may be primed to produce anti-inflammatory molecules.
  • the present disclosure provides compositions and methods directed to one or more MSCs that have been modified ex vivo to increase production of one or more anti-inflammatory or immunomodulatory molecules via binding of CD44 with a ligand.
  • the term “ligand” and grammatical variation thereof means a natural or artificial molecule(s) which bind to CD44 directly or indirectly, and that is effective to promote production of anti-inflammatory or immunomodulatory molecules when ligated to the CD44 present on a cell.
  • the CD44 is ligated ex vivo with a molecule that is effective to promote production of anti-inflammatory or immunomodulatory molecules by a cell.
  • CD44 ligands include naturally occurring ligands (such as an extracellular matrix component) or artificial ligands that are effective to promote production of anti-inflammatory or immunomodulatory molecules by a cell.
  • CD44 ligands that are effective to promote production of anti-inflammatory or immunomodulatory molecules by a cell include, but are not limited to, hyaluronic acid (HA), osteopontin (OPN), bone morphometric proteins (BMPs), TGF (and other tissue reperative cytokines), collagens (e.g., Type I and VI), serglycins, galectins (e.g. gal-8), Siglecs, matrix metalloproteinases (MMPs), ARHGEF1, Ezrin (via PIP2), epidermal growth factor receptor (Hyaluronan-dependent), fibrin and fibrinogen, fibronectin, FYN, Lck, selectins, and Src.
  • HA hyaluronic acid
  • OPN osteopontin
  • BMPs bone morphometric proteins
  • TGF and other tissue reperative cytokines
  • collagens e.g., Type I and VI
  • serglycins e.g.,
  • the CD44 ligands that are effective to promote production of anti-inflammatory and/or immunomodulatory molecules by a cell are selectins. In some embodiments, the CD44 ligands that are effective to promote production of anti- inflammatory and/or immunomodulatory molecules by a cell include, but are not limited to, E- selectin, L-selectin, and P-selectin. [0121] In some embodiments, the CD44 ligand that is effective to promote production of anti-inflammatory and/or immunomodulatory molecules by a cell is hyaluronic acid (HA).
  • HA hyaluronic acid
  • the HA is a high molecular weight HA (HW HA) that exhibits pro-inflammatory effects in vivo, such as HA with a molecular weight of at least 100,000 daltons.
  • the CD44 is ligated with a molecule with a specific binding affinity for CD44, such as for example, an antibody or antigen binding fragment thereof derived from any animal source, including, but not limited to monoclonal antibodies, polyclonal antibodies, phagemids, aptamers, Camel Ig (a camelid antibody (VHH)), Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibody (“scFv”), bis- scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”), and single-domain antibody (scFv”), and single-domain antibody (
  • the CD44 is ligated with a monoclonal or polyclonal antibody effective to initiate or enhance production of anti- inflammatory cytokines in the CD44+ cells.
  • the CD44 is ligated with a ligand that is effective to enhance the binding of HA to the CD44.
  • the CD44 is ligated with a monoclonal or polyclonal antibody that is effective to enhance the binding of HA to the CD44.
  • antibodies that enhance the binding of HA to CD44 include, but are not limited to, IRAWB14 antibody (see, e.g, Zheng, Z., et al., Monoclonal Antibodies to CD44 and Their Influence on Hyaluronan Recognition, The Journal of Cell Biology, Vol. 130, No. 2, 485-495).
  • the cell bearing CD44 is activated with a small molecule that is effective in enhancing the capacity of CD44 to bind to HA.
  • the CD44 ligand may be a modified ligand.
  • the CD44 ligand may be a hybrid of, or conjugated to, one or more other molecules.
  • an E-selectin may be an E-selectin or L-selectin chimera with IgG (i.e., an E-Ig chimera or L-Ig chimera).
  • the cells are treated with the CD44 ligand in amounts and for durations effective to promote production of anti-inflammatory and/or immunomodulatory molecules in the CD44+ cells.
  • the cells are treated for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, or at least 48 hours.
  • the cells are treated with the CD44 ligand for between about 30 minutes and about 48 hours, such as between about 30 minutes and about 2 hours, between about 30 minutes and 90 minutes, and between about 30 minutes and one hour.
  • the CD44 is ligated ex vivo with HA for a period of time sufficient to prime the CD44+ cells to initiate or enhance production of anti-inflammatory cytokines.
  • the CD44 is altered prior to, or concurrent with, ligation to promote interaction with a ligand.
  • the interactions of CD44 with ligands may be regulated via certain naturally occurring modifications to the intracellular or extracellular regions of CD44.
  • Those naturally occurring modifications may block or reduce the ability of CD44 to bind to a ligand.
  • the naturally occurring modifications of CD44 are altered ex vivo to permit and/or promote binding of a ligand to CD44.
  • terminal sialic acids on CD44 O-glycans or N-glycans which are known to block HA binding, are removed by treatment with one or more sialidases, prior to or concurrent with ligation of HA.
  • the glycans decorating CD44 are altered to promote binding of one or more ligands, such as selectins.
  • CD44 expressing cells are treated ex vivo with one or more glycosidases and/or glycosyltransferases to construct on the CD44 a glycan structure that is effective for ligand binding.
  • CD44 expressing cells are treated ex vivo with one or more fucosyltransferases, for example, to enforce expression of HCELL, which binds to E-selectin and L-selectin.
  • a modification of a cell’s glycocalyx is achieved by use of a glycosyltransferase to install a chemically reactive group, orthogonal functional group, or molecular tag that is attached to the donor nucleotide sugar.
  • fucosyltransferase-mediated installation of a modified-fucose by use of a donor GDP-fucose wherein the fucose has been modified by methods known in the art with a chemically reactive group, or orthogonal functional group, or molecular tag (e.g., biotinylated GDP-fucose, azido- GDP-fucose, etc.) thereby allowing for subsequent linkage of other molecules onto the installed fucose within cell surface lactosaminyl glycans (examples of this approach include, but are not limited to, use of biotinylated GDP-fucose with subsequent complexing using streptavidin- conjugated molecules and/or use of “click chemistry” wherein the azido-containing fucose molecule is then complexed to an alkyne-containing molecule).
  • a chemically reactive group, or orthogonal functional group, or molecular tag e.g., biotinyl
  • molecules covalently linked to the donor nucleotide fucose can be stereospecifically added in a distinct pattern onto cell surface lactosaminyl glycans to endow CD44 with the ability to bind to desired ligand.
  • the CD44 is modified ex vivo to provide structures receptive to ligands.
  • the CD44 is modified ex vivo via exofucosylation to enforce hematopoietic cell E-Selectin/L-Selectin Ligand (HCELL) expression.
  • the modified CD44 may be ligated with one or more of a selectin (e.g., E-selectin and L-selectin) and monoclonal antibodies (mAbs) (e.g., the mAbs “CSLEX-1” and “HECA452”) effective to increase production of anti-inflammatory or immunomodulatory molecules in the CD44 + cells.
  • a selectin e.g., E-selectin and L-selectin
  • mAbs monoclonal antibodies
  • the CD44 modifying enzymes e.g., glycosidases, glycosyltransferases and fucosyltransferases are obtained from any convenient source, e.g., purified from eukaryotic or prokaryotic cells or obtained from commercial sources, including R&D Systems, SigmaAldrich, SCHsciences, and CarbExplore Research.
  • ligation of the CD44 is effective to produce elevated levels of at least one anti-inflammatory or immunomodulatory molecule in the CD44 + cells.
  • anti-inflammatory molecule and grammatical variation thereof means any molecule produced by a cell that acts to dampen inflammation, for example, by suppressing or restraining the action(s) of inflammatory effectors in a cell or tissue.
  • immunomodulatory molecule and grammatical variation thereof means any molecule produced by a cell that modulates an innate or adaptive immune response, excluding deleterious molecules that exacerbate an inflammatory disease or condition (e.g., pro-inflammatory molecules).
  • the anti-inflammatory molecule is an anti-inflammatory cytokine.
  • cytokine includes, but is not limited to, leukocyte-generated peptides (e.g., lymphocyte-generated lymphokines, monocyte-produced monokines), chemokines, interferons, interleukins, adipocyte-secreted adipokines and muscle-generated myokines.
  • the anti-inflammatory molecules include, but are not limited to, Interleukin-10 (IL- 10), TGF- ⁇ , IDO, nitric oxide (NO) metabolites, PGE2 and combinations thereof.
  • ligation of the CD44 is effective to elevate production of at least one anti- inflammatory molecule at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to native population of cells, e.g., an untreated population of cells, such as an untreated population of CD44 + cells.
  • ligation of the CD44 is effective to elevate production of at least one immunomodulatory molecule at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to a native population of cells, e.g., an untreated population of cells, such as an untreated population of CD44 + cells.
  • the anti-inflammatory and/or immunomodulatory molecule is released from the CD44 + cells via secretome.
  • the anti-inflammatory and/ or immunomodulatory molecule is released from the CD44 + cells via extracellular vesicle (e.g., exosome). In some embodiments, the anti-inflammatory and/or immunomodulatory molecule is released from the CD44 + cells in soluble form. In some embodiments, anti-inflammatory and/or immunomodulatory molecules are produced by the CD44 + cells via secretome/release of trophic agents.without priming. [0134] In some embodiments, the anti-inflammatory or immunomodulatory molecules produced by the CD44 + cells are effective to promote tissue repair in a subject.
  • the term “subject” and grammatical variation thereof includes, but is not limited to, any mammal, such as a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
  • the production of elevated level of anti-inflammatory cytokines (such as IL-10) by the CD44 + cells are effective to facilitate regenerative healing of damaged tissue in a subject.
  • the production of elevated level of anti-inflammatory cytokines (such as IL-10) by the CD44 + cells are effective to facilitate regenerative healing via one or more of promoting the production and deposition of extracellular matrix components in damaged tissue of a subject, modulating fibroblast function, modulating myofibroblast differentiation, and modulating endothelial progenitor cell survival and function.
  • ligation of the CD44 is effective to produce elevated levels of at least one anti-inflammatory or immunomodulatory molecules in the CD44 + cells that is effective to promote tissue repair in a subject.
  • MSCs Mesenchymal stem/stromal cells
  • Osteoporosis is a progressive generalized skeletal disorder, primarily affecting women due to menopause-associated estrogen loss, whereby osteoclast-mediated bone resorption outpaces osteoblast-mediated bone formation. This process develops within the bone medullary cavity (“marrow cavity”) and predominantly erodes trabecular bone, markedly disrupting skeletal integrity/architecture, resulting in bone weakness and fragility (i.e., spontaneous and/or low- impact) fractures. Globally, one-in-three women over age 50 will suffer an osteoporosis-related fracture mounting to ⁇ 10 million fractures/year, yielding a world-wide rate of a fracture every 3 seconds.
  • osteoporosis is incurable.
  • the principal goal of all osteoporosis therapy is to prevent new fragility fractures.
  • osteoclast-mediated bone resorption e.g., bisphosphonates; denosumab (a monoclonal antibody that inhibits osteoclast development by targeting the cytokine “receptor activator of nuclear factor- ⁇ b ligand” (RANKL) thereby blunting its binding to the osteoclast receptor RANK), and/or stimulating bone formation (“osteoanabolics”: e.g., parathyroid hormone (PTH) analogs; romosozumab (a mAb that targets sclerostin and thereby neutralizes its inhibition of Wnt signaling, leading to both osteoanabolic and antiresorptive effects)).
  • antiresorptives e.g., bisphosphonates
  • denosumab a monoclonal antibody that inhibits osteoclast development by targeting the cytokine “receptor activator of nuclear factor- ⁇ b ligand” (RANKL) thereby blunting its binding to the osteoclast receptor RANK
  • MSCs Mesenchymal stem/stromal cells
  • MSCs are readily harvested from marrow, rapidly expand in tissue culture, and, in particular, bone marrow-derived MSCs (“BM-MSCs”) are distinctly specialized for osteogenesis and osteoregeneration.
  • BM-MSCs bone marrow-derived MSCs
  • osteoblasts are post-mitotic cells whose replenishment depends on BM-MSC differentiation into osteoblasts.
  • MSCs also secrete bioactive molecules that dampen inflammation and support tissue repair.
  • the combination of MSC osteogenic and paracrine-mediated anti-inflammatory properties raises hope that marrow delivery of culture-expanded BM-MSCs at affected skeletal sites could drive bone regeneration, preventing and/or reversing osteoporosis.
  • osteoporosis is a “generalized” disease (i.e., not anatomically localized)
  • IV intravascular
  • BM-MSC autografts are preferable to allografts to avoid immunorejection of osteolineage progeny, but, at odds with this clinical benefit are safety concerns: MSCs originating from older persons or from a diseased tissue source could pose a potential heightened risk of malignancy and other undesired tissue formation, these cells are also more likely to be senescent and/or biologically compromised, and, more intrinsically, functional derangements in BM-MSCs of osteoporotic patients might be contributory to the pathobiology.
  • autoBM- MSCs autologous BM-MSCs
  • osteotropism marrow migration/colonization
  • Distinct glycan motifs within the glycocalyx comprised of defined oligosaccharide clusters containing specific monosaccharides arranged in a characteristic spatial arrangement/configuration, license specific cellular properties impacting multiple biologic events (including cell migration, immune responses, pathogen invasiveness, cell/tissue development, and carcinogenesis).
  • These oligosaccharides are assembled within the Golgi apparatus by action of glycosyltransferases that covalently link, in step-wise fashion, the component monosaccharides in a regio- and stereospecific manner upon a pertinent “acceptor” glycan structure.
  • This chemo-enzymatic reaction can be performed directly on a living cell’s surface (i.e., exogenously) using pertinent glycosyltransferases together with their respective donor nucleotide sugars under reaction conditions that are non-toxic and preserve native cell biology.
  • This technique is called “glycosyltransferase-programmed stereosubstitution” (GPS) and it engenders exceptionally specific and definitive glycocalyx motif-editing.
  • One well-characterized glycocalyx motif is the structure known as “sialylated Lewis X” (sLeX; CD15s), a tetrasaccharide comprised of sialic acid (“NeuAc”), galactose (“Gal”), fucose (“Fuc”), and N-acetylglucosamine (“GlcNAc”) covalently assembled as follows: NeuAc- ⁇ (2,3)-Gal- ⁇ (1,4)-[Fuc- ⁇ (1,3)]-GlcNAc- ⁇ 1-R).
  • sLeX is the canonical binding determinant for E- selectin (CD62E), a Ca2+-dependent lectin constitutively expressed on specialized bone marrow microvessels that serve as gateways for entry of circulating cells into the marrow.
  • E- selectin CD62E
  • Ca2+-dependent lectin constitutively expressed on specialized bone marrow microvessels that serve as gateways for entry of circulating cells into the marrow.
  • Hematopoietic stem/progenitor cells HSPCs
  • sLeX-E-selectin interactions mediate HSPC osteotropism, a prerequisite for successful hematopoietic stem cell transplantation.
  • Natively, MSCs do not express sLeX, and, accordingly, they lack osteotropic capacity.
  • the MSC glycocalyx contains terminal ⁇ (2,3)-sialylated type 2 lactosamines (i.e., NeuAc- ⁇ (2,3)- Gal- ⁇ (1,4)-GlcNAc-R) missing only fucose ⁇ (1,3)-linked to GlcNAc to compose sLeX.
  • this trisaccharide acceptor is displayed uniquely on a glycovariant of the cell membrane glycoprotein CD44.
  • Glycoengineering of human MSC surface “standard” CD44 via ⁇ (1,3)-fucosyltransferase-mediated installation of Fuc results in temporary expression ( ⁇ 48 hours) of sLeX motifs on CD44.
  • HCELL Hematopoietic Cell E-/L-selectin Ligand
  • Osteoporosis was defined via central DXA scan at the lumbar spine (LS) and at the femoral neck (FN) meeting the criteria of a T-score of -2 ⁇ 5 or lower, or T-score ⁇ -1 ⁇ 0 with history of at least one hip, vertebral or forearm fragility fracture. Detailed eligibility criteria are provided in the protocol.
  • Fuc- autoBM-MSC products were released only if meeting technical specifications that included characteristic MSC immunophenotypic profile, >90% cell viability, 100% sLeX expression, sterility (assessed by aerobic and anaerobic microbiologic assays, and endotoxin-negativity), and genetic stability as evaluated by G-band karyotyping and/or CGH-arrays.
  • Fuc-autoBM-MSCs Administration [0159] Patients were hospitalized for infusion.
  • Fuc-autoBM-MSCs (5x10 6 /mL in saline) were administered via peripheral venous catheter at 4-6 mL/min. The first four patients received 2x10 6 cells/Kg, and subsequent six patients received 5x10 6 cells/Kg. Vital signs were monitored during infusion and for 24 hours before discharge. [0160] Radiologic Studies [0161] All patients underwent X-ray studies of dorsal and lumbar spine (AP and lateral), and of pelvis and hips (AP), at baseline, and at 12 months and 24 months post-Fuc-autoBM-MSC infusion.
  • DXA Dual-energy X-ray Absorptiometry
  • QCT Quantitative Computed Tomography
  • QTS Quality of Trabecular Structure
  • High resolution peripheral QCT General Electric 64-MDCT was performed at right distal radius at baseline and at months 4, 12 and 24 post-infusion.
  • Trabecular bone microstructure was quantitatively analyzed by using Quibim Precision Software tool, as previously described 15,16 . This tool provides bone volume percentage, trabecular thickness, separation, and irregularity analysis, and combines these values into a single score (the “QTS”). Higher QTS values indicate better bone quality and lower values indicate a higher fracture risk.
  • BTA Bone Tissue Area
  • Bone biomarker levels were evaluated as percent change from baseline levels (“delta % change”), with values summarized as mean + SEM.
  • delta % change For BTA and QTS score analyses, variance homogeneity was assessed by Levene test followed by Welch's t-test or Student's t-test for non-homogeneous or homogeneous variances, respectively. Kolmogorov-Smirnov test was undertaken to analyze data distribution (non-parametric, p ⁇ 0.05). For assessing changes in areal BMD and volumetric BMD (QTS), delta % change was calculated and Student 2-tailed t-test was employed.
  • Fragility Fracture Incidence is a swimmer’s plot depicting fragility fractures pre- and post-Fuc-AutoBM-MSC infusion. Only one patient (#5) sustained a fragility fracture within 2-years post-infusion.
  • (Right): Incidence of fragility fractures pre-infusion and post-infusion for all years of observation for all patients (p 0.005, Welch ⁇ s t-test).
  • Biomarkers of bone turnover At baseline, and 4, 6, 9, 12, 18 and 24 months post- infusion, serum levels were measured for bone neoformation biomarkers P1NP, osteocalcin (BGLAP), and bone ALP (Fig.2A, B), and for bone resorption biomarkers ⁇ -CTx and NTX (Fig. 3). For each patient, the percent change from baseline values was assessed for all biomarkers and mean + SEM was calculated (Fig. 2B; Fig 3). There was a trend toward increases in bone formation markers P1NP and BGLAP, even when excluding patients (#4, #9, #10) who received teriparatide at any time post-infusion (Fig.2B).
  • FIG.4A Histomorphometric analysis of bone tissue biopsies: Iliac crest biopsies were obtained at baseline and at day +120 post-Fuc-autoBM-MSC administration. Baseline and post- infusion analysis performed per patient (Fig.4A) showed statistically significant increases in mean BTA values in seven patients (#1, #2, #3, #4, #5, #8, #10), decreases in two (#6, #7), and no change in one patient (#9).
  • the most sensitive and specific bone neoformation biomarker is P1NP, and the mean level of this biomarker increased prominently in 8/10 patients, with one patient (#10) having a sharp drop from baseline P1NP levels solely due to discontinuation of teriparatide at 5.5 months post-Fuc-autoBM-MSC infusion ( Figure 2A, B).
  • Osteoporosis is treatable, yet ⁇ 30% of patients that suffer from an osteoporosis- related fracture in the US avoid antiosteoporosis medications, and treatment rates worsen with age.
  • osteoporosis treatment gap highlights the critical unmet medical needs of patients suffering from this disease_ENREF_2: new osteoporosis treatments are desired that are safe, readily adherable/tolerable, and, could engender durable bone regeneration without necessitating recurrent-dosing, life-long therapy.
  • >1500 clinical trials have been undertaken to assess the impact of MSCs for treatment of various medical conditions, but no clinical studies have examined the utility of MSCs in treatment of osteoporosis, nor have examined use of MSCs derived exclusively from older patients for any medical condition.
  • glycan assembly is a highly ordered process, executed by exceptionally precise glycosyltransferase-mediated step-wise addition of relevant monosaccharides onto specific precursors, the “acceptor glycans”.
  • a living cell’s glycocalyx can be custom-modified to display a desired glycan motif via glycosyltransferase- programmed stereo- and regio-specific installation of the relevant monosaccharide(s) on a pertinent acceptor glycan.
  • This compositional editing critically depends on creation of glycosyltransferases that can function at the cell surface (i.e., exogenously) utilizing reaction conditions that are non-toxic to the target cell and preserve the cell’s innate biologic phenotype.
  • HCELL a potent molecular effector of osteotropism.
  • the resultant enforced HCELL expression is transient (duration ⁇ 48 hours) due to natural cell surface CD44 turn-over, and, thereafter, the MSC glycocalyx reverts to its usual composition.
  • exofucosylation does not alter MSC osteogenic capacity as evaluated by in vitro assays, and, also, by in vivo studies of intravascularly administered HCELL+ human MSCs into immunodeficient mice.
  • BM-MSCs were utilized in this study as they have been reported to possess higher osteogenic potential compared to MSCs derived from other tissues (e.g., fat).
  • MSC infusion be an “add-on” to existing therapies in each patient.
  • Autologous, as opposed to allogeneic, BM-MSCs were used to avoid potential immunorejection because osteoblasts are highly immunogenic, potently stimulating T cell alloreactivity. Consistent with this notion, efforts to treat osteogenesis imperfecta (a congenital skeletal disease) using intravascularly administered allogeneic MSCs (without glycocalyx editing) attained only a brief, and relatively modest, osteogenic effect.
  • MSCs are the precursors of osteoblasts
  • MSC-based therapy for osteoporosis there is a paucity of studies related to MSC-based therapy for osteoporosis.
  • osteoporosis and “mesenchymal stem cell” (or “mesenchymal stromal cell” or “MSC”), apart from our study, only one other trial has been registered in CT.gov to assess the applicability of intravenously administered MSCs for osteoporosis treatment: that Phase 1 trial utilized (allogeneic) umbilical cord-derived (UC-derived) MSCs and the trial was suspended (without associated explanation; NCT04501354).
  • MSCs derived from older patients could harbor age-associated “clonal mutations” prompting malignant transformation, and such mutations could also theoretically arise during culture-expansion.
  • MSCs can abet tumorigenicity by contributing to elaboration of the “tumor microenvironment” (TME) that supports tumor growth, tumor immune-evasiveness, and therapy resistance.
  • TEE tumor microenvironment
  • trabecular bone is far more metabolically active than cortical bone, and, accordingly, osteoporosis predominantly disintegrates trabecular bone.
  • This beneficial effect could be secondary to direct differentiation of administered MSCs into osteoblasts (as observed in preclinical human-mouse MSC xenotransplantation studies), and/or an MSC-mediated paracrine influence supporting osteoblast integrity/bioactivity and/or an MSC-based anti-osteoclastic effect and/or an MSC- induced anti-inflammatory effect dampening bone injury and thereby sustaining bone neoformation.
  • osteoporosis treatment gap highlights the critical unmet medical needs of patients suffering from this disease: _ENREF_2 new osteoporosis treatments are desired that are safe, readily adherable/tolerable, and, optimally, could engender durable bone regeneration without necessitating recurrent-dosing, life-long therapy.
  • the capability to perform precision glycocalyx motif-editing to install sLeX display (i.e., enforce HCELL expression) on MSCs has expressly enabled a heretofore unattainable analysis of the biology of culture-expanded human bone marrow-derived MSCs within human beings suffering from osteoporosis, thereby providing a more accurate and complete understanding, as well as a greater appreciation, of how these cells may transform patient care.
  • the finding that administration of glycocalyx motif-edited human MSCs is feasible and safe, together with the promising clinical responses observed following just a single infusion of Fuc- autoBM-MSCs, provides rationale for conducting larger trials to further evaluate the antiosteoporosis efficacy of this treatment approach.
  • Osteoporos Int 33 2049-2102.10.1007/s00198-021-05900-y. Johnell, O., and Kanis, J.A. (2006). An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int 17, 1726-1733.10.1007/s00198-006-0172- 4. Compston, J.E., McClung, M.R., and Leslie, W.D. (2019). Osteoporosis. Lancet 393, 364-376. 10.1016/S0140-6736(18)32112-3. Walker, M.D., and Shane, E. (2023).
  • Endogenous bone marrow MSCs are dynamic, fate- restricted participants in bone maintenance and regeneration.
  • Fiji an open-source platform for biological- image analysis. Nat Methods 9, 676-682.10.1038/nmeth.2019. Alberich-Bayarri, A., Marti-Bonmati, L., Angeles Perez, M., Sanz-Requena, R., Lerma-Garrido, J.J., Garcia-Marti, G., and Moratal, D. (2010). Assessment of 2D and 3D fractal dimension measurements of trabecular bone from high-spatial resolution magnetic resonance images at 3 T. Med Phys 37, 4930-4937.10.1118/1.3481509.
  • a method of treating or ameliorating a disease in a subject in need thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases together with the pertinent requisite donor nucleotide sugars to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; introducing the glycocalyx-modified MSCs into the subject.
  • MSCs culture-expanded mesenchymal stem cells
  • a method of treating or ameliorating a bone disease or condition in a subject in need thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the bone disease or condition; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases together with the pertinent requisite donor nucleotide sugars to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; introducing the glycocalyx-modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effective for bone regeneration.
  • MSCs culture-expanded mesenchymal stem cells
  • a method making immunomodulatory/restorative/reparative mesenchymal stem cells comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and/or wherein the subject is greater than 30 years old.
  • An immunomodulatory/restorative/reparative mesenchymal stem cell produced by the process of: obtaining a population of MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and/or wherein the subject is greater than 30 years old.
  • a method of preparing a secretome comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and/or wherein the subject is greater than 30 years old; and harvesting the secretome (e.g., exosomes and other extracellular vesicles) from the MSCs.
  • a secretome comprising immunomodulatory/regenerative/reparative/restorative vesicles and bioactive factors produced by the process of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and/or wherein the subject is greater than 30 years old; and harvesting the secretome (e.g., extracellular vesicles) from the MSCs.
  • a pharmaceutical composition comprising a population of MSCs produced by the process of: (a) obtaining a population of culture-expanded MSCs originating from the subject (i.e., autologously-derived MSCs); (b) modifying the glycocalyx of the culture-expanded MSCs, ex vivo, using glycosyltransferase(s) and attendant nucleotide sugar donor(s) to enforce cell surface expression of sLeX on the MSC surface to produce glycocalyx-modified MSCs; (c) Introducing the glycocalyx-modified MSCs into the subject intravascularly so as to induce a tissue reparative or tissue restorative effect in the affected tissue(s), wherein one or both of the following apply: [0215] (i) The MSCs are obtained from one of the disease-affected tissues of the subject [0216] (ii) The subject is over 30 years of age.
  • any preceding embodiment wherein the skeletal disease or condition is caused by one or more of congenital/genetic diseases, trauma, infection, degenerative diseases, metabolic diseases, neoplastic diseases, medications, iatrogenic effects, dietary deficiencies/malnutrition, endocrinologic conditions, gastrointestinal conditions/malabsorption states, and pregnancy/lactation-induced bone loss.
  • the method or composition of any preceding embodiment wherein the skeletal disease or condition is osteoporosis caused by one or more of an autoimmune disorder, a genetic disorder, digestive/gastrointestinal disorder, metabolic condition, kidney disease, malnutrition, Vitamin D deficiency, medical procedure, medications, cancer, hematologic/blood disorder, neurological/nervous system disorder, bone marrow disorder, endocrine/hormonal disorder, menopause, and pregnancy/lactation.
  • the skeletal condition is one or more of fracture and trauma.
  • the MSCs are glycocalyx-modified ex vivo by exofucosylation.
  • glycocalyx- modified MSCs are effective to increase plasma levels of bone neoformation marker N-terminal propeptide of type I procollagen (P1NP).
  • P1NP bone neoformation marker N-terminal propeptide of type I procollagen
  • a method of treating or ameliorating a bone disease or condition in a subject in need thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from a skeletal disease or condition, and then differentiating the MSCs in vitro into osteoblasts; modifying the glycocalyx of the osteoblasts, ex vivo, using one or more glycosyltransferases to enforce cell surface expression of sLeX on the osteoblasts to produce glycocalyx-modified osteoblasts; and introducing the glycocalyx-modified osteoblasts into the subject, wherein the glycocalyx-modified osteoblasts are effective for bone regeneration.
  • MSCs culture-expanded mesenchymal stem cells
  • a method for treating a disease in a human subject wherein the endothelial beds of the tissue(s) affected by the disease express E-selectin comprising the steps of: ( a) obtaining a population of culture-expanded MSCs originating from the subject (i.e., autologously-derived MSCs); ( b) modifying the glycocalyx of the culture-expanded MSCs, ex vivo, using glycosyltransferase(s) and attendant nucleotide sugar donor(s) to enforce cell surface expression of sLeX on the MSC surface to produce glycocalyx-modified MSCs; ( c) Introducing the glycocalyx-modified MSCs into the subject intravascularly so as to induce a tissue reparative or tissue restorative effect in the affected tissue(s), wherein one or both of the following apply: ( i) The MSCs are obtained from one of the disease-affected tissues of the subject (ii) The subject is over 30

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Abstract

La présente divulgation concerne, entre autres, des compositions et des méthodes de traitement de maladies et d'états pathologiques (tels que des maladies du squelette) à l'aide de cellules souches mésenchymateuses modifiées par glycocalyx. Selon certains aspects, l'invention concerne des compositions et des méthodes destinées à améliorer la colonisation tissulaire de CSM humaines administrées de manière intravasculaire et, par conséquent, optimiser le ou les avantages biologiques souhaités pour toute indication clinique dans laquelle des MSC sont utilisées.
PCT/US2025/036118 2024-07-01 2025-07-01 Compositions et méthodes de traitement de maladies humaines avec des cellules souches mésenchymateuses modifiées par glycocalyx Pending WO2026010977A1 (fr)

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Citations (4)

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WO1998032450A1 (fr) * 1997-01-24 1998-07-30 Osiris Therapeutics, Inc. Regeneration osseuse dans le cadre de l'osteoporose a l'aide de cellules mesenchymateuses de moelle osseuse humaine
WO2016109543A1 (fr) * 2014-12-30 2016-07-07 The Brigham And Women's Hospital, Inc. Méthodes pour améliorer la thérapie cellulaire
WO2021236564A2 (fr) * 2020-05-18 2021-11-25 Robert Sackstein Compositions et méthodes de traitement de troubles inflammatoires
WO2024152043A1 (fr) * 2023-01-13 2024-07-18 The Brigham And Women's Hospital Compositions et procédés d'identification et d'isolement de cellules souches et progénitrices hématopoïétiques humaines

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WO1998032450A1 (fr) * 1997-01-24 1998-07-30 Osiris Therapeutics, Inc. Regeneration osseuse dans le cadre de l'osteoporose a l'aide de cellules mesenchymateuses de moelle osseuse humaine
WO2016109543A1 (fr) * 2014-12-30 2016-07-07 The Brigham And Women's Hospital, Inc. Méthodes pour améliorer la thérapie cellulaire
WO2021236564A2 (fr) * 2020-05-18 2021-11-25 Robert Sackstein Compositions et méthodes de traitement de troubles inflammatoires
WO2024152043A1 (fr) * 2023-01-13 2024-07-18 The Brigham And Women's Hospital Compositions et procédés d'identification et d'isolement de cellules souches et progénitrices hématopoïétiques humaines

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SHIM JEONGHYUN, KIM KYOUNG-TAE, KIM KWANG GI, CHOI UN-YONG, KYUNG JAE WON, SOHN SEIL, LIM SANG HEON, CHOI HYEMIN, AHN TAE-KEUN, CH: "Safety and efficacy of Wharton's jelly-derived mesenchymal stem cells with teriparatide for osteoporotic vertebral fractures: A phase I/IIa study", STEM CELLS TRANSLATIONAL MEDICINE, ALPHAMED PRESS, INC., US, vol. 10, no. 4, 1 April 2021 (2021-04-01), US , pages 554 - 567, XP093389947, ISSN: 2157-6564, DOI: 10.1002/sctm.20-0308 *

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