Patent Application
20230015661
Current techniques and tools for cell-based therapies are often prone to unwanted and dangerous side effects, such as uncontrolled proliferation, limited engineering capability, and anti-DNA immune responses. While cell-based therapies have great potential to address critical needs in the treatment of human diseases, clinical success often faces obstacles, such as cell heterogeneity, limited engineering capability, inconsistent efficacy, poor quality control or reproducibility in large-scale manufacturing, and patient safety concerns.
The present disclosure is based, at least in part, on the generation of bioengineered enucleated cells to improve therapeutic functions and produce cell-like entities that are controllable and safe.
As elaborated on below and exemplified in the working examples, the methods for bioengineering enucleated cells designed for therapeutic use and the use of the cells offer several benefits over previous cell-based therapeutics, including, e.g., safety, defined lifespan, no risk of nuclear-encoded gene transfer to host, and effective delivery of therapeutic cargo. Other advantages of the presently claimed disclosure are described herein.
Provided herein are methods of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising an enucleated cell genetically engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous peptide. In some embodiments, the composition further comprises a therapeutic agent. In some embodiments, the therapeutic agent comprises at least one of a small RNA, a small molecule drug, a peptide, a virus, or combinations thereof. In some embodiments, the therapeutic agent comprises a chemotherapeutic agent.
Provided herein are methods of governing immune activation in a subject, the method comprising: administering to the subject an enucleated cell, wherein the enucleated cell is genetically engineered to activate the immune system. In some embodiments, the enucleated cell is genetically engineered to express at least one exogenous protein. In some embodiments, the exogenous protein is a cell surface protein. In some embodiments, the exogenous protein is an immune activating protein. In some embodiments, the exogenous protein comprises a cytokine, IL-12, calreticulin, phosphatidylysine, phagocytosis prey-binding domain, annexin 1, OX40/OC40L, 4-1BB, B7 family members, or combinations thereof.
Provided herein are methods of governing immune recognition in a subject, the method comprising: administering to the subject an enucleated cell, wherein the enucleated cell is genetically engineered to evade recognition by the immune system. In some embodiments, the enucleated cell is genetically engineered to deplete the enucleated cell of immune recognition molecules. In some embodiments, the immune recognition molecules comprise HLA antigens, proteoglycans, sugar moieties, embryonic antigens, or combinations thereof. In some embodiments, the enucleated cell is genetically engineered to express at least one exogenous protein. In some embodiments, the exogenous protein is a cell surface protein. In some embodiments, the exogenous protein is an immune evasion molecule. In some embodiments, the exogenous protein comprises a cytokine, IL-1, IL-4, IL-6, IL-8, IL-10, TGF-β, IGF-2, VEGF, TNF-alpha, CD47, HLA-E, HLA-G, HLA-E/G, PD-1, PD-L1, TIGIT, CD112R, CTLA-4, a chemokine, chemokine ligand 1, C—C motif chemokine receptor 7, an NK inhibitor receptor, HLA-class I-specific inhibitory receptor, killer cell immunoglobulin-like receptor (KIR), NKG2A, lymphocyte activation gene-3 (LAG-3), or combinations thereof.
Provided herein are methods of identifying the presence of a disease condition in a subject, the method comprising: administering to the subject an enucleated cell genetically engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous peptide, wherein the genetically engineered enucleated cell identifies the presence or location of a disease condition. In some embodiments, the exogenous protein is an inflammation homing receptor. In some embodiments, the inflammation homing receptor directs the enucleated cell to damaged and/or inflamed tissue.
In some embodiments, the enucleated cell is derived from a natural killer (NK) cell, a macrophage, a neutrophil, a fibroblast, and adult stem cell, a mesenchymal stromal cell (MSC), an inducible pluripotent stem cell, or combinations thereof. In some embodiments, the enucleated cell is derived from a mesenchymal stromal cell (MSC).
In some embodiments, the exogenous DNA molecule comprises single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or combinations thereof. In some embodiments, the exogenous RNA molecule comprises messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), a RNA virus, or combinations thereof. In some embodiments, the exogenous protein comprises a cytokine, a growth factor, a hormone, an antibody, an enzyme, or combinations thereof.
In some embodiments, the administering comprises intravenous administration, subcutaneous administration, intraperitoneal administration, rectal administration, oral administration, or combinations thereof. In some embodiments, the administering comprises intratumoral administration.
In some embodiments, the disease comprises inflammation, an infection, a cancer, a neurological disease, an autoimmune disease, a cardiovascular disease, an ophthalmologic disease, a skeletal disease, a metabolic disease, or combinations thereof. In some embodiments, the cancer comprises multiple myeloma, glioblastoma, lymphoma, leukemia, mesothelioma, sarcoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, or combinations thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
This disclosure describes methods and uses for cell-based therapies with genetically engineered enucleated cells. In practice, cells can be genetically engineered to improve therapeutic functions and are enucleated to produce cell-like entities that are controllable and safe (
Bioengineered enucleated cells can be designed for therapeutic use by performing important cellular functions after enucleation, having a defined lifespan, exhibiting therapeutic functions, and being amenable to multi-layered engineering and large-scale manufacturing. As used herein, “enucleation” is the rendering of a cell to a non-replicative state, either through inactivation or removal of the nucleus. In some embodiments, cells can be treated with cytochalasin to soften the cortical actin cytoskeleton. The nucleus is then physically extracted from the cell body by high-speed centrifugation in gradients of Ficoll to generate an enucleated cell. Because enucleated cells and intact nucleated cells sediment to different layers in the Ficoll gradient, enucleated cells can be easily isolated and prepared for therapeutic purposes or fusion to other cells (e.g., nucleated or enucleated cells). In some embodiments, the enucleation process is clinically scalable to process tens of millions of cells.
In some embodiments, enucleated cells can be used as a disease-homing vehicle to deliver clinically relevant cargos/payloads to treat various diseases (e.g., any of the diseases described herein). In some embodiments, enucleated cells loaded with cargos, payloads, or biomolecules can be referred to as “cargocytes”. In some embodiments, cargocytes can refer to bioengineered enucleated cells designed for therapeutic use. In some embodiments, enucleated cells possess significant therapeutic value because they remain viable, do not differentiate into other cell types, secrete bioactive proteins, can physically migrate/home for 3-4 days, can be extensively engineered ex vivo to perform specific therapeutic functions, and can be fused to the same or other cell types to transfer desirable production, natural or engineered. Therefore, enucleated cells have wide utility as a cellular vehicle to deliver therapeutic biomolecules and disease-targeting cargos including, but not limited to, chemotherapeutic drugs (e.g., doxorubicin), genes, viruses, bacteria, mRNAs, shRNAs, siRNA, peptides, plasmids, and nanoparticles. In some embodiments, enucleated cells enable the generation of a safe (e.g., no unwanted DNA is transferred to the subject), and controllable (e.g., cell death occurs in precisely 3-4 days) cell-based carrier that can be genetically engineered to deliver specific disease-fighting and health promoting cargos to humans or animals.
In some embodiments, an enucleated cell (e.g., cargocyte) is genetically engineered and designed for therapeutic use. As used herein, “genetically engineered,” in reference to cells, refers to a cell that comprises a nucleic acid sequence (e.g., DNA, RNA, or mRNA) that is not present in, or is present at a different level than, an otherwise similar cell under similar conditions that is not engineered (e.g., compared to RBCs, which are derived from erythroblasts, an enucleated cell (e.g., cargocyte) can be derived from any type of nucleated cell, including, but not limited to iPSC (induced pluripotent stem cells), any immortalized cell, stem cells, primary cells an exogenous DNA molecule, or an exogenous RNA molecule), or a cell that comprises a polypeptide expressed from said nucleic acid (e.g., an exogenous protein, or an exogenous polypeptide). In some embodiments, a genetically engineered cell has been altered from its native state by the introduction of an exogenous nucleic acid, or is the progeny of such an altered cell. In some embodiments, a genetically engineered cell comprises an exogenous nucleic acid (e.g., DNA, RNA, or mRNA). In some embodiments, the enucleated cell is engineered to express at least one (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous polypeptide, or an exogenous protein, or combinations thereof. In some embodiments, the enucleated cell is engineered to simultaneously express at least two or more (e.g., three or more, four or more, five or more, or six or more) of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous polypeptide, or an exogenous protein, or combinations thereof. In some embodiments, the exogenous DNA molecule is a single-stranded DNA, a double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or combinations thereof. In some embodiments, the exogenous RNA molecule is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), a RNA virus, or combinations thereof. In some embodiments, the exogenous protein is a cytokine, a growth factor, a hormone, an antibody, an enzyme, or combinations thereof.
In some embodiments, enucleated cells can be derived from a variety of different cell types. In some embodiments, enucleated cells can be derived from any nucleated cell type that maintains a nucleus throughout its lifespan or does not naturally enucleate. In some embodiments, an enucleated cell can be derived from a normal cell line. In some embodiments, an enucleated cell can be derived from a cancer cell line. In some embodiments, an enucleated cell can be derived from therapeutic cells obtained from the immune system. For example, an enucleated cell can be derived from a mesenchymal stromal cell (MSC), a natural killer (NK) cell, a macrophage, a neutrophil, a lymphocyte, a mast cell, a basophil, an eosinophil, and/or a fibroblast. In some embodiments, an enucleated cell is derived from a mesenchymal stromal cell (MSC). In some embodiments, an enucleated cell is derived from hTERT-immortalized adipose-derived MSCs (hT-MSC) wherein MSCs have proven therapeutic potential in clinical studies and the immortalized phenotype provides a homogenous cell population with consistent characteristics, which facilitates further bioengineering. In some embodiments, an enucleated cell can be derived from an adult stem cell and/or an inducible pluripotent stem cell (iPSC).
Some cell types do not have a nucleus, e.g., red blood cells. Further, exosomes and small cellular membrane vesicles derived from therapeutic cells can act as delivery vesicles, but are markedly different than the enucleated cells of this disclosure. Enucleated cells of this disclosure (e.g., cargocytes) are different from RBCs, exosomes, and small cellular membrane vesicles. These types of delivery vesicles do not have the cellular organelles needed to produce and secrete exogenous proteins (e.g., ER/Golgi, mitochondrial, endosome, lysosome, cytoskeleton, etc.). Thus, enucleated cells of the disclosure can function like nucleated cells and exhibit critical biological functions such as adhesion, tunneling nanotube formation, actin-mediated spreading (2D and 3D), migration, chemoattractant gradient sensing, mitochondrial transfer, mRNA translation, protein synthesis, and secretion of exosomes and other bioactive molecules. One or more of these functions may not be exhibited by exosomes, small cellular membrane vesicles, RBCs, or other similar delivery-only vesicles.
In some embodiments, enucleation efficiency describes the percentage of cells in a population that have been successfully enucleated through the methods described here or otherwise known in the art. In some embodiments, the enucleation efficiency of cells can be over 95% (e.g., 96%, 97%, 98%, 99%, or 100%) efficient. In some embodiments, a recovery rate refers to the percentage of viable cells out of an input population. In some embodiments, enucleated cells can be generated with an at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) recovery rate. In some embodiments, over 95% enucleation efficiency is achieved for hT-MSCs. In some embodiments, the methods of the disclosure yield an 80-90% recovery rate.
As used herein, the term “substantially the same”, when used herein with respect to cell structure, can refer to a cell relative to a reference cell sharing at least the functional subcellular organelles. For example, an enucleated cell may exhibit substantially the same cell structure as a parental cell if the two cells contain the same functional subcellular organelles. In some embodiments, the enucleated cell contains the same functional subcellular organelles as a parental cell wherein the functional subcellular organelles comprise at least one of the Golgi, Endoplasmic Reticulum, mitochondria, lysosomes, ribosomes, endosomes, or combinations thereof.
The term “substantially the same”, when used herein with respect to cell function, can refer to a cell relative to a reference cell exhibiting similar functional characteristics. For example, an enucleated cell may retain same surface marker protein expression. In some embodiments, the enucleated cell has similar zeta potential as a parental (e.g., nucleated) cell. In some embodiments, the enucleated cell membrane receptors and migration and invasion machineries are fully functional, exhibiting similar functionality as a parental cell. In some embodiments, the enucleated cell actively produces and secretes the same extracellular vesicles as those produced by a parental cell.
In some embodiments, enucleated cells readily attach to tissue culture plates with well-organized cytoskeletal structure. In some embodiments, enucleated cells are viable for up to 72 hours post-enucleation. In some embodiments, enucleated cells can contain crucial and functional subcellular organelles, including, but not limited to, Golgi, Endoplasmic Reticulum (ER), mitochondria, lysosomes, and endosomes (
In some embodiments, cell-based therapeutics use normal or engineered nucleated cells. In some embodiments, cell-based therapies irradiate cells prior to patient injection in order to prevent cell proliferation and induced lethal DNA-damage. However, this approach induces mutations and produces significant amounts of reactive oxygen species that irreversibly damage cellular proteins and DNA, which can release large amounts of damaged/mutated DNA into the body of a subject. Such products can be dangerous if they integrate into other cells and/or induce an unwanted anti-DNA immune response. Irradiated cells are also dangerous because they can transfer their mutated DNA and genes to host cells by cell-cell fusion. Compared to cellular irradiation, removing the entire nucleus from a cell is a less damaging and significantly safer method for limiting cellular lifespan that precludes any introduction of nuclear DNA into a subject. Furthermore, many stem cells such as mesenchymal stem cells (MSCs) are highly resistant to radiation-induced death, and therefore can not be rendered safe using this method.
In some embodiments, therapeutic cells can be engineered with a drug-inducible suicide switch to limit cellular lifespan. However, activation of the switch in vivo requires injecting a subject with potent and potentially harmful drugs with unwanted side effects. While this method induces suicide in culture cells (<95%), it is expected to be inefficient when translated into the clinic. Moreover, the death of the therapeutic cell released large amounts of DNA (e.g., normal or genetically altered DNA), which can integrate into host cells or induce a dangerous systemic anti-DNA immune response. If the cell mutates and loses/inactivates the suicide switch, it becomes an uncontrollable mutant cell. In addition, these cells can fuse with host cells in the subject, and therefore transfer mutant DNA. Such fused cells are dangerous because not all host cells inherit the suicide gene, but can inherit some of the therapeutic cell's genes/DNA during chromosomal reorganization and cell hybridization. In addition, for the same reason, therapeutic cells with suicide switches can not be used as cell fusion partners in vitro.
Another method to limit therapeutic cell lifespan is heat-induced death. However, this causes severe damage that terminates crucial biological functions necessary for therapeutic use. Unlike enucleated cells, these cells can still transfer DNA to the subject since they retain their nucleus and all genetic material. Numerous chemicals inhibit cell proliferation and/or cause cell death prior to therapeutic use, including, but not limited to, chemotherapeutic drugs or mitomycin C. However, such drugs have significant off-target effects that significantly damage the cell, and are unwanted for clinical applications due to high toxicities. Many anti-proliferative and death-inducing drugs do not effectively inhibit 100% if the cells due to resistance, and unlike enucleated cells, many drug effects are revisable. Thus, this approach is not suitable to prevent cell growth of immortalized/cancer cells in vivo.
The present disclosure provides methods for producing engineered enucleated cells (e.g., cargocytes) designed for therapeutic use. In some embodiments, enucleated cells are produced with either natural or inducible expression and/or uptake of biomolecules with therapeutic functions including, but not limited to, DNA, RNA (e.g., mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, plasmids, viruses, and small molecule drugs. In some embodiments, bioengineering approaches improve enucleated cell function. In some embodiments, parental cells (e.g., nucleated cells) are genetically engineered before enucleation (e.g., pre-enucleation). In some embodiments, parental cells are genetically engineered after enucleation (e.g., post-enucleation).
In some embodiments, enucleated cells (e.g., cargocytes) are engineered to produce biomolecules (secreted, intracellular, and natural and inducible) exogenously including, but not limited to, DNA/genes, RNA (e.g., mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, and plasmids, bacteria, viruses, small molecule drugs, ions, cytokines, growth factors, and hormones. In some embodiments, enucleated cells produce therapeutic levels of a bioactive protein or an immune stimulator.
In some embodiments, parental cells are genetically engineered to produce biomolecules (secreted, intracellular, and natural and inducible) exogenously before enucleation. In some embodiments, parental cells are genetically engineered to produce biomolecules exogenously including, but not limited to, DNA/genes, RNA (e.g., mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, and plasmids, bacteria, viruses, small molecule drugs, ions, cytokines, growth factors, and hormones. In some embodiments, parental cells are genetically engineered to produce therapeutic levels of a bioactive protein or an immune stimulator. In some embodiments, parental cells are genetically engineered to produce tumor trophic proteins.
In some embodiments, enucleated cells can be used as a vehicle to deliver therapeutic biologics (e.g., therapeutic cargos) including, but not limited to, DNA/genes, RNA (e.g., mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, plasmids, viruses, and small molecule drugs. Unlike nucleated cells, enucleated cells can be loaded with high doses of DNA-damaging/gene targeting agents for delivery to patients as a therapeutic against cancer or other diseases. In some embodiments, the DNA-damaging/gene targeting agents include, but are not limited to, DNA-damaging chemotherapeutic drugs, DNA-integrating viruses, oncolytic viruses, and gene therapy applications.
In some embodiments, therapeutic enucleated cells (natural or engineered) can be used as fusion partners to other cells (therapeutic or natural) to enhance and/or transfer biomolecules (secreted, intracellular, and natural and inducible) including, but not limited to, DNA/genes, RNA (mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, and plasmids, bacteria, viruses, small molecule drugs, ions, cytokines, growth factors, and hormones. Unlike nucleated cells, the fusion of enucleated cells to the same or another cell type of similar or different origin generates a unique cell hybrid that lacks problematic nuclear transfer, while maintaining desirable therapeutic attributes including, but not limited to, cell surface proteins, signal transduction molecules, secreted proteins, and epigenetic changes.
In some embodiments, enucleated cells can be used as biosensors and signal transduction indicators of biological processes and disease states. In some embodiments, because enucleated cells cannot undergo DNA damage-induced apoptotic death, they can be used in combination with apoptotic-inducing and/or DNA toxic/targeting agents for treatment of cancer and other diseases.
Enucleated cells are smaller than their nucleated counterparts and for this reason can migrate better through small openings in the vasculature and tissue parenchyma. In addition, removing the large dense nucleus alleviates a major physical barrier allowing the cell to move freely through small openings in the vessels and tissue parenchyma. Therefore, enucleated cells have improved bio-distribution in the body and movement into target tissues. In some embodiments, an enucleated cell is at least 1 μm in diameter. In some embodiments, an enucleated cell is greater than 1 μm in diameter. In some embodiments, an enucleated cell is 1-100 μm in diameter (e.g., 1-90 μm, 1-80 μm, 1-70 μm, 1-60 μm, 1-50 μm, 1-40 μm, 1-30 μm, 1-20 μm, 1-10 μm, 1-5 μm, 5-100 μm, 5-90 μm, 5-80 μm, 5-70 μm, 5-60 μm, 5-50 μm, 5-40 μm, 5-30 μm, 5-20 μm, 5-10 μm, 10-100 μm, 10-90 μm, 10-80 μm, 10-70 μm, 10-60 μm, 10-50 μm, 10-40 μm, 10-30 μm, 10-20 μm, 20-100 μm, 20-90 μm, 20-80 μm, 20-70 μm, 20-60 μm, 20-50 μm, 20-40 μm, 20-30 μm, 30-100 μm, 30-90 μm, 30-80 μm, 30-70 μm, 30-60 μm, 30-50 μm, 30-40 μm, 40-100 μm, 40-90 μm, 40-80 μm, 40-70 μm, 40-60 μm, 40-50 μm, 50-100 μm, 50-90 μm, 50-80 μm, 50-70 μm, 50-60 μm, 60-100 μm, 60-90 μm, 60-80 μm, 60-70 μm, 70-100 μm, 70-90 μm, 70-80 μm, 80-100 μm, 80-90 μm, or 90-100 μm). In some embodiments, some enucleated cells can advantageously be small enough to allow for better biodistribution or to be less likely to be trapped in the lungs of a subject.
In some embodiments, a genetically engineered enucleated cell has a defined life span of less than 1 hour to 14 days (e.g., less than 1 hour, less than 6 hours, less than 12 hours, less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, less than 10 days, less than 11 days, less than 13 days, less than 14 days, 1 to 14 days, 1 to 12 days, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 14 days, 2 to 12 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 14 days, 3 to 12 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 14 days, 4 to 12 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 14 days, 5 to 12 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 14 days, 6 to 12 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 14 days, 7 to 12 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 14 days, 8 to 12 days, 8 to 10 days, 8 to 9 days, 9 to 14 days, 9 to 12 days, 9 to 10 days, 10 to 14 days, 10 to 12 days, or 12 to 14 days). In some embodiments, the lifespan of a population of genetically engineered enucleated cells can be evaluated by determining the average time at which a portion of the genetically engineered enucleated cell population (e.g., at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the population) is determined to be dead. Cell death can be determined by any method known in the art. In some embodiments, the viability of genetically engineered enucleated cells, e.g., at one or more time points, can be evaluated by determining whether morphometric or functional parameters are intact (e.g., by trypan-blue dye exclusion, evaluating for intact cell membranes, evaluating adhesion to plastics (e.g., in adherent enucleated cells), evaluating genetically engineered enucleated cell migration, negative staining with apoptotic markers, and the like). In some embodiments, the life span of a genetically engineered enucleated cell may be related to the life span of the cell from which it was obtained.
In some embodiments, a genetically engineered enucleated cell has been altered from its native state by depleting the enucleated cell of immune recognition molecules. For example, these immune recognition molecules can be HLA antigens, proteoglycans, sugar moieties, embryonic antigens, or combinations thereof. In some embodiments, the enucleated cell is genetically engineered to express at least one exogenous protein. In some embodiments, the exogenous protein is a cell surface protein. In some embodiments, the exogenous protein is an immune evasion molecule. An immune evasion molecule can be a molecule expressed by a cell, which allows the cell to avoid the innate immune system and to evade immune responses. In some embodiments, an immune evasion molecule is a cytokine, IL-10, CD47, HLA-E/G, PD-1, LAG-3, CTLA-4, or combinations thereof. In some embodiments, the exogenous protein is an immune activating protein. In some embodiments, an immune activating protein is a cytokine, IL-12, calreticulin, phosphatidylysine, phagocytosis prey-binding domain, annexin 1, OX40/OC40L, 4-1BB, B7 family members, or combinations thereof.
In some embodiments of any of the methods and compositions described herein, a nucleated cell (e.g., an eukaryotic cell, a mammalian cell (e.g., a human cell, a canine cell, a feline cell, an equine cell, a porcine cell, a primate cell, a rodent cell (e.g., a mouse cell, a guinea pig cell, a hamster cell, or a mouse cell), an immune cell, or any nucleated cell described herein), is treated with cytochalasin to soften the cortical actin cytoskeleton. The nucleus is then physically extracted from the cell body by high-speed centrifugation in gradients of Ficoll to generate an enucleated cell. In some embodiments, the nucleus is removed by density gradient centrifugation. As used herein, the term “enucleated cell” can refer to a previously nucleated cell (e.g., any cell described herein) that consists of the inner mass of a cell and the cell organelles. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. In some embodiments, the nucleated cell is a primary cell. In some embodiments, the nucleated cell is an immune cell (e.g., a T cell, a B cell, a macrophage, a natural killer cell, a neutrophil, a mast cell, a basophil, a dendritic cell, a monocyte, a myeloid-derived suppressor cell, an eosinophil. In some embodiments, the nucleated cell is a phagocyte or a leukocyte. In some embodiments, the nucleated cell is a stem cell (e.g., an adult stem cell, an embryonic stem cell, an inducible pluripotent stem cell (iPS)). In some embodiments, the nucleated cell is a progenitor cell. In some embodiments, the nucleated cell is a cell line. In some embodiments, the nucleated cell is a suspension cell. In some embodiments, the nucleated cell is an adherent cell. In some embodiments, the nucleated cell is a cell that has been immortalized by expression of an oncogene. In some embodiments, the nucleated cell is immortalized by the expression of human telomerase reverse transcriptase (hTERT). In some embodiments, the nucleated cell is a mesenchymal stromal cell (MSC). In some embodiments, the nucleated cell is an hTERT-immortalized adipose-derived MSC (hTERT-MSC). In some embodiments, the nucleated cell is a patient derived cell (e.g., an autologous patient-derived cell, or an allogenic patient-derived cell).
Methods of culturing a cell (e.g., any of the cells described herein) are well known in the art. Cells can be maintained in vitro under conditions that favor growth, proliferation, viability, and differentiation. In some embodiments, the nucleated cells (e.g., MSCs) are cultured in 3D-hanging drops (e.g., 3D MSCs) then enucleated to generate 3D enucleated cells.
In some embodiments of any of the compositions and methods provided herein, the enucleated cell is frozen for later use. Various methods of freezing cells are known in the art, including, but not limited to, the use of a serum (e.g., Fetal Bovine Serum) and dimethyl sulfoxide (DMSO). In some embodiments of any of the compositions and methods provided herein, the enucleated cell is thawed prior to use.
Methods of Introducing a Biomolecule into a Enucleated Cell
Various methods are known in the art that can be used to introduce a biomolecule (e.g., a RNA molecule (e.g., mRNA, miRNA, siRNA, shRNA, lncRNA), a DNA molecule (e.g., a plasmid), a protein, a peptide into an enucleated cell. Non-limiting examples of methods that can be used to introduce a biomolecule into an enucleated cell include: liposome mediated transfer, an adenovirus, an adeno-associated virus, a herpes virus, a retroviral based vector, a lentiviral vector, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetofection, nanoparticle transfection, or combinations thereof. In some embodiments of any of the compositions and methods provided herein, a therapeutic agent, a virus, an antibody, or a nanoparticle is introduced into the enucleated cells.
As used herein, the terms “immune evasion” or “to evade immune recognition” refer to a fundamental process in tumor formation and progression. During tumor development, a chronic inflammatory microenvironment reduces the anti-tumoral immune response and favors the escape of tumor from immune elimination. Inflammatory immune cells include tumor-associated macrophages (TAMs), cytotoxic T (CD8) lymphocytes (CTLs), Th (CD4) lymphocytes, natural killer (NK) cells, regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). Among them, Treg cells, MDSCs and macrophages are mainly involved in the immunosuppressive action of key molecules, such as transforming growth factor beta (TGF-β), prostaglandin E2, indoleamine 2, 3-dioxygenase and interleukin-10 (IL-10). Several growth factors, namely TGF-β, insulin-like growth factor 2 (IGF-2) and vascular endothelial growth factor (VEGF), cytokines (e.g., IL-1, IL-4, IL-6, IL-8, IL-10) and tumor-necrosis factor alpha, chemokines (e.g., chemokine (C—X—C motif) ligand 1 and C—C motif chemokine receptor 7) have been reported to be closely involved in tumor progression, invasion and immune evasion.
Further, immune evasion occurs through the selection of immune evasion molecules (e.g., tumor variants) that become resistant to an immune attack primarily mediated by T cells and natural killer (NK) cells. For example, an immune evasion molecule can be a cytokine (e.g., IL-1, IL-4, IL-6, IL-8, IL-10), TGF-0, IGF-2, VEGF, TNF-alpha, CD47, HLA-E, HLA-G, HLA-E/G, PD-1, PD-L1, TIGIT, CD112R, CTLA-4, chemokines (e.g., chemokine ligand 1, C—C motif chemokine receptor 7), or any combinations thereof. In some embodiments, an immune evasion molecule can be an NK inhibitor receptor (e.g., HLA-class I-specific inhibitory receptors, e.g., killer cell immunoglobulin-like receptor (KIR), NKG2A, or lymphocyte activation gene-3 (LAG-3)).
The generation of bioengineered enucleated cells offer to improve therapeutic functions and produce cell-like entities that are controllable and safe. The bioengineered enucleated cells designed to evade recognition by the immune system and further therapeutic use offer several benefits over previous cell-based therapeutics, including, e.g., safety, defined lifespan, no risk of nuclear-encoded gene transfer to host, and effective delivery of therapeutic cargo. In some embodiments, enucleated cells can be genetically engineered to express a cytokine (e.g., IL-1, IL-4, IL-6, IL-8, IL-10), TGF-β, IGF-2, VEGF, TNF-alpha, CD47, HLA-E, HLA-G, HLA-E/G, PD-1, PD-L1, TIGIT, CD112R, CTLA-4, chemokines (e.g., chemokine ligand 1, C—C motif chemokine receptor 7), or any combinations thereof. In some embodiments, enucleated cells can be genetically engineered to express an NK inhibitor receptor (e.g., HLA-class I-specific inhibitory receptors, e.g., killer cell immunoglobulin-like receptor (KIR), NKG2A, or lymphocyte activation gene-3 (LAG-3)).
In some embodiments, a method of governing immune recognition in a subject includes, administering to the subject an enucleated cell, wherein the enucleated cell is genetically engineered to evade recognition by the immune system. In some embodiments, the enucleated cell is genetically engineered to deplete the enucleated cell of immune recognition molecules. In some embodiments, the immune recognition molecules include, but are not limited to, HLA antigens, proteoglycans, sugar moieties, embryonic antigens, or combinations thereof. In some embodiments, the enucleated cell is genetically engineered to express at least one exogenous protein. In some embodiments, the exogenous protein is a cell surface protein. In some embodiments, the exogenous protein is an immune evasion molecule. In some embodiments, the exogenous protein includes a cytokine (e.g., IL-1, IL-4, IL-6, IL-8, IL-10), TGF-β, IGF-2, VEGF, TNF-alpha, CD47, HLA-E, HLA-G, HLA-E/G, PD-1, PD-L1, TIGIT, CD112R, CTLA-4, chemokines (e.g., chemokine ligand 1, C—C motif chemokine receptor 7), or any combinations thereof. In some embodiments, the exogenous protein includes an NK inhibitor receptor (e.g., HLA-class I-specific inhibitory receptors, e.g., killer cell immunoglobulin-like receptor (KIR), NKG2A, or lymphocyte activation gene-3 (LAG-3)).
As used herein, the term “immune activation” refers to the transition of leukocytes (e.g., macrophages, neutrophils, NK cells) and other cell types involved in the immune system. Activation of the immune system is a pathologically appropriate response to invading pathogens. Immune activation provides a beneficial role in control and clearance of invading pathogens. Also, surveillance and activity of the immune system contributes of control and suppression of pathogen replication and spread. Further, cancer immunotherapy uses the immune system and its components to mount an anti-tumor response through immune activation. Immune activation proteins can include, but are not limited to, a cytokine, IL-12, calreticulin, phosphatidylysine, phagocytosis prey-binding domain, annexin 1, OX40/OC40L, 4-1BB, CD70, GITRL, LIGHT, CD30L, B7 family members, or combinations thereof.
The bioengineered enucleated cells designed for immune activation and further therapeutic use offer several benefits over previous cell-based therapeutics, and further allow better understanding of the activation and regulation of innate immune signaling in the immune response to pathogens and cancer. In some embodiments, enucleated cells can be genetically engineered to express a cytokine, IL-12, calreticulin, phosphatidylysine, phagocytosis prey-binding domain, annexin 1, OX40/OC40L, 4-1BB, CD70, GITRL, LIGHT, CD30L, B7 family members, or combinations thereof.
In some embodiments, a method of governing immune activation in a subject includes, administering to the subject an enucleated cell, wherein the enucleated cell is genetically engineered to activate the immune system. In some embodiments, the enucleated cell activates an immune response in the subject. In some embodiments, the enucleated cell is genetically engineered to express at least one exogenous protein. In some embodiments, the exogenous protein is a cell surface protein. In some embodiments, the exogenous protein is an immune activating protein. In some embodiments, the exogenous protein comprises a cytokine, IL-12, calreticulin, phosphatidylysine, phagocytosis prey-binding domain, annexin 1, OX40/OC40L, 4-1BB, B7 family members, or combinations thereof
In some embodiments, the present disclosure provides pharmaceutical compositions that include an enucleated cell and a pharmaceutically acceptable carrier. In some embodiments, the composition can be used as a disease-homing vehicle to deliver clinically relevant cargos/payloads to treat various diseases. In some embodiments, the composition can be used for treating or diagnosing a disease.
In some embodiments, the composition includes one or more enucleated cells genetically engineered to express at least one exogenous protein. In some embodiments, the exogenous protein is a cell surface protein. In some embodiments, the exogenous protein is an immune evasion molecule. In some embodiments, the composition includes an immune evasion molecule, for example, a cytokine (e.g., IL-1, IL-4, IL-6, IL-8, IL-10), CD47, HLA-E/G, PD-1, LAG-3, CTLA-4, PD-L1, TIGIT, CD112R, and NK inhibitor receptors, such as HLA-class I-specific inhibitory receptors (e.g., killer cell immunoglobin-like receptor (KIR), NKG2A, and lymphocyte activation gene-3 (LAG-3)), or combinations thereof. In some embodiments, the exogenous protein is an immune activating protein. In some embodiments, the composition includes an immune activating protein, for example, a cytokine, IL-12, calreticulin, phosphatidylysine, phagocytosis prey-binding domain, annexin 1, OX40/OC40L, 4-1BB, B7 family members, or combinations thereof.
In some embodiments, a pharmaceutical composition can include a buffer, a diluent, a solubilizer, an emulsifier, a preservative, an adjuvant, an excipient, or any combination thereof. In some embodiments, a composition can be formulated for parenteral administration. For example, a pharmaceutical composition provided herein may be provided in a sterile injectable form (e.g., a form that is suitable for subcutaneous injection or intravenous infusion). For example, in some embodiments, a pharmaceutical composition is provided in a liquid dosage form that is suitable for injection.
In some embodiments, the pharmaceutical composition is formulated with a pharmaceutically acceptable parenteral vehicle. For example, such vehicles can include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. In some embodiments, a formulation is sterilized by known or suitable techniques. In some embodiments, a pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient, which can include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
In some embodiments of any of the methods provided herein, the pharmaceutical composition is administered with one or more additional therapies (e.g., chemotherapy (e.g., a chemotherapeutic agent (e.g., doxorubicin, paclitaxel, cyclophosphamide), cell-based therapy, radiation therapy, immunotherapy, a small molecule, an inhibitory nucleic acid (e.g., antisense RNA, antisense DNA, miRNA, siRNA, lncRNA), an exosome-based therapy, gene therapy or surgery). In some embodiments, the one or more additional therapies include immune checkpoint blockade, wherein immune checkpoint inhibitors are administered. In some embodiments, the immune checkpoint inhibitors can include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD47 inhibitors, B7 inhibitors, CD137 inhibitors, or CTLA-4 inhibitors. In some embodiments, the immune checkpoint inhibitor can include a PD-1 inhibitor including, but not limited to, Pembrolizumab, Nivolumab, or Cemiplimab. In some embodiments, the immune checkpoint inhibitor can include a PD-L1 inhibitor including, but not limited to, Atezolizumab, Avelumab, or Durvalumab. In some embodiments, the immune checkpoint inhibitor can include a LAG-3 inhibitor including, but not limited to, relatlimab. In some embodiments, the immune checkpoint inhibitor can include a CTLA-4 inhibitor including, but not limited to, Ipilimumab. In some embodiments, the composition including the enucleated cell is administered simultaneously with the one or more additional therapies. In some embodiments, the composition including the enucleated cell is administered separately from the one or more additional therapies.
In some embodiments of any of the compositions provided herein, the composition further includes one or more additional therapies (e.g., chemotherapy (e.g., a chemotherapeutic agent (e.g., doxorubicin, paclitaxel, cyclophosphamide), cell-based therapy, radiation therapy, immunotherapy, a small molecule, an inhibitory nucleic acid (e.g., antisense RNA, antisense DNA, miRNA, siRNA, lncRNA) or surgery). In some embodiments, the one or more additional therapies include immune checkpoint blockade, wherein immune checkpoint inhibitors are administered. In some embodiments, the immune checkpoint inhibitors can include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD47 inhibitors, B7 inhibitors, CD137 inhibitors, or CTLA-4 inhibitors. In some embodiments, the composition can further include a PD-1 inhibitor including, but not limited to, Pembrolizumab, Nivolumab, or Cemiplimab. In some embodiments, the composition can further include a PD-L1 inhibitor including, but not limited to, Atezolizumab, Avelumab, or Durvalumab. In some embodiments, the composition can further include a LAG-3 inhibitor including, but not limited to, relatlimab. In some embodiments, the composition can further include a CTLA-4 inhibitor including, but not limited to, Ipilimumab. In some embodiments, a composition can also contain one or more additional therapeutically active substances.
In some embodiments, a pharmaceutical composition can include one population of enucleated cells, wherein substantially all the enucleated cells are genetically engineered to express the same molecule, such as the same exogenous DNA molecule, exogenous RNA molecule, exogenous polypeptide, or exogenous protein. In some embodiments, the one population of enucleated cells is engineered to express one biomolecule (e.g., cargo). In some embodiments, the one population of enucleated cells is engineered to express two or more biomolecules (e.g., two biomolecules, three biomolecules, four biomolecules, or five biomolecules). In some embodiments, the one population of enucleated cells is engineered to express two biomolecules, wherein the two exogenous molecules introduced to express the two biomolecules could be the same type of molecule. For example, the one population of enucleated cells engineered to express two biomolecules could be loaded with two different exogenous DNA molecules. In embodiments wherein the one population of enucleated cells is engineered to express two or more biomolecules, each exogenous molecule introduced to express the payload could be different. For example, in one population of enucleated cells engineered to express two biomolecules, wherein one molecule expressing one biomolecule could be an exogenous DNA molecule, and a second molecule expressing a second biomolecule could be an exogenous RNA molecule.
In some embodiments, a pharmaceutical composition can include different populations of enucleated cells, wherein each population is engineered to express a different exogenous molecule (e.g., an exogenous DNA molecule, an exogenous RNA molecule, an exogenous polypeptide, or an exogenous protein, or combinations thereof). For example, a pharmaceutical composition can include one population of enucleated cells genetically engineered to express a cytokine (e.g., IL-1, IL-4, IL-6, IL-8, IL-10), and a second population of enucleated cells genetically engineered to express a checkpoint inhibitor (PD-1 inhibitors, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD47 inhibitors, B7 inhibitors, CD137 inhibitors, or CTLA-4 inhibitors). Additional examples include, but are not limited to a pharmaceutical composition including one population of enucleated cells genetically engineered to express IL-12, and a second population of enucleated cells genetically engineered to express a PD-1 inhibitor. An additional example includes, a pharmaceutical composition including one population of enucleated cells genetically engineered to express CXCR4, a second population of enucleated cells genetically engineered to express CCR2, and a third population of enucleated cells genetically engineered to express PGSL-1/FUT-7. In some embodiments, a pharmaceutical composition can include different populations of enucleated cells, wherein one population of enucleated cells is engineered to express one biomolecule, and a second population of enucleated cells is engineered to express two or more biomolecules. In some embodiments, a pharmaceutical composition can include two populations of enucleated cells, wherein each population is engineered to express two or more biomolecules.
In some embodiments, combination therapies, whether the composition includes one or more population(s) of enucleated cells engineered to express one or more biomolecules, or wherein the composition includes one or more population(s) of enucleated cells engineered to express one or more biomolecules and further includes a separate therapeutic, exhibits synergism as a therapeutic. Synergism, in some contexts, can mean that the combination of biomolecules and/or therapies produces a more beneficial effect (e.g., stronger, longer lasting, better tolerated, etc.) than expected based on the responses to each biomolecule and/or therapy alone. In some embodiments, a combination therapy, wherein an enucleated cell and a checkpoint inhibitor is administered, can produce synergistic effects of treating a disease. For example, when enucleated cells genetically engineered to express IL-12 are administered with the PD-1 checkpoint inhibitor, it has been shown to significantly reduce tumor growth and improve survival in a mouse model (e.g.,
The present disclosure provides methods for the use of enucleated cells (natural or engineered) to enhance and/or transfer biomolecules (secreted, intracellular, and natural and inducible) including, but not limited to, DNA/genes, RNA (mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, and plasmids, bacteria, viruses, small molecule drugs, ions, cytokines, growth factors, and hormones.
Enucleated cells (e.g., cargocytes) are smaller in diameter while lacking rigid nuclei and are expected to pass through small constrictions such as capillaries or interstitial spaces more effectively than nucleated parental cells. For example, enucleated cells have been shown to pass through microvasculature better than nucleated parental cells, therefore better facilitating in vivo homing to damaged or inflamed tissue.
In some embodiments, the enucleated cell is genetically engineered to express an “inflammation homing receptor”, wherein “inflammation homing receptor” herein, refers to an adhesion molecule on leukocytes that binds to endothelial cells in blood vessels. Inflammation homing receptors are used by white blood cells to guide them to sites of tissue inflammation in the body. These diverse tissue-specific adhesion molecules on lymphocytes (e.g., homing receptors) and on endothelial cells (e.g., vascular addressins) contribute to the development of specialized immune responses. In some embodiments, an inflammation homing receptor is an α4β7, VCAM-1, CD34, GLYCAM-1, LFA-1, CD44, and combinations thereof.
In some embodiments, the enucleated cell is genetically engineered to express a “firefly luciferase”, wherein “firefly luciferase” herein, refers to a light-emitting enzyme and bioluminescent reporter for studying gene regulation and function. It is a very sensitive genetic reporter due to the absence of endogenous luciferase activity in mammalian cells or tissues. Firefly luciferase is a 62,000 Dalton protein, which is active as a monomer and does not require subsequent processing for its activity. The enzyme catalyzes ATP-dependent D-luciferin oxidation to oxyluciferin, producing light emission centered at 560 nm. Light emitted from the reaction is directly proportional to the number of luciferase enzyme molecules.
In some embodiments, the enucleated cell is genetically engineered to express only an inflammation homing receptor. In some embodiments, the enucleated cell is genetically engineered to express only firefly luciferase. In some embodiments, the enucleated cell is genetically engineered to express both an inflammation homing receptor and firefly luciferase.
In some embodiments, an enucleated cell is used to identify the presence of a disease condition in a subject by administering to the subject an enucleated cell genetically engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous peptide, wherein the genetically engineered enucleated cell identifies the presence or location of a disease condition. In some embodiments, the exogenous protein is an inflammation homing receptor. In some embodiments, the inflammation homing receptor directs the enucleated cell to damaged and/or inflamed tissue.
The present methods include the use of enucleated cells for treating or diagnosing a disease (e.g., a cancer (e.g., multiple myeloma, glioblastoma, lymphoma, a solid cancer, a leukemia), an infection (e.g., viral infections, such as but not limited to, human immunodeficiency virus (HIV)-infection, Severe Acute Respiratory Syndrome or COVID-19 infection (coronavirus infection), parasitic infection, such as but not limited to, Chagas disease, or bacterial infection, such as but not limited to, tuberculosis), a neurological disease (e.g., Parkinson's Disease, Huntington's Disease, Alzheimer's Disease) an autoimmune disease (e.g., diabetes, Crohn's disease, multiple sclerosis, sickle cell anemia), a cardiovascular disease (e.g., acute myocardial infarction, heart failure, refractory angina), a ophthalmologic disease, a skeletal disease, a metabolic disease (e.g., phenylketonuria, glycogen storage deficiency type 1A, Gaucher disease)) in a subject. In some embodiments, the subject is in need of, or has been determined to be in need of, such an enucleated cell treatment. As used herein, the term “subject” refers to any mammal. In some embodiments, the subject may be a rodent (e.g., a mouse, a rat, a hamster, a guinea pig), a canine (e.g., a dog), a feline (e.g., a cat), an equine (e.g., a horse), an ovine, a bovine, a porcine, a primate, e.g., a simian (e.g., a monkey), an ape (e.g., a gorilla, a chimpanzee, an orangutan, a gibbon), or a human. As used herein, treating includes “prophylactic treatment” which means reducing the incidence of or preventing (or reducing risk of) a sign or symptom of a disease in a subject at risk for the disease, and “therapeutic treatment”, which means reducing signs or symptoms of a disease, reducing progression of a disease, reducing severity of a disease, re-occurrence in a subject diagnosed with the disease. As used herein, the term “treat” means to ameliorate at least one clinical parameter of the disease.
As used herein, the term “administration,” “administering” and variants thereof means introducing a composition or agent into a subject and includes concurrent and sequential introduction of a composition or agent. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. Administration includes self-administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject. Administration can be carried out by any suitable route.
In some embodiments of any of the methods provided herein, the composition is administered at least once (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70 80, 90, 100 times) during a period of time (e.g., every 2 days, twice a week, once a week, every week, three times per month, two times per month, one time per month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year).
In some embodiments, a method of treating a disease in a subject includes administering to the subject a therapeutically effective amount of a composition comprising an enucleated cell genetically engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous peptide. In some embodiments, the composition further comprises a therapeutic agent. In some embodiments, the therapeutic agent comprises at least one of a small RNA, a small molecule drug, a peptide, a virus, or combinations thereof. In some embodiments, the therapeutic agent comprises a chemotherapeutic agent.
A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing form the spirit and scope of the invention.
Embodiment 1. A method of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising an enucleated cell genetically engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous peptide.
Embodiment 2. The method of embodiment 1, wherein the enucleated cell is engineered to express two or more exogenous DNA molecules, exogenous RNA molecules, exogenous proteins, or exogenous peptides, or combinations thereof.
Embodiment 3. The method of embodiment 1, wherein the enucleated cell is engineered to express three or more exogenous DNA molecules, exogenous RNA molecules, exogenous proteins, or exogenous peptides, or combinations thereof.
Embodiment 4. The method of embodiment 1, wherein the enucleated cell is engineered to express four or more exogenous DNA molecules, exogenous RNA molecules, exogenous proteins, or exogenous peptides, or combinations thereof.
Embodiment 5. The method of embodiment 1, wherein the enucleated cell is derived from a natural killer (NK) cell, a macrophage, a neutrophil, a fibroblast, and adult stem cell, a mesenchymal stromal cell (MSC), an inducible pluripotent stem cell, or combinations thereof.
Embodiment 6. The method of embodiment 1, wherein the enucleated cell is derived from a mesenchymal stromal cell (MSC).
Embodiment 7. The method of embodiment 1, wherein the exogenous DNA molecule comprises single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or combinations thereof.
Embodiment 8. The method of embodiment 1, wherein the exogenous RNA molecule comprises messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), a RNA virus, or combinations thereof.
Embodiment 9. The method of embodiment 1, wherein the exogenous protein comprises a cytokine, a growth factor, a hormone, an antibody, an enzyme, or combinations thereof.
Embodiment 10. The method of embodiment 1, wherein the enucleated cell of the composition is selected using fluorescence activated cell sorting (FACS).
Embodiment 11. The method of embodiments 1-10, wherein the enucleated cell further comprises a therapeutic agent.
Embodiment 12. The method of embodiment 11, wherein the therapeutic agent comprises at least one of a small RNA, a small molecule drug, a peptide, a virus, or combinations thereof
Embodiments 13. The method of embodiment 11, wherein the therapeutic agent comprises a chemotherapeutic agent.
Embodiment 14. The method of embodiments 1-13, wherein the administering comprises intravenous administration, subcutaneous administration, intraperitoneal administration, rectal administration, oral administration, or combinations thereof.
Embodiment 15. The method of embodiments 1-14, wherein the composition exhibits minimal accumulation in non-target tissues.
Embodiment 16. The method of embodiments 1-15, wherein the administering is within the site of disease.
Embodiment 17. The method of embodiment 1-16, wherein the disease comprises inflammation, an infection, cancer, a neurological disease, an autoimmune disease, a cardiovascular disease, an ophthalmologic disease, a skeletal disease, a metabolic disease, or combinations thereof.
Embodiment 18. The method of embodiments 1-17, wherein the disease comprises inflammation.
Embodiment 19. The method of embodiments 1-18, wherein the disease comprises cancer.
Embodiment 20. The method of embodiment 19, wherein the cancer comprises multiple myeloma, glioblastoma, lymphoma, leukemia, mesothelioma, sarcoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, or combinations thereof.
Embodiment 21. The method of embodiments 1-20, wherein the administering comprises intratumoral administration.
Embodiment 22. The method of embodiments 1-21, wherein the method inhibits cancer progression.
Embodiment 23. The method of embodiments 1-22, wherein the method reduces tumor growth.
Embodiment 24. The method of embodiments 1-23, wherein the method produces complete tumor regression.
Embodiment 25. The method of embodiments 1-24, wherein the method improves subject likelihood of survival.
Embodiment 26. The method of embodiments 1-25, wherein the method produces a systemic anti-tumor immune response.
Embodiment 27. The method of embodiments 1-26, wherein the composition of enucleated cells is over 90% pure.
Embodiment 28. The method of embodiments 1-27, wherein the composition is over 95% pure.
Embodiment 29. The method of embodiments 1-28, wherein the composition is over 98% pure.
Embodiment 30. The method of embodiments 1-29, wherein the composition is over 99% pure.
Embodiment 31. A method of genetically engineering an enucleated cell, the method comprising:
enucleating a nucleated cell; and
introducing into the enucleated cell an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, an exogenous peptide and/or a therapeutic agent, wherein the genetically engineered enucleated cell retains functional translation and secretory machinery of a parental cell in vivo.
Embodiment 32. The method of embodiment 31, wherein the introducing step occurs before enucleation of the nucleated cell.
Embodiment 33. The method of embodiment 31, wherein the introducing step occurs after the enucleation of the nucleated cell.
Embodiment 34. The method of embodiments 31-33, wherein the enucleating step is over 95% efficient.
Embodiment 35. The method of embodiments 31-34, wherein the method has at least an 80% recovery rate.
Embodiment 36. The method of embodiments 31-35, wherein the method has at least an 85% recovery rate.
Embodiment 37. The method of embodiments 31-36, wherein the introducing step comprises viral transduction.
Embodiment 38. The method of embodiments 31-37, wherein the introducing step comprises using at least one of liposome mediated transfer, an adenovirus, an adeno-associated virus, a herpes virus, a retroviral based vector, lipofection, a lentiviral vector, or combinations thereof.
Embodiment 39. The method of embodiments 31-38, wherein the exogenous DNA molecule comprises single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or combinations thereof.
Embodiment 40. The method of embodiments 31-38, wherein the exogenous RNA molecule comprises messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), a RNA virus, or combinations thereof.
Embodiment 41. The method of embodiments 31-38, wherein the exogenous protein comprises a cytokine, a growth factor, a hormone, an antibody, an enzyme, or combinations thereof.
Embodiment 42. The method of embodiments 31-41, further comprising cryopreserving the genetically engineered enucleated cell.
Embodiment 43. The method of embodiment 42, wherein the genetically engineered enucleated cell is more likely to recover from cryopreservation compared to a parental cell.
Embodiment 44. A genetically engineered enucleated cell produced by introducing into an enucleated cell a least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, an exogenous peptide, or a therapeutic agent.
Embodiment 45. The genetically engineered enucleated cell of embodiment 44, the enucleated cell is engineered to express two or more exogenous DNA molecules, exogenous RNA molecules, exogenous proteins, or exogenous peptides, or combinations thereof.
Embodiment 46. The genetically engineered enucleated cell of embodiment 44, the enucleated cell is engineered to express three or more exogenous DNA molecules, exogenous RNA molecules, exogenous proteins, or exogenous peptides, or combinations thereof.
Embodiment 47. The genetically engineered enucleated cell of embodiment 44, the enucleated cell is engineered to express four or more exogenous DNA molecules, exogenous RNA molecules, exogenous proteins, or exogenous peptides, or combinations thereof.
Embodiment 48. The genetically engineered enucleated cell of embodiment 44, wherein the enucleated cell is derived from a mesenchymal stromal cell (MSC).
Embodiment 49. The genetically engineered enucleated cell of embodiment 48, wherein the enucleated cell is derived from an hTERT-immortalized adipose-derived MSC (hT-MSC).
Embodiment 50. The genetically engineered enucleated cell of embodiment 49, wherein the enucleated cell secretes similar extracellular vesicles (EVs) as compared to a parental hT-MSC cell.
Embodiment 51. The genetically engineered enucleated cell of embodiment 44, wherein the introducing step comprises viral transduction.
Embodiment 52. The genetically engineered enucleated cell of embodiment 44, wherein the introducing step comprises at least one of liposome mediated transfer, adenovirus, adeno-associated virus, herpes virus, a retroviral based vector, lipofection, a lentiviral vector, or combinations thereof.
Embodiment 53. The genetically engineered enucleated cell of embodiment 44, which exhibits substantially the same cell structure as a parental cell.
Embodiment 54. The genetically engineered enucleated cell of embodiment 53, wherein the genetically enucleated cell contains functional subcellular organelles.
Embodiment 55. The genetically engineered enucleated cell of embodiment 54, wherein the functional subcellular organelles comprise at least one of the Golgi, endoplasmic reticulum, mitochondria, lysosomes, endosomes, ribosomes, or combinations thereof.
Embodiment 56. The genetically engineered enucleated cell of embodiment 44, which exhibits substantially the same cell function as a parental cell.
Embodiment 57. The genetically engineered enucleated cell of embodiment 56, wherein the genetically enucleated cell has substantially the same zeta potential than that of a parental cell.
Embodiment 58. The genetically engineered enucleated cell of embodiment 56, wherein the genetically enucleated cell contains functional membrane receptors.
Embodiment 59. The genetically engineered enucleated cell of embodiment 56, wherein the genetically enucleated cell contains functional migration and invasion machinery.
Embodiment 60. The genetically engineered enucleated cell of embodiment 56, wherein the genetically enucleated cell can actively produce and secrete substantially the same extracellular vesicles as those produced by a parental cell.
Embodiment 61. The genetically engineered enucleated cell of embodiments 44-60, wherein the genetically enucleated cell produces therapeutic bioactive proteins in vivo.
Embodiment 62. The genetically engineered enucleated cell of embodiments 44-61, wherein the genetically enucleated cell is engineered to express a cell surface protein.
Embodiment 63. The genetically engineered enucleated cell of embodiment 44, wherein the cell surface protein comprises CXCR4, CCR2, PSGL-1, CD44, CD90, CD105, CD106, CD166, Stro-1, or combinations thereof.
Embodiment 64. The genetically engineered enucleated cell of embodiments 44-63, wherein the diameter of the genetically engineered enucleated cell is smaller than that of a parental cell.
Embodiment 65. The genetically engineered enucleated cell of embodiments 44-64, wherein the diameter of the genetically engineered enucleated cell is about 1 micrometers to 100 micrometers.
Embodiment 66. The genetically engineered enucleated cell of embodiments 44-65, which was derived from cells cultured in hanging drop cell culture.
Embodiment 67. The genetically engineered enucleated cell of embodiments 44-66, wherein the genetically enucleated cell is viable for up to 72 hours post-enucleation.
Embodiment 68. The genetically engineered enucleated cell of embodiments 44-67, wherein the genetically enucleated cell retains MSC surface marker protein expression for at least 48 hours.
Embodiment 69. The genetically engineered enucleated cell of embodiments 44-68, wherein the genetically enucleated cell responds to an extracellular signal.
Embodiment 70. The genetically engineered enucleated cell of embodiment 69, wherein the extracellular signal is a chemokine.
Embodiment 71. The genetically engineered enucleated cell of embodiments 44-70, wherein the genetically enucleated cell is capable of chemotaxis.
Embodiment 72. The genetically engineered enucleated cell of embodiments 44-71, wherein the genetically enucleated cell is capable of protein secretion.
Embodiment 73. The genetically engineered enucleated cell of embodiments 44-72, wherein the genetically enucleated cell is capable of homing.
Embodiment 74. The genetically engineered enucleated cell of embodiments 44-73, wherein the genetically enucleated cell is capable of delivering a target product at a target site in vivo.
Embodiment 75. A method of governing immune recognition and/or activation in a subject, the method comprising:
administering to the subject an enucleated cell, wherein the enucleated cell is genetically engineered to evade recognition by the immune system and/or activate the immune system.
Embodiment 76. The method of embodiment 75, wherein the enucleated cell evades immune recognition in the subject.
Embodiment 77. The method of embodiment 76, wherein the enucleated cell is genetically engineered to deplete the enucleated cell of immune recognition molecules.
Embodiment 78. The method of embodiment 77, wherein the immune recognition molecules comprise HLA antigens, proteoglycans, sugar moieties, embryonic antigens, or combinations thereof.
Embodiment 79. The method of embodiment 76, wherein the enucleated cell is genetically engineered to express at least one exogenous protein.
Embodiment 80. The method of embodiment 79, wherein the exogenous protein is a cell surface protein.
Embodiment 81. The method of embodiment 80, wherein the exogenous protein is an immune evasion molecule.
Embodiment 82. The method of embodiments 79-81, wherein the exogenous protein comprises a cytokine, IL-10, CD47, HLA-E/G, PD-1, LAG-3, CTLA-4, or combinations thereof.
Embodiment 83. The method of embodiment 75, wherein the enucleated cell activates an immune response in the subject.
Embodiment 84. The method of embodiment 83, wherein the enucleated cell is genetically engineered to express at least one exogenous protein.
Embodiment 85. The method of embodiment 84, wherein the exogenous protein is a cell surface protein.
Embodiment 86. The method of embodiment 85, wherein the exogenous protein is an immune activating protein.
Embodiment 87. The method of embodiments 84-86, wherein the exogenous protein comprises a cytokine, IL-12, calreticulin, phosphatidylysine, phagocytosis prey-binding domain, annexin 1, OX40/OC40L, 4-1BB, B7 family members, or combinations thereof.
Embodiment 88. The method of embodiments 75-87, further comprising treating a disease in the subject.
Embodiment 89. The method of embodiment 88, wherein the disease comprises an inflammation disorder and/or a cancer.
Embodiment 90. The method of embodiment 89, wherein the cancer comprises multiple myeloma, glioblastoma, lymphoma, leukemia, mesothelioma, sarcoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, or combinations thereof.
Embodiment 91. A method of identifying the presence of a disease condition in a subject, the method comprising:
administering to the subject an enucleated cell genetically engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous peptide,
wherein the genetically engineered enucleated cell identifies the presence or location of a disease condition.
Embodiment 92. The method of embodiment 91, wherein the exogenous protein is an inflammation homing receptor.
Embodiment 93. The method of embodiment 92, wherein the inflammation homing receptor directs the enucleated cell to damaged and/or inflamed tissue.
Embodiment 94. The method of embodiment 91-93, wherein the enucleated cell further comprises a firefly luciferase.
Embodiment 95. The method of embodiment 94, wherein the firefly luciferase emits detectable light.
Embodiment 96. The method of embodiment 91-95, wherein the disease comprises inflammation, an infection, cancer, a neurological disease, an autoimmune disease, a cardiovascular disease, an ophthalmologic disease, a skeletal disease, a metabolic disease, or combinations thereof.
Embodiment 97. The method of embodiment 96, wherein the disease comprises a cancer.
Embodiment 98. The method of embodiment 97, wherein the cancer comprises multiple myeloma, glioblastoma, lymphoma, leukemia, mesothelioma, sarcoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, or combinations thereof.
The disclosure is further described in the following examples, which do not limit the scope of the disclosure.
First, parental cells (e.g., nucleated cells) were genetically engineered before enucleation of MSCs. G-protein-coupled receptors such as CXCR4 transduce extracellular stimuli into intracellular signals and regulate important cellular functions. The hT-MSCs were engineered with stable CXCR4 expression (MSCCXCR4) via lentivirus infection and drug selection. After enucleation, enucleated cells derived from MSCCXCR4 show stable surface expression of CXCR4 by flow cytometry for up to 48 hours, were shown to be viable for up to 72 hours post-enucleation (
Next, the enucleated cells were engineered to produce therapeutic levels of biologics in vivo. Here, enucleated cells were tested if they can produce bioactive proteins in a tumor microenvironment. The hT-MSCs and enucleated cells were transfected with mouse IL-12 (mIL-12) mRNA (MSC-IL-12 and Cargocyte-IL-12) (
While nucleated cells like MSCs have been engineered to deliver therapeutic biologics, enucleated cell behavior in vivo is more controllable and predictable because they cannot proliferate or engraft into tissues, and do not have transcriptional machinery that can be activated in the disease microenvironment. In the case of delivering IL-12, IFN-γ is a major downstream effector that can significantly activate gene transcription of undesirable immunosuppressive factors such as PD-L1 and IDO1 on the cell vehicles. When stimulated with human IFN-γ in vitro, both hT-MSCs and irradiated hT-MSCs produced dramatically increased PD-L1 and IDO1 mRNAs, whereas genetically engineered enucleated cells did not (
First, since enucleated cells are smaller and lack rigid nuclei (
Next, the enucleated cells were designed and engineered with specific chemokine receptors and adhesion molecules corresponding to a diseased tissue, which was hypothesized to increase enucleated cells homing to the target site in vivo. An acute inflammation mouse model was used, in which bacterial-derived lipopolysaccharide (LPS) was intradermally (i.d.) injected into the pinna to induce acute, local inflammation. Saline was i.d. injected into the contralateral ear as a control. This model allows examination of therapeutic cell homing quantitatively between an inflamed and non-inflamed contralateral tissue within the same animal. It was found that SDF-1α, Ccl2, and P-Selectin but not E-Selectin were upregulated in inflamed ears compared to controls, starting 6 hours post-LPS injection (
Also, enucleated cells were engineered with leukocyte homing molecules combined with their innate tumor trophic properties, which was hypothesized to significantly improve enucleated cells homing to tumors. It was determined if CCP cargocytes can home to chemokines produced by E0771 murine BC conditioned media (CM). E0771 cells are an established murine BC line that produce SDF-1aα and CCL2 in vitro and in tumors in vivo. The engineered enucleated cells migrates towards E0771 CM, which is significantly enhanced through genetic engineering with CXCR4 and CCR2 chemoattractant receptors (
It was then tested if the engineering strategy improved in vivo homing. 3D-cultured MSCs were labeled with DiD and i.v. injected into mice 6 hours after i.d. LPS injection (
Next, the ability of bioengineered enucleated cells (e.g., cargocytes) to deliver anti-inflammatory biologics to treat inflamed tissue was investigated. IL-10 is a potent anti-inflammatory cytokine, but clinical applications need more efficient and specific delivery methods. Enucleated cells transfected with human IL-10 mRNA (Cargocyte-IL-10) produced IL-10 for up to 72 hours in vitro, similar to transfected parental cells (MSC-IL-10), while non-engineered hT-MSCs did not secrete detectable IL-10 (
Then the therapeutic efficacy of engineered enucleated cells in the inflamed tissue was examined. Histologically, inflamed ears from PBS-treated mice showed severe hemorrhage and edema with moderate amounts of mixed leukocytes, while mice treated with 3D-CargocytesTri-E IL-10 had minimal hemorrhage and edema and mild amounts of mixed leukocytes (
Finally, it was shown that engineered human MSC-derived enucleated cells in mouse models have not produced any overt negative health effects in over 300 mice following i.t. or i.v. administration, as determined by clinical observation and gross examination of tissues by a board-certified veterinary pathologist (C.N.A.). BALB/c mice i.v. injected with bioengineered enucleated cells (e.g., cargocytes) had no significant change in the plasma concentration of pro-inflammatory cytokines IL-6, IL-1β, TNF-α and IFN-γ at 4 hours or 24 hours post-injection. Moreover, as a prototype for clinical use, we labeled cell nuclei with Histone 2B-GFP and generated Cargocytes of 99.999% purity through FACS, without loss of viability or migration ability (
Further, the therapeutic delivery of bioengineered enucleated cells in a disease model of acute pancreatitis (AP) was tested. AP is a severe disease with significant morbidity and mortality that currently lacks effective treatments. Caerulein is a decapeptide analog of hormone Cholecystokinin (CCK), which can stimulate exocrine pancreatic secretion and induce AP in pre-clinical mouse models. Previous studies suggested frequent systemic administration of high doses of anti-inflammatory cytokine IL-10 in pre-clinical AP models can greatly attenuate the inflammation and mitigate the disease. However, repeated high doses of IL-10 are not cost-effective in clinical applications and may also lead to unwanted severe complications such as anemia, suggesting a specific and efficient delivery vehicle may be necessary. In early stage of caerulein-induced AP, chemokines such as Ccl2 and SDF-1α, and adhesion molecules such as E-/P-Selectins and Vcam1, are all significantly upregulated in the inflamed mouse pancreas (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a continuation of U.S. application Ser. No. 17/880,141, filed Aug. 3, 2022, which is a continuation of International Application PCT/US2021/016919, with an international filing date of Feb. 5, 2021, which claims priority to U.S. Provisional Application Ser. No. 62/971,526, filed on Feb. 7, 2020; U.S. Provisional Application Ser. No. 62/993,967, filed on Mar. 24, 2020; and U.S. Application Ser. No. 62/994,598, filed on Mar. 25, 2020, each of which is incorporated herein by reference in its entirety.
This invention was made with Government support under Grant Nos. CA182495, and CA097022 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62994598 | Mar 2020 | US | |
| 62993967 | Mar 2020 | US | |
| 62971526 | Feb 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17880141 | Aug 2022 | US |
| Child | 17944604 | US | |
| Parent | PCT/US2021/016919 | Feb 2021 | US |
| Child | 17880141 | US |
