Manufacturing systems and methods for cell therapy platforms

Abstract

Methods for obtaining enucleated cells from nucleated cells are described herein. Methods for cell processing, comprising providing a composition containing nucleated cells and enucleating at least a portion of nucleated cells to produce an enucleated cell fraction, are also described herein. Methods for cell processing, comprising expressing heterologous gene products, are also described herein. Pharmaceutical compositions containing enucleated cells are also provided.

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Application No. 63 / 495,716, filed on 12 April 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application contains a sequence listing submitted electronically in XML format, which is incorporated herein by reference as a whole. The XML copy created on April 10, 2024, is named 53712-731_601_SL.xml and has a size of 42,803 bytes.

[0003] Description of federally funded research and development. This disclosure was made with the support of the U.S. government under grant number 1 R43 HL158351-01 from the National Institutes of Health. Therefore, the government has certain rights with respect to this disclosure. [Overview of the Initiative]

[0004] In some embodiments, a method for delivering a therapeutic agent to target cells of a subject is described herein, the method comprising introducing a plurality of enucleated cells containing the therapeutic agent into a subject or sample of a subject in vivo or ex vivo under conditions sufficient to deliver the therapeutic agent to the target cells of a subject, wherein the plurality of enucleated cells are obtained from a cryopreserved composition or a cryopreserved composition, and the therapeutic agent is delivered to the target cells in an amount approximately equal to or greater than the amount of the therapeutic agent delivered to otherwise equivalent target cells of a subject by otherwise equivalent enucleated cells that are neither cryopreserved nor cryopreserved. In some embodiments, the method further comprises preparing a fluid composition containing a plurality of enucleated cells from a cryopreserved composition. In some embodiments, the cryopreserved composition is cryopreserved in liquid nitrogen. In some embodiments, the cryopreserved composition is cryopreserved for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 1 month, at least about 1 month, or at least about 1 year. In some embodiments, the cryopreserved composition is stored at a maximum of about -80°C before being cryopreserved. In some embodiments, the cryopreserved composition is stored at a temperature of about -80°C or lower for at least about 24 hours. In some embodiments, the method further includes preparing a fluid composition containing a plurality of enucleated cells from the cryopreserved composition. In some embodiments, the cryopreserved composition is stored at a temperature of about 4°C or lower. In some embodiments, the cryopreserved composition remains cryopreserved for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 1 month, at least about 1 month, or at least about 1 year. In some embodiments, multiple enucleated cells from the cryopreserved composition are suspended in xenofree medium. In some embodiments, multiple enucleated cells from the cryopreserved composition are suspended in cryoculture medium.In some embodiments, the freezing medium contains at least 2%, at least 5%, or at least 10% DMSO. In some embodiments, the freezing medium contains CryoStor® medium. In some embodiments, the CryoStor® medium is CryoStor® CS5 or CryoStor® CS10. In some embodiments, the freezing medium contains DMSO, sucrose, sodium hydroxide, potassium hydroxide, or a combination thereof. In some embodiments, the freezing medium contains about 2% to about 15% DMSO. In some embodiments, the freezing medium contains about 0.5% to about 2% sucrose. In some embodiments, the freezing medium contains about 1% sucrose. In some embodiments, the freezing medium contains about 0.5% to about 1% sodium hydroxide. In some embodiments, the freezing medium contains about 0.6% sodium hydroxide. In some embodiments, the freezing medium contains about 0.05% to about 0.5% potassium hydroxide. In some embodiments, the freezing medium contains about 0.1% potassium hydroxide. In some embodiments, preparing the fluid composition involves thawing the frozen composition. In some embodiments, thawing the cryopreserved composition is carried out at room temperature or 37°C. In some embodiments, the method further includes reconstituting a plurality of enucleated cells from the cryopreserved composition after thawing. In some embodiments, phosphate buffer solution (PBS) is used to reconstitute a plurality of enucleated cells from the cryopreserved composition. In some embodiments, sodium lactate solution is used to reconstitute a plurality of enucleated cells from the cryopreserved composition. In some embodiments, physiological saline is used to reconstitute a plurality of enucleated cells from the cryopreserved composition. In some embodiments, the therapeutic agent comprises a virus, an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, an exogenous peptide, or any combination thereof. In some embodiments, the therapeutic agent comprises a virus. In some embodiments, the virus is adeno-associated virus (AAV), adenovirus, reovirus, coxsackievirus, retrovirus, poxvirus, baculovirus, or herpesvirus.In some embodiments, the virus includes an oncolytic virus. In some embodiments, the oncolytic virus is adenovirus, human immunodeficiency virus, marabavirus, measles virus, Newcastle disease virus, poliovirus, Seneca Valley virus, parvovirus, Semryqui Forest virus, varicella stomatitis virus, Sindbis virus, or any combination thereof. In some embodiments, the amount of virus delivered to the subject is measured by the viral titer in the target cells. In some embodiments, the viral titer measured in the target cells is greater than the viral titer measured in otherwise equivalent target cells. In some embodiments, the viral titer measured in the target cells is approximately equal to the viral titer measured in otherwise equivalent target cells. In some embodiments, the exogenous protein includes a cytokine or a cytokine receptor-binding fragment thereof. In some embodiments, the amount of cytokine or a cytokine receptor-binding fragment delivered to the subject is measured by the secretion of cytokine or a cytokine receptor-binding fragment thereof from multiple enucleated cells. In some embodiments, the secretion of the measured cytokine or its cytokine receptor-binding fragment is approximately equal to or greater than the secretion of the cytokine or its cytokine receptor-binding fragment by otherwise equivalent, non-cryopreserved enucleated cells. In some embodiments, the secretion of the measured cytokine or its cytokine receptor-binding fragment is approximately equal to or greater than the secretion of the cytokine or its cytokine receptor-binding fragment by otherwise equivalent, cryopreserved nucleated cells. In some embodiments, the exogenous protein includes an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor includes an inhibitor specific to PD-L1, PD-1, or a combination thereof. In some embodiments, the exogenous protein includes an antigen. In some embodiments, the exogenous protein includes an immunomodulatory protein. In some embodiments, the therapeutic agent includes an exogenous RNA molecule.In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment, a chemokine, or any combination thereof. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment. In some embodiments, the cytokine or its cytokine receptor-binding fragment includes interleukin-12 (IL-12), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-7 (IL-7), interleukin-21 (IL-21), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-15 (IL-15), or any combination thereof. In some embodiments, the exogenous RNA molecule encodes a chemokine. In some embodiments, the chemokine includes stromal cell-derived factor 1α (SDF1α), CC-motif chemokine ligand 2 (CCL2), CC-motif chemokine ligand 3 (CCL3), CC-motif chemokine ligand 5 (CCL5), CC-motif chemokine ligand 8 (CCL8), CC-motif chemokine ligand 1 (CCL1), CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), CC-motif chemokine ligand 11 (CCL11), CXC-motif chemokine ligand 12 (CXCL12), or any combination thereof. In some embodiments, the exogenous RNA molecule encodes an immune checkpoint inhibitor, antigen, or immunomodulatory protein. In some embodiments, the immune checkpoint inhibitor includes an inhibitor specific to PD-L1, PD-1, or a combination thereof. In some embodiments, the method further includes treating a disease or condition in a subject. In some embodiments, the disease is cancer. In some embodiments, cancer includes solid tumors. In some embodiments, cancer is lung cancer, cancer metastasis in lung tissue, liver cancer, or cancer metastasis in liver tissue. In some embodiments, liver cancer is hepatocellular carcinoma or cholangiocarcinoma. In some embodiments, cancer is lung cancer.In some embodiments, lung cancer is small cell lung cancer, non-small cell lung cancer, or bronchial carcinoid. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, lung cancer is bronchial carcinoid. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. In some embodiments, the method further comprises intravenous administration to a plurality of enucleated cells. In some embodiments, the target cells include cancer cells. In some embodiments, the target cells include solid tumor cells. In some embodiments, the target cells include lung cells. In some embodiments, the target cells include hepatocytes.

[0005] In some embodiments, compositions comprising a plurality of enucleated cells formulated from a cryopreserved composition or a cryopreserved composition, wherein the cryopreserved composition or the cryopreserved composition comprises a plurality of cryopreserved or cryopreserved enucleated cells, and at least a subset of the plurality of enucleated cells comprises (i) a therapeutic agent and (ii) intracellular organelles sufficient to release the therapeutic agent in vivo or ex vivo in an amount approximately equal to or greater than the amount released by otherwise identical, non-cryopreserved and non-cryopreserved enucleated cells. In some embodiments, the plurality of enucleated cells have a diameter including about 70% or less of the average diameter of the nucleated parent cell. In some embodiments, the plurality of enucleated cells have a diameter including about 1 micrometer (μm) to about 100 μm. In some embodiments, the plurality of enucleated cells have a diameter including about 5 μm to about 25 μm. In some embodiments, the plurality of enucleated cells have a diameter including about 8 μm. In some embodiments, the therapeutic agent comprises a virus, an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein or exogenous peptide, or any combination thereof. In some embodiments, the therapeutic agent comprises a virus. In some embodiments, the virus is adeno-associated virus (AAV), adenovirus, reovirus, coxsackievirus, retrovirus, poxvirus, baculovirus, or herpesvirus. In some embodiments, the virus comprises an oncolytic virus. In some embodiments, the oncolytic virus is adenovirus, human immunodeficiency virus, marabavirus, measles virus, Newcastle disease virus, poliovirus, Seneca Valley virus, parvovirus, Semryqui Forest virus, varicella stomatitis virus, Sindbis virus, or any combination thereof. In some embodiments, the amount of virus released is measured by the viral titer in target cells. In some embodiments, the viral titer measured in target cells is greater than the viral titer measured in otherwise equivalent target cells. In some embodiments, the viral titer measured in target cells is approximately equal to the viral titer measured in otherwise equivalent target cells.In some embodiments, the therapeutic agent comprises a cytokine or a cytokine receptor-binding fragment thereof. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released in vivo or ex vivo is a measure of the secretion of cytokine or cytokine receptor-binding fragment from multiple enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of cytokine or cytokine receptor-binding fragment by otherwise equivalent enucleated cells that were neither cryopreserved nor cryopreserved. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of cytokine or cytokine receptor-binding fragment by otherwise equivalent cryopreserved or cryopreserved nucleated cells. In some embodiments, the exogenous protein comprises an immune checkpoint inhibitor. In some embodiments, the exogenous protein comprises an antigen. In some embodiments, the exogenous protein comprises an immunomodulatory protein. In some embodiments, the immune checkpoint inhibitor comprises an inhibitor specific to PD-L1, PD-1, or a combination thereof. In some embodiments, the therapeutic agent comprises an exogenous RNA molecule. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment, a chemokine, or any combination thereof. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment. In some embodiments, the cytokine or its cytokine receptor-binding fragment includes IL-12, IFN-α, IFN-β, IFN-γ, IL-7, IL-21, TNF-α, GM-CSF, IL-15, or any combination thereof. In some embodiments, the exogenous RNA molecule encodes a chemokine. In some embodiments, the chemokine includes SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11, CXCL12, or any combination thereof.In some embodiments, the exogenous RNA molecule encodes an immune checkpoint inhibitor, an antigen, or an immunomodulatory protein. In some embodiments, the immune checkpoint inhibitor includes inhibitors specific to PD-L1, PD-1, or a combination thereof. In some embodiments, each enucleated cell lacks a nucleus and includes one or more structural features of a nucleated cell. In some embodiments, one or more structural features include one or more tunnel nanotubes. In some embodiments, the intracellular organelle includes the Golgi apparatus, the endoplasmic reticulum, or any combination thereof.

[0006] In some embodiments, pharmaceutical compositions comprising the compositions described herein and pharmaceutically acceptable excipients, diluents, or carriers are described herein. In some embodiments, the pharmaceutical compositions are in unit dose form. In some embodiments, the pharmaceutical compositions are formulated for administration to a subject by intrathecal, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladderal, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or any combination thereof. In some embodiments, the pharmaceutical compositions are formulated for intravenous administration. In some embodiments, the pharmaceutical compositions further comprise at least one additional activator. In some embodiments, the at least one additional activator comprises cytokines, growth factors, hormones, enzymes, small molecules, compounds, or any combination thereof.

[0007] In some embodiments, a kit is described herein. The kit comprises a composition or pharmaceutical composition as described herein, and a container for storing the composition or pharmaceutical composition. In some embodiments, the kit further comprises a resuspension buffer. In some embodiments, the resuspension buffer comprises PBS. In some embodiments, the resuspension buffer comprises physiological saline. In some embodiments, the resuspension buffer comprises a sodium lactate solution. In some embodiments, the kit further comprises instructions for a method of delivering the composition or pharmaceutical composition to target cells of interest, the method comprising introducing the composition or pharmaceutical composition to target cells of interest in vivo or ex vivo under conditions sufficient to deliver the therapeutic agent to the target cells. In some embodiments, the method further comprises treating a disease or condition of interest by administering the therapeutic agent to target cells of interest. In some embodiments, the disease or condition of interest includes cancer. In some embodiments, cancer includes solid tumors. In some embodiments, cancer is lung cancer, cancer metastases in lung tissue, liver cancer, or cancer metastases in liver tissue. In some embodiments, introducing a composition or pharmaceutical composition into target cells of a subject includes administering the composition or pharmaceutical composition to the subject in the intrathecal cavity, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary gastrointestinal route, or any combination thereof. In some embodiments, the kit further comprises at least one additional activator, the at least one additional activator comprising cytokines, growth factors, hormones, enzymes, small molecules, compounds, or any combination thereof.

[0008] The embodiments disclosed herein provide a method for delivering a therapeutic agent to target cells of a subject, comprising: a) preparing a fluid formulation from a cryopreserved composition, wherein the cryopreserved composition comprises the cryopreserved multiple enucleated cells, and at least a subset of the multiple enucleated cells comprises the therapeutic agent; and b) introducing the fluid formulation into a subject or sample of a subject in an amount approximately equal to or greater than the amount of the therapeutic agent delivered to otherwise identical, non-cryopreserved enucleated cells of a subject, under conditions sufficient to deliver the therapeutic agent to the target cells of a subject in vivo or ex vivo. In some embodiments, the cryopreserved composition is cryopreserved in liquid nitrogen. In some embodiments, the cryopreserved composition is cryopreserved for at least about 24 hours. In some embodiments, the cryopreserved composition is cryopreserved for at least about 7 days. In some embodiments, the cryopreserved composition is cryopreserved for at least about 1 month. In some embodiments, the cryopreserved composition is cryopreserved for at least about 1 year. In some embodiments, the cryopreserved composition is placed at a maximum of about -80°C before being cryopreserved. In some embodiments, the cryopreserved composition is placed at a maximum of about -80°C for at least about 24 hours. In some embodiments, multiple enucleated cells from the cryopreserved composition are suspended in xenofree medium. In some embodiments, multiple enucleated cells from the cryopreserved composition are suspended in CryoStor® medium. In some embodiments, the CryoStor® medium is CryoStor® CS10 medium. In some embodiments, preparing the fluid composition includes thawing the cryopreserved composition. In some embodiments, thawing the cryopreserved composition is carried out at room temperature. In some embodiments, thawing the cryopreserved composition is carried out at 37°C. In some embodiments, thawing the cryopreserved composition is carried out in a water bath at 37°C. In some embodiments, the method further includes reconstituting multiple enucleated cells from the cryopreserved composition after thawing.In some embodiments, reconstitution of multiple enucleated cells from a cryopreserved composition is performed using phosphate-buffered solution (PBS). In some embodiments, reconstitution of multiple enucleated cells from a cryopreserved composition is performed using a sodium lactate solution. In some embodiments, the therapeutic agent comprises a virus, an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, an exogenous peptide, or a combination thereof. In some embodiments, the therapeutic agent comprises a virus. In some embodiments, the virus is adeno-associated virus (AAV), adenovirus, reovirus, coxsackievirus, retrovirus, poxvirus, baculovirus, or herpesvirus. In some embodiments, the virus comprises an oncolytic virus. In some embodiments, the oncolytic virus is adenovirus, human immunodeficiency virus, marabavirus, measles virus, Newcastle disease virus, poliovirus, Seneca Valley virus, parvovirus, Semryqui Forest virus, varicella stomatitis virus, Sindbis virus, or a combination thereof. In some embodiments, the amount of virus delivered to a subject in vivo or ex vivo is a measure of viral titer in target cells. In some embodiments, the viral titer measured in target cells is greater than the viral titer measured in otherwise identical target cells. In some embodiments, the viral titer measured in target cells is approximately equal to the viral titer measured in otherwise identical target cells. In some embodiments, the therapeutic agent comprises a cytokine or a cytokine receptor-binding fragment thereof. In some embodiments, the amount of cytokine or a cytokine receptor-binding fragment delivered to a subject in vivo or ex vivo is a measure of secretion of cytokine or a cytokine receptor-binding fragment from multiple enucleated cells. In some embodiments, the secretion of cytokine or a cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of cytokine or a cytokine receptor-binding fragment from otherwise identical, non-cryopreserved enucleated cells.In some embodiments, the secretion of the cytokine or its cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of the cytokine or its cytokine receptor-binding fragment by otherwise identical cryopreserved nucleated cells. In some embodiments, the therapeutic agent includes an exogenous RNA molecule. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment, a chemokine, or a combination thereof. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment. In some embodiments, the cytokine or its cytokine receptor-binding fragment includes interleukin-12 (IL-12), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-7 (IL-7), interleukin-21 (IL-21), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-15 (IL-15), or a combination thereof. In some embodiments, the exogenous RNA molecule encodes a chemokine. In some embodiments, the chemokine includes stromal cell-derived factor 1α (SDF1α), CC-motif chemokine ligand 2 (CCL2), CC-motif chemokine ligand 3 (CCL3), CC-motif chemokine ligand 5 (CCL5), CC-motif chemokine ligand 8 (CCL8), CC-motif chemokine ligand 1 (CCL1), CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), CC-motif chemokine ligand 11 (CCL11), CXC-motif chemokine ligand 12 (CXCL12), or a combination thereof. In some embodiments, the method further includes treating a disease or condition in a subject. In some embodiments, the disease is cancer. In some embodiments, the cancer is lung cancer, cancer metastasis in lung tissue, liver cancer, or cancer metastasis in liver tissue. In some embodiments, liver cancer is hepatocellular carcinoma or cholangiocarcinoma. In some embodiments, cancer is lung cancer.In some embodiments, lung cancer is small cell lung cancer, non-small cell lung cancer, or bronchial carcinoid. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, lung cancer is bronchial carcinoid. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. In some embodiments, treating a disease or condition in a subject involves intravenous administration of a fluid formulation to the subject. In some embodiments, the target cells of the subject include lung cells. In some embodiments, the target cells of the subject include hepatocytes.

[0009] Embodiments disclosed herein provide formulations comprising a plurality of enucleated cells formulated from a cryopreserved composition, the cryopreserved composition comprising a plurality of cryopreserved enucleated cells, at least a subset of the plurality of enucleated cells comprising (i) a therapeutic agent and (ii) an intracellular organelle sufficient to release the therapeutic agent in vivo or ex vivo in an amount substantially equal to or greater than the amount of therapeutic agent released by otherwise identical, non-cryopreserved enucleated cells. In some embodiments, each enucleated cell of the plurality of enucleated cells comprises a diameter including about 70% or less of the average diameter of the nucleated parent cell. In some embodiments, each enucleated cell of the plurality of enucleated cells comprises a diameter including about 1 micrometer (μm) to about 100 μm. In some embodiments, each enucleated cell of the plurality of enucleated cells comprises a diameter including about 5 μm to about 25 μm. In some embodiments, each nucleated cell of the plurality of enucleated cells comprises a diameter including about 8 μm. In some embodiments, the therapeutic agent comprises a virus, an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein or exogenous peptide, or any combination thereof. In some embodiments, the therapeutic agent comprises a virus. In some embodiments, the virus is adeno-associated virus (AAV), adenovirus, reovirus, coxsackievirus, retrovirus, poxvirus, baculovirus, or herpesvirus. In some embodiments, the virus comprises an oncolytic virus. In some embodiments, the oncolytic virus is adenovirus delta-24, human immunodeficiency disease virus, marabavirus, measles virus, Newcastle disease virus, poliovirus, Seneca Valley virus, parvovirus, Semryqui Forest virus, varicella stomatitis virus, Sindbis virus, or a combination thereof. In some embodiments, the amount of virus released in vivo or ex vivo is a measure of viral titer in target cells. In some embodiments, the viral titer measured in target cells is greater than the viral titer measured in otherwise identical target cells. In some embodiments, the viral titer measured in target cells is approximately equal to the viral titer measured in otherwise identical target cells.In some embodiments, the therapeutic agent comprises a cytokine or a cytokine receptor-binding fragment thereof. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released in vivo or ex vivo is a measure of the secretion of cytokine or cytokine receptor-binding fragment from multiple enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of cytokine or cytokine receptor-binding fragment by otherwise identical, non-cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of cytokine or cytokine receptor-binding fragment by otherwise identical, non-cryopreserved nucleated cells. In some embodiments, the therapeutic agent comprises an exogenous RNA molecule. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment, a chemokine, or a combination thereof. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment. In some embodiments, the cytokine or its cytokine receptor-binding fragment includes IL-12, IFN-α, IFN-β, IFN-γ, IL-7, IL-21, TNF-α, GM-CSF, IL-15, or a combination thereof. In some embodiments, the exogenous RNA molecule encodes a chemokine. In some embodiments, the chemokine includes SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11, CXCL12, or a combination thereof. In some embodiments, each enucleated cell lacks a nucleus and includes one or more structural features of a nucleated cell. In some embodiments, one or more structural features include one or more tunnel nanotubes. In some embodiments, the intracellular organelle includes the Golgi apparatus, the endoplasmic reticulum, or a combination thereof.

[0010] Embodiments disclosed herein provide a) a formulation of any one of the embodiments described above, and b) a pharmaceutical formulation comprising a pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments, the pharmaceutical formulation is in unit dose form. In some embodiments, the pharmaceutical formulation is formulated for administration to a subject by intrathecal, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or a combination thereof. In some embodiments, the pharmaceutical formulation is formulated for intravenous administration. In some embodiments, the pharmaceutical formulation further comprises at least one additional activator. In some embodiments, the at least one additional activator comprises cytokines, growth factors, hormones, enzymes, small molecules, compounds, or any combination thereof.

[0011] Embodiments disclosed herein provide a kit comprising a formulation described in any one of the embodiments described above, or a pharmaceutical formulation described in any one of the embodiments described above, and a) a container for storing the formulation or pharmaceutical formulation. In some embodiments, the kit further comprises a resuspension buffer. In some embodiments, the resuspension buffer comprises phosphate-buffered saline (PBS). In some embodiments, the resuspension buffer comprises a sodium lactate solution. In some embodiments, the kit further comprises instructions for a method of delivering the formulation or pharmaceutical formulation to target cells of interest, the method comprising introducing the formulation or pharmaceutical formulation into target cells of interest under conditions sufficient to deliver the therapeutic agent to the target cells in vivo or ex vivo. In some embodiments, the instructions further comprise a method of delivering the formulation or pharmaceutical formulation to target cells of interest, the method comprising introducing the formulation or pharmaceutical formulation into target cells of interest under conditions sufficient to deliver the therapeutic agent to the target cells in vivo or ex vivo. In some embodiments, the method further comprises treating a disease or condition of interest by administering the therapeutic agent to target cells of interest in vivo. In some embodiments, the disease or condition of interest includes cancer. In some embodiments, cancer is lung cancer, cancer metastasis in lung tissue, liver cancer, or cancer metastasis in liver tissue. In some embodiments of any one of the kits described above, introducing the formulation or pharmaceutical formulation into target cells of the subject includes administering the formulation or pharmaceutical formulation to the subject in the intrathecal cavity, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary gastrointestinal route, or a combination thereof. In some embodiments of any one of the kits described above, the kit further comprises at least one additional activator, the at least one additional activator comprising cytokines, growth factors, hormones, enzymes, small molecules, compounds, or any combination thereof.

[0012] Built-in by reference All publications, patents, and patent applications described in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that any incorporated publications and patents or patent applications conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting materials.

[0013] Some novel features of the inventive concepts disclosed herein are described in this disclosure. A better understanding of the features and advantages of the inventive concepts disclosed herein can be obtained by reference to the following detailed description, which shows non-limiting exemplary embodiments in which the principles of the disclosed inventive concepts are utilized, and the accompanying drawings.

Brief Description of the Drawings

[0014] [Figure 1] A flowchart showing non-limiting steps of a process for treating a composition of enucleated cells or a pharmaceutical composition for the delivery of a therapeutic agent, according to one embodiment of the present disclosure. [Figure 2] Shows the timeline of production of enucleated cells for the delivery of single domain antibodies according to various embodiments, compared to the typical timeline of biological drug development. [Figure 3A] Shows the workflow of an experiment to evaluate the adhesion of fresh enucleated cells and cryopreserved enucleated cells to fibronectin-coated plates. [Figure 3B] Images of fresh enucleated cells (left, e.g., before freezing) and cryopreserved enucleated cells (right, e.g., after thawing) 24 hours after seeding. Images were acquired with a Nikon Eclipse Ti microscope. [Figure 4A] Shows the workflow of an experiment to evaluate the secretion of IL-12 by fresh enucleated cells and cryopreserved enucleated cells. Three enucleated cells are seeded each. [Figure 4B]Levels of IL-12 (nanograms / milliliter) secreted from fresh enucleated cells, enucleated cells cryopreserved in 90% FBS + 10% DMSO, and enucleated cells cryopreserved in CryoStor® CS10 are shown. The data shown are the average of three samples per group of enucleated cells examined, and the error bars indicate the standard deviation of the data. [Figure 5A] The workflow of an experiment to evaluate the in vivo functional capacity of fresh enucleated cells and cryopreserved enucleated cells is shown. [Figure 5B] Levels of IL-12 (ng / mL) in the supernatant obtained from cultures containing either fresh enucleated cells or cryopreserved enucleated cells are shown. [Figure 5C] Levels of IL-12 picograms / mL (pg / mL) on days 1, 2, and 3 after injection in the plasma of mice injected with either fresh enucleated cells or cryopreserved enucleated cells are shown. The data shown are the average of three mice per group of enucleated cells examined, and the error bars indicate the standard error of the mean of the data. [Figure 5D] Levels of IFNγ (pg / mL) in the plasma of mice injected with fresh enucleated cells or cryopreserved enucleated cells on days 1, 2, and 3 after injection are shown. The data shown are the average of three mice per group of enucleated cells examined, and the error bars indicate the standard error of the mean of the data. [Figure 5E] Fold changes in the expression of IL-12 mRNA in the lungs of mice injected with either fresh enucleated cells or cryopreserved enucleated cells on days 1, 2, and 3 after injection are shown. Fold changes are calculated using the delta-delta Ct method that compares the expression level of the target gene to the expression level of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT). The data shown are the average of three mice per group of enucleated cells examined, and the error bars indicate the standard error of the mean of the data. [Figure 5F]This shows the multiplicative changes in IFN-γ expression in the lungs of mice injected with either fresh or cryopreserved enucleated cells on days 1, 2, and 3 post-injection. Multiplicative changes are calculated using the delta-delta Ct method, comparing the expression level of the target gene to the expression level of hypoxanthine phosphoribosyltransferase (HPRT) of a housekeeping gene. The data shown are the mean of 3 mice per group of enucleated cells examined, and error bars indicate the standard error of the mean data. [Figure 5G] This shows the ploidy of IL-12 mRNA expression in the livers of mice injected with either fresh or cryopreserved enucleated cells on days 1, 2, and 3 post-injection. The ploidy is calculated using the delta-delta Ct method, comparing the expression level of the target gene to the expression level of hypoxanthine phosphoribosyltransferase (HPRT) of a housekeeping gene. The data shown are the mean of 3 mice per group of enucleated cells examined, and the error bars indicate the standard error of the mean data. [Figure 5H] This shows the multiplicative changes in IFN-γ expression in the livers of mice injected with either fresh or cryopreserved enucleated cells on days 1, 2, and 3 post-injection. Multiplicative changes are calculated using the delta-delta Ct method, comparing the expression level of the target gene to the expression level of hypoxanthine phosphoribosyltransferase (HPRT) of a housekeeping gene. The data shown are the mean of 3 mice per group of enucleated cells examined, and the error bars indicate the standard error of the mean data. [Figure 5I] This shows the percentage of single DiD-labeled enucleated cells present in the lungs of mice injected with either fresh or cryopreserved enucleated cells, on days 1, 2, and 3 post-injection. Data are presented as the frequency of single cells and as the total number of DiD+ events. The data shown are the mean of 3 mice per group of enucleated cells examined, and error bars indicate the standard error of the mean of the data. [Figure 6A]This document describes the workflow for an experiment evaluating the delivery of VSV to mouse lungs using fresh or cryopreserved enucleated cells. Five mice are injected with either fresh or cryopreserved enucleated cells. [Figure 6B] This shows the VSV titer (plaque-forming units per gram of lung tissue) present in the lungs of mice injected with either fresh VSV-infected enucleated cells or cryopreserved VSV-infected enucleated cells. The data shown are the mean of 5 mice per group of enucleated cells examined, and the error bars represent the standard error of the mean of the data. [Figure 6C] This shows the VSV titer (PFU / mL) of the supernatant collected 48 hours after seeding either fresh VSV-infected enucleated cells (e.g., before freezing) or cryopreserved VSV-infected enucleated cells (e.g., after thawing). [Figure 7A] This image shows human Wharton jelly (mesenchymal stem cell) MSCs (MSCs) transfected with mouse IL-12 mRNA and seeded either fresh or after freezing and thawing. Secreted mouse IL-12 was analyzed by ELISA from conditioned media under various conditions. n=3. [Figure 7B] Human umbilical cord MSCs (MSCs) seeded with transfected mouse IL-12 mRNA are shown, either fresh or after freezing and thawing. Secreted mouse IL-12 was analyzed by ELISA from conditioned media under each condition. n=3. [Figure 8A] The figure shows C57BL / 6 mice subcutaneously inoculated with 1 × 10⁶ EO771 tumor cells. After 12 days, the mice were stratified by tumor volume. Every 3 days, mice were treated intratumorally (it) with mouse IL-12 mRNA-transfected or untransfected cryopreserved human bone marrow enucleated cells, and simultaneously treated intraperitoneally (ip) with anti-PD1 antibody. Tumor volume was measured three times a week, and mice that did not meet health criteria were sacrificed. n is indicated in the figure. [Figure 8B]This figure shows C57BL / 6 mice subcutaneously inoculated with 1 × 10⁶ EO771 tumor cells. After 12 days, the mice were stratified by tumor volume. Every 3 days, mice were treated intratumorally (it) with either mouse IL-12 mRNA-transfected or untransfected cryopreserved human bone marrow enucleated cells, and simultaneously treated with anti-PD1 antibody by intraperitoneal (ip) injection. Kaplan-Meier curves for the same mice as in Figure 8A. n is indicated in the figure.

[0015] Novel features of this disclosure are described in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description illustrating exemplary embodiments. [Modes for carrying out the invention]

[0016] Quality control of enucleated cell platforms for biomedical applications is a challenge, and this challenge is further exacerbated by large-scale manufacturing. Many of the advantages of enucleated cells disclosed herein stem from the absence of a nucleus, including the elimination of the need for in vivo gene transfer and the limitation of in vivo lifespan. However, existing large-scale manufacturing techniques result in the inclusion of nucleated parent cell portions in the resulting therapeutic compositions, thus undermining the advantages of the enucleated cell platform.

[0017] In addition to advancements in manufacturing scalability and quality control, the enucleation cell platform described herein offers certain advantages over existing cell-based therapeutic platforms and is unparalleled in suitability for large-scale use as a therapeutic composition. Further disclosures relating to the enucleation cells described herein are found in U.S. Patent No. 10,927,349, which is incorporated herein by reference as a whole. Furthermore, further usefulness and advantages of the enucleation cells disclosed herein are found in International Application No. PCT / US2022 / 018007, filed on 25 February 2022 and published as WO / 20221 / 83057 A1, and U.S. Patent Application No. 17 / 885,867, filed on 11 August 2022 and published as WO / 20211 / 63222 A1, each incorporated herein by reference as a whole.

[0018] For example, cell delivery platforms have specific therapeutic applications (such as addressing disease relapses), and existing manufacturing timelines for these may limit the scalability and speed required to address disease relapses in individuals. Existing therapeutic cell therapies requiring extensive manipulation require at least 12 months for development. On the other hand, enucleated cells disclosed herein can be extensively manipulated before and after enucleation (e.g., using targeting regions specific to target tissue, immune system evasion regions to reduce in vivo phagocytosis, etc.), and then stored for extended periods without sacrificing their restored viability by preferred means disclosed herein (e.g., cryopreservation). When a new pathogen or a new strain of a known pathogen is identified, the enucleated cells (already manipulated to express appropriate targeting regions, immune system evasion regions, immune activators, etc.) can be restored to their biological activity (e.g., rehydration, thawing, etc.) and further manipulated to express or deliver therapeutic agents for the prevention or treatment of relapsing diseases or conditions. These advantages are shown in Figure 2, which illustrates that the enucleated cell manufacturing process of this disclosure takes approximately 2 months, compared to a preferred timeline of 12 months or more.

[0019] Existing erythrocyte or platelet therapeutic platforms are enucleated by erythrocyte generation. In this erythrocyte generation, blood cells ultimately differentiate, and some intracellular organelles and ribosomes responsible for protein synthesis and secretion are eliminated. Therefore, the resulting erythrocytes or platelets lose cell-like functionality (e.g., protein expression, secretion, cell motility, chemokine sensing, homing ability, etc.) that may be important for therapeutic applications (e.g., production, delivery, or secretion of therapeutic agents in vivo) after enucleation by erythrocyte generation. In contrast, the enucleated cells described herein retain one or more intracellular organelles endogenous to the parent cell after enucleation. In some embodiments, all of the one or more intracellular organelles are retained. In some embodiments, fewer than all of the one or more intracellular organelles are retained. In some embodiments, the Golgi apparatus and / or endoplasmic reticulum, which are involved in protein synthesis and secretion, are retained. By retaining one or more intracellular organelles, enucleated cells are at least partially able to synthesize or release biomolecules disclosed herein (e.g., single-domain antibodies, or portions thereof, targeting portions, immune-evading portions, etc.) in the absence of a nucleus.

[0020] Enucleated cells disclosed herein may originate from substantially any nucleated cell (referred to herein as “parent” cell). In some embodiments, the parent cell is an immune cell. In some embodiments, the immune cell is a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, or lymphocyte (B cell and T cell). In some embodiments, the parent cell is a stem cell. In some embodiments, the parent cell is an adult stem cell. In some embodiments, the parent cell is a mesenchymal stromal cell (MSC). In some embodiments, the enucleated cells originate from inducible pluripotent stem cells (iPSCs). In some embodiments, the parent cell is not a erythrocyte. In some embodiments, the parent cell is not an erythrocyte progenitor cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial progenitor cell.

[0021] A method for producing enucleated cells in high volume and with high purity is described herein. Herein, the produced enucleated cells can be formulated into compositions or pharmaceutical compositions for the treatment of a disease or pathological condition in a subject requiring treatment. Figure 1 shows a non-limiting example of the production of enucleated cells (100) as described herein. Nucleated cells (101) can be isolated from a subject and cultured in vitro for clonal proliferation. In some embodiments, nucleated cells (101) can also be immortalized or derived from a cell line. In some embodiments, nucleated cells can be manipulated to contain heterologous polynucleotides (103) (102). The nucleated cells can then be enucleated by continuous flow centrifugation (104). Using continuous flow centrifugation for cell enucleation is an improvement over currently available enucleation methods, and enucleation performed by continuous flow centrifugation improves the volume (yield, etc.) or purity of enucleated cells obtained from nucleated cells. After obtaining a composition (105) of enucleated cells (which may contain residual nucleated cells), the enucleated cells can be further purified from the composition by selecting an enucleated cell marker (106) or by inducing cell death of the remaining residual nucleated cells (107) to obtain a portion of enucleated cells (108). The portion of enucleated cells can be subjected to cryo-hibernation (109), cryopreservation (110), freeze-drying (111), or a combination thereof, and can be formulated into a composition or pharmaceutical composition for delivering a therapeutic agent to treat a target disease or condition.

[0022] In some embodiments, methods for delivering a therapeutic agent to target cells of a subject are described herein. In some embodiments, the target cells of a subject include lung cells. In some embodiments, the target cells of a subject include hepatocytes. In some embodiments, the method involves introducing a plurality of enucleated cells containing the therapeutic agent into a subject or a sample of a subject in vivo or ex vivo, under conditions sufficient to deliver the therapeutic agent to the target cells of a subject. In some embodiments, the plurality of enucleated cells are obtained from a cryopreserved composition or a cryopreserved composition. In some embodiments, the therapeutic agent is delivered to the target cells in an amount approximately equal to or greater than the amount of the therapeutic agent delivered to otherwise equivalent target cells of a subject by enucleated cells that are otherwise equivalent, not cryopreserved, and not cryopreserved. In some embodiments, the method involves preparing a fluid formulation containing a plurality of enucleated cells from a cryopreserved composition or a cryopreserved composition. In some embodiments, the cryopreserved composition is cryopreserved for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 1 month, at least about 1 month, or at least about 1 year, or indefinitely. In some embodiments, cryopreservation includes storing a plurality of enucleated cells at a temperature of about -80°C. In some embodiments, the cryopreserved composition is stored at a maximum of about -80°C. In some embodiments, the cryopreserved composition is stored at a maximum of about -80°C for at least 24 hours. In some embodiments, cryopreservation includes storing a plurality of enucleated cells at a temperature of about -20°C. In some embodiments, cryopreservation includes storing a plurality of enucleated cells in liquid nitrogen. In some embodiments, cryopreservation includes storing a plurality of enucleated cells in contact with a cryopreserved medium described herein. In some embodiments, the cryopreserved medium includes a xenofree medium. In some embodiments, a plurality of enucleated cells from the cryopreserved composition are suspended in a xenofree medium. In some embodiments, multiple enucleated cells from a cryopreserved composition are suspended in a frozen culture medium.In some embodiments, the freezing medium contains about 2% DMSO. In some embodiments, the freezing medium contains about 5% DMSO. In some embodiments, the freezing medium contains about 10% DMSO. In some embodiments, the freezing medium contains at least 5% or at least 10% DMSO. In some embodiments, the freezing medium contains CryoStor® medium. In some embodiments, the CryoStor® medium is CryoStor® CS5. In some embodiments, the CryoStor® medium is CryoStor® CS10. In some embodiments, the method comprises thawing a plurality of enucleated cells, the plurality of enucleated cells exhibiting equivalent cellular function or vitality compared to a plurality of enucleated cells that have not been cryopreserved. In some embodiments, thawing comprises contacting the plurality of enucleated cells in a water bath. In some embodiments, thawing comprises contacting the plurality of enucleated cells at room temperature. In some embodiments, thawing the cryopreserved composition is carried out at room temperature. In some embodiments, thawing comprises contacting the plurality of enucleated cells at 37°C. In some embodiments, thawing the cryopreserved composition is carried out at 37°C.

[0023] As a non-limiting example, Figure 3A shows a workflow that begins with thawing vials of cryopreserved enucleated cells in a 37°C water bath and ends with imaging the cells to confirm the enucleation efficiency. The left branch of Figure 3A is shown as the image before cryopreservation, as shown in Figure 3B (left). The right branch of Figure 3A is shown as the image after cryopreservation, as shown in Figure 3B (right).

[0024] In some embodiments, multiple enucleated cells contain a therapeutic agent. In some embodiments, the therapeutic agent includes a virus, an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, an exogenous peptide, or any combination thereof. In some embodiments, the therapeutic agent contains a virus. In some embodiments, the virus is adeno-associated virus (AAV), adenovirus, reovirus, coxsackievirus, retrovirus, poxvirus, baculovirus, or herpesvirus. In some embodiments, the virus includes an oncolytic virus. In some embodiments, the oncolytic virus is adenovirus, human immunodeficiency virus, marabavirus, measles virus, Newcastle disease virus, poliovirus, Seneca Valley virus, parvovirus, Semryqui Forest virus, varicella stomatitis virus, Sindbis virus, or any combination thereof. In some embodiments, the amount of virus delivered to the target is measured by the viral titer in the target cells. In some embodiments, the viral titer measured in the target cells is greater than the viral titer measured in otherwise equivalent target cells. In some embodiments, the viral titer measured in target cells is approximately equal to the viral titer measured in otherwise equivalent target cells. In some embodiments, the exogenous protein comprises a cytokine or its cytokine receptor-binding fragment. In some embodiments, the amount of cytokine or its cytokine receptor-binding fragment delivered to the target is measured by the secretion of the cytokine or its cytokine receptor-binding fragment from multiple enucleated cells. In some embodiments, the measured secretion of cytokine or its cytokine receptor-binding fragment is approximately equal to or greater than the secretion of cytokine or its cytokine receptor-binding fragment by otherwise equivalent, non-cryopreserved enucleated cells. In some embodiments, the measured secretion of cytokine or its cytokine receptor-binding fragment is approximately equal to or greater than the secretion of cytokine or its cytokine receptor-binding fragment by otherwise equivalent, cryopreserved nucleated cells. In some embodiments, the exogenous protein comprises an immune checkpoint inhibitor.In some embodiments, the exogenous protein includes an antigen. In some embodiments, the exogenous protein includes an immunomodulatory protein. In some embodiments, the immune checkpoint inhibitor includes an inhibitor specific to PD-L1, PD-1, or a combination thereof. In some embodiments, the therapeutic agent includes an exogenous RNA molecule. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment, a chemokine, or any combination thereof. In some embodiments, the exogenous RNA molecule encodes a cytokine or its cytokine receptor-binding fragment. In some embodiments, the cytokine or its cytokine receptor-binding fragment includes interleukin-12 (IL-12), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-7 (IL-7), interleukin-21 (IL-21), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-15 (IL-15), or any combination thereof. In some embodiments, the exogenous RNA molecule encodes a chemokine. In some embodiments, the chemokine includes stromal cell-derived factor 1α (SDF1α), CC-motif chemokine ligand 2 (CCL2), CC-motif chemokine ligand 3 (CCL3), CC-motif chemokine ligand 5 (CCL5), CC-motif chemokine ligand 8 (CCL8), CC-motif chemokine ligand 1 (CCL1), CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), CC-motif chemokine ligand 11 (CCL11), CXC-motif chemokine ligand 12 (CXCL12), or any combination thereof. In some embodiments, the exogenous RNA molecule encodes an antigen. In some embodiments, the exogenous RNA molecule encodes an immunomodulatory protein. In some embodiments, the exogenous RNA molecule encodes an immune checkpoint inhibitor.In some embodiments, the immune checkpoint inhibitor comprises an inhibitor specific to PD-L1, PD-1, or a combination thereof. In some embodiments, the therapeutic agent comprises a cytokine or a cytokine receptor-binding fragment thereof. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released in vivo or ex vivo is a measure of the secretion of cytokine or cytokine receptor-binding fragment from multiple enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of cytokine or cytokine receptor-binding fragment by otherwise equivalent, non-cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of cytokine or cytokine receptor-binding fragment by otherwise equivalent, cryopreserved nucleated cells. In some embodiments, the exogenous RNA molecule encodes an antigen. In some embodiments, the exogenous RNA molecule encodes an immunomodulatory protein.

[0025] In some embodiments, a plurality of nucleated cells encoding a therapeutic agent can treat the disease or condition described herein. In some embodiments, the method further includes treating a disease or condition in a subject. In some embodiments, the disease is cancer. In some embodiments, cancer includes solid tumors. In some embodiments, cancer is lung cancer, cancer metastasis in lung tissue, liver cancer, or cancer metastasis in liver tissue. In some embodiments, liver cancer is hepatocellular carcinoma or cholangiocarcinoma. In some embodiments, cancer is lung cancer. In some embodiments, lung cancer is small cell lung cancer, non-small cell lung cancer, or bronchial carcinoid. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, lung cancer is bronchial carcinoid. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

[0026] In some embodiments, the enucleation methods disclosed herein yield a composition containing enucleated cells (also referred herein as the “enucleated cell fraction” of the composition). In some embodiments, the composition further comprises approximately equal to or less than about 1 percent (%) by volume of residual nucleated cells (also referred herein as the “nucleated cell fraction” of the composition). In some embodiments, the nucleated cell fraction comprises approximately equal to or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or 0.9% by volume of the composition. In some embodiments, the nucleated cell fraction comprises, on a volume basis, 0.1% to about 0.2%, about 0.1% to about 0.3%, about 0.1% to about 0.4%, about 0.1% to about 0.5%, about 0.1% to about 0.6%, about 0.1% to about 0.7%, about 0.1% to about 0.8%, about 0.1% to about 0.9%, or about 0.1% to about 1.0% of the composition. In some embodiments, the nucleated cell fraction comprises, on a volume basis, about 0.2% to about 0.3%, about 0.2% to about 0.4%, about 0.2% to about 0.5%, about 0.2% to about 0.6%, about 0.2% to about 0.7%, about 0.2% to about 0.8%, about 0.2% to about 0.9%, or about 0.2% to about 1.0% of the composition. In some embodiments, the nucleated cell fraction comprises about 0.3% to about 0.4%, about 0.3% to about 0.5%, about 0.3% to about 0.6%, about 0.3% to about 0.7%, about 0.3% to about 0.8%, about 0.3% to about 0.9%, or about 0.3% to about 1.0% by volume of the composition. In some embodiments, the nucleated cell fraction comprises about 0.4% to about 0.5%, about 0.4% to about 0.6%, about 0.4% to about 0.7%, about 0.4% to about 0.8%, about 0.4% to about 0.9%, or about 0.4% to about 1.0% by volume of the composition. In some embodiments, the nucleated cell fraction comprises about 0.5% to about 0.6%, about 0.5% to about 0.7%, about 0.5% to about 0.8%, about 0.5% to about 0.9%, or about 0.5% to about 1.0% of the composition by volume.In some embodiments, the nucleated cell fraction comprises about 0.7% to about 0.8%, about 0.7% to about 0.9%, or about 0.7% to about 1.0% of the composition by volume. In some embodiments, the nucleated cell fraction comprises about 0.8% to about 0.9%, or about 0.8% to about 1.0% of the composition by volume. In some embodiments, the nucleated cell fraction comprises about 0.9% to about 1.0% of the composition by volume. In some embodiments, the nucleated cell fraction is eliminated by induced cell death after enucleation. In some embodiments, induced cell death is employed using a biomolecular suicide switch that is expressed in response to external stimuli, such as exposure to a small molecule drug (e.g., limitid) or a prodrug (e.g., ganciclovir).

[0027] Furthermore, pharmaceutical compositions and formulations comprising the compositions described herein and pharmaceutically acceptable carriers, excipients, diluents, or spray inhalants are also described herein. The pharmaceutical compositions are provided in the form of pharmaceutical formulations. In some embodiments, the pharmaceutical formulations are formulated for administration to a subject as a combination therapy (e.g., a prodrug, adjuvant, additional therapeutic agent, or other therapy) or as a monotherapy. In some embodiments, the pharmaceutical formulations are formulated for systemic administration or site-of-action administration (e.g., intratumoral administration).

[0028] A kit comprising a composition disclosed herein and packaging material configured to deliver the composition to an individual is disclosed herein. The kit disclosed herein may comprise a composition comprising an enucleated cell fraction and a nucleated cell fraction of less than 0.1%. In some embodiments, the kit further comprises instructions for further manipulating the enucleated cells in the enucleated cell fraction, for example, to produce or secrete a therapeutic agent disclosed herein. In some embodiments, the kit further comprises a stimulus used to induce the expression or activity of a biomolecular suicide switch in the nucleated cell fraction of the composition. In any case, the instructions may further comprise instructions for a method of formulating the resulting composition into a pharmaceutical formulation for administration to an object disclosed herein.

[0029] composition Disclosed herein are compositions comprising enucleated cells that can be extensively manipulated to express an activator or a portion thereof in the absence of a nucleus. Such enucleated cells are viable cell-like entities capable of synthesizing, releasing (e.g., secreting), or delivering an activator to target cells or tissues in the absence of a nucleus. The compositions disclosed herein can be stored in a suspension biological stage for any period by means of cryopreservation, freezing, or freeze-drying, and once biological activity is restored, this does not affect the viability of the enucleated cells. In some embodiments, the compositions disclosed herein are cryopreserved. Furthermore, the compositions disclosed herein contain approximately equal to or less than 0.1% nucleated cells (e.g., parent cells that were not enucleated during the enucleation treatment), making the compositions disclosed herein optimal for therapeutic use. The enucleated cells (referred to herein as “cytoplasm”) may further contain native cell surface molecules retained from the parent cells. In some embodiments, the enucleated cells further contain exogenous molecules, examples of which include targeted moieties, transmembrane moieties, and additional therapeutic agents (e.g., other than activators), examples of which are disclosed herein.

[0030] (a) Enucleated cells The enucleated cells of this disclosure are obtained from or derived from corresponding nucleated cells (hereinafter referred to as “parent cells”). The parent cells may be derived from a variety of different cell types, including eukaryotic cells. For example, enucleated cells may be derived from adult stem cells, mesenchymal stromal cells (MSCs), natural killer (NK) cells, macrophages, myoblasts, neutrophils, endothelial cells, endothelial progenitor cells and / or fibroblasts. In some embodiments, enucleated cells are derived from mesenchymal stromal cells. In some embodiments, enucleated cells are derived from inducible pluripotent stem cells (iPSCs). In some embodiments, the parent cells are derived from cells immortalized using a preferred method. In some embodiments, enucleated cells include or retain one or more structural features of the parent cell, including intracellular organelles, one or more tunnel nanotubes, or a combination thereof. In some embodiments, enucleated cells include one or more intracellular organelles for synthesizing or secreting exogenous polypeptides (e.g., therapeutic agents) in the absence of a nucleus. In some embodiments, one or more intracellular organelles include the Golgi apparatus, the endoplasmic reticulum, or a combination thereof. In some embodiments, enucleated cells contain or express any one of the therapeutic agents described herein.

[0031] In some embodiments, the cells may originate from any organism having one or more cells. Non-limiting examples of cells include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of single-cell eukaryotes, protist cells, plant-derived cells (e.g., plant crops, fruits, vegetables, grains, soybeans, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), and algal cells (e.g., Botryococcus braunii, Conamidrim). This includes cells such as *Cypripedium macranthos*, *Nannochloropsis gaditana*, *Chlorella pyrenoides*, and *Sargassum fuciformis*, seaweed (e.g., kelp), fungal cells (e.g., yeast cells, mushroom cells), animal cells, invertebrate cells (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), vertebrate cells (e.g., fish, amphibians, reptiles, birds, mammals), and mammalian cells (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Cells may not originate from naturally occurring organisms (e.g., cells may be synthesized and manufactured, and are sometimes called artificial cells). In some embodiments, cells are somatic cells. In some embodiments, cells are stem cells or progenitor cells. In some embodiments, cells are mesenchymal stem cells or mesenchymal progenitor cells. In some embodiments, cells are hematopoietic stem cells or hematopoietic progenitor cells. In some embodiments, cells are muscle cells, skin cells, blood cells, or immune cells. Other non-exclusive examples of cells include lymphoid cells (examples include B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells); myeloid cells (examples include granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells); endocrine cells (including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophilic cells), adrenal (chromaffin cells), pineal (pineal cells) cells); nervous system cells (including glial cells (astrocytes, microglia), giant neurosecretory cells, astrocytes,This includes Bettcher cells and pituitary gland cells (gonadotropins, corticotropes, thyroid-stimulating hormone-secreting cells, growth hormone-secreting cells, mammary gland-stimulating hormone-secreting cells); respiratory system cells (including pneumocytes (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells); circulatory system cells (including cardiomyocytes, pericytes); digestive system cells (including stomach cells (gastrocnemiocytes, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells); enteroendocrine cells (and These include enterochromophilic cells, APUD cells, liver cells (hepatocytes, Kupffer cells), cartilage / bone / muscle cells; osteocytes (including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts)); chondrocytes (including chondrocytes, chondrocytes); skin cells (including hair follicle cells, keratinocytes, melanocytes (nevus cells)); muscle cells (including cardiomyocytes); urinary system cells (including podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells, renal proximal urine); This includes tubular brush border cells and macula densa cells; germline cells (including sperm, Sertoli cells, Leydig cells, and oocytes); and other cells (including adipocytes, fibroblasts, tendinocytes, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, cuticle hair stem cells, cuticle root sheath cells, root sheath cells of the Huxley layer, root sheath cells of the Henle layer, outer root sheath cells, hair matrix cells (stem cells), moist stratified barrier epithelial cells, cornea, tongue, oral cavity, esophagus, anal canal, and periphery. The epithelial cells include surface epithelial cells of stratified squamous epithelium of the urethra and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urethral epithelial cells (lining the inside of the urethra, vagina, bladder, and ureters), exocrine epithelial cells, salivary gland mucus cells (secreting polysaccharide-rich secretions), salivary gland serous cells (secreting glycoprotein enzyme-rich secretions), von Ebner's gland cells of the tongue (washing the taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), ceruminous gland cells of the ear (secreting earwax), eccrine gland dark cells (secreting glycoproteins), and eccrine gland clear cells (secreting small molecules).Apocrine sweat gland cells (odorous secretions, sex hormone sensitive), Mohl's gland cells of the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretions), Bowman's gland cells of the nose (cleansing the olfactory epithelium), Brunner's gland cells of the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting seminal components containing fructose to help sperm swim), prostate cells (secreting seminal components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubrication secretion), Littley's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), isolation of the respiratory and digestive tracts. Goblet cells (mucus secretion), mucus cells lining the inside of the stomach (mucus secretion), gastric gland enzyme progenitor cells (pepsinogen secretion), gastric gland acid-secreting cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Paneth cells of the small intestine (lysozyme secretion), type II pneumocytes of the lungs (surfactant secretion), Clara cells of the lungs, hormone-secreting cells, anterior pituitary cells, growth hormone-secreting cells, mammogenic hormone-secreting cells, thyroid-stimulating hormone-secreting cells, gonadotropins, corticotropes, pituitary intermediate cells, giant cell neurosecreting cells, cells of the intestinal and respiratory tracts, thyroid cells, Thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, eosinophilic cells, adrenal cells, chromaffin cells, Leydig cells of the testis, endometrial cells of follicular cells, luteal cells of ruptured follicles, granulosa lutein cells, follicular membrane lutein cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands and urogenital tract), kidney, type I pneumocytes (lining the inside of the air spaces in the lungs), pancreatic duct cells (acinate central cells), non-striatal duct cells (of sweat glands, salivary glands, mammary glands, etc.), ductal cells (of seminal vesicles, prostate, etc.), closed interior Epithelial cells lining the inside of body cavities, ciliated cells with propulsive function, extracellular matrix secretory cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes, megakaryocytes (platelet progenitor cells), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (central nervous system), neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes,This includes stem cells and differentiation-determining progenitor cells of the blood and immune systems (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transduction cells, autonomic nerve cells, sensory organ and peripheral neuron supporting cells, neurons and glial cells of the central nervous system, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (spermatocyte stem cells), sperm, nurse cells, ovarian follicular cells, Sertoli cells (in the testes), thymic epithelial cells, stromal cells, and interstitial kidney cells.

[0032] In some embodiments, the cells are eukaryotic cells. Non-limiting examples of eukaryotic cells include mammals (e.g., rodents, non-human primates, or humans), non-mammalian animals (e.g., fish, birds, reptiles, or amphibians), invertebrates, insects, fungi, or plant cells. In some embodiments, the eukaryotic cells are yeast cells such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cells are higher eukaryotes such as mammals, birds, plants, or insect cells. In some embodiments, the nucleated cells are primary cells. In some embodiments, the nucleated cells are immune cells (e.g., lymphocytes (e.g., T cells, B cells), macrophages, natural killer cells, neutrophils, mast cells, basophils, dendritic cells, monocytes, bone marrow-derived suppressor cells, eosinophils). In some embodiments, the nucleated cells are phagocytes or leukocytes. In some embodiments, nucleated cells are stem cells (e.g., adult stem cells (e.g., hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, testicular cells), embryonic stem cells, or inducible pluripotent stem cells (iPS cells)). In some embodiments, nucleated cells are progenitor cells. In some embodiments, nucleated cells are derived from a cell line. In some embodiments, nucleated cells are suspension cells. In some embodiments, nucleated cells are adherent cells. In some embodiments, nucleated cells are cells immortalized by the expression of oncogenes. In some embodiments, nucleated cells are immortalized by the expression of human telomerase reverse transcriptase (hTERT) or any oncogene. In some embodiments, nucleated cells are patient- or subject-derived cells (e.g., autologous patient-derived cells or allogeneic patient-derived cells). In some embodiments, nucleated cells are transfected with a vector (e.g., a viral vector (e.g., a retroviral vector (e.g., a lentiviral vector), an adeno-associated virus (AAV) vector, a vesicular virus vector (e.g., a vesicular stomatitis virus (VSV) vector), or a hybrid viral vector) or plasmid) before the nucleated cells are denucleated using any denucleation technique described herein or known in the art.

[0033] In some embodiments, the cytoplasm is derived from the subject's own cells. In some embodiments, the cytoplasm is derived from the subject's allogeneic cells.

[0034] In some embodiments, the cytoplasm is derived from immune cells. In some embodiments, the cytoplasm is derived from natural killer (NK) cells, neutrophils, macrophages, lymphocytes, fibroblasts, adult stem cells (e.g., hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, mesenchymal stem cells, mesenchymal stromal cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, skin stem cells, or testicular cells), mast cells, basophils, eosinophils, endothelial cells, endothelial cell progenitor cells, or inducible pluripotent stem cells.

[0035] In some embodiments, parent cells may be enucleated or manipulated for therapeutic purposes. In some embodiments, parent cells may be treated with cytochalasin to soften the cortical actin cytoskeleton. In some embodiments, the nuclei are then physically extracted from the cell bodies by high-speed centrifugation in a polysaccharide gradient to produce enucleated cells. In some embodiments, the polysaccharide is ficol to generate a ficol gradient to produce enucleated cells. Since enucleated cells and intact nucleated cells precipitate into different layers in the ficol gradient, enucleated cells may be isolated or prepared for therapeutic purposes or for fusion with other cells (nucleated or enucleated cells). By utilizing the methods described herein, the enucleation process can be made clinically scalable to process tens of millions of cells. In some embodiments, enucleated cells may be used as disease-homing vehicles for delivering clinically relevant cargo or payloads to treat the various diseases or conditions described herein.

[0036] In some embodiments, the enucleated cell contains at least one therapeutic agent. In some embodiments, the enucleated cell disclosed herein expresses the therapeutic agent in the absence of a nucleus along with one or more intracellular organelles. In some embodiments, the therapeutic agent is exogenous to the enucleated cell or its parent (nucleated) cell. In some embodiments, the enucleated cell expresses the therapeutic agent on its surface. In some embodiments, the therapeutic agent is secreted by the enucleated cell into the extracellular space (e.g., the microenvironment) of the target tissue. In some embodiments, the therapeutic agent is the enucleated cell's cargo (e.g., encapsulated by the enucleated cell).

[0037] In some embodiments, enucleated cells are obtained from a first subset of multiple nucleated cells. In some embodiments, enucleated cells are present in a composition further comprising a second subset of multiple nucleated cells. In some embodiments, the second subset of nucleated cells comprises less than about 0.1% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 0.5% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 1% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 5% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 10% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 15% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 20% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 25% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 30% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 40% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 50% of the composition by volume.

[0038] In one embodiment, a nucleated cell (e.g., a parent cell before enucleation to obtain the enucleated cells described herein) contains a heterologous polynucleotide encoding a heterologous gene product configured to induce cell death in the nucleated cell. In some embodiments, the heterologous polynucleotide includes a promoter. In some embodiments, the promoter is configured to activate the transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the promoter includes an inducible promoter. In some embodiments, the inducible promoter is configured to activate the transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product when induced.

[0039] In some embodiments, the enucleated cells described herein can be preserved by cryopreservation, cryopreservation, freeze-drying, or a combination thereof. In some embodiments, cryopreserved enucleated cells are viable after thawing in the same way as otherwise equivalent, uncryopreserved enucleated cells. In some embodiments, freeze-dried enucleated cells are viable in the same way as otherwise equivalent, unfreeze-dried enucleated cells. In some embodiments, cryopreserved enucleated cells are viable in the same way as otherwise equivalent, unfreeze-dried enucleated cells.

[0040] In some embodiments, enucleated cells, or compositions containing enucleated cells, may be cryopreserved (e.g., stored at freezing temperature) or cryopreserved (e.g., stored at a temperature between ambient temperature and freezing temperature). The duration of cryopreservation or cryopreservation may be approximately equal to or longer than 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, enucleated cells exhibit viability after cryopreservation or cryopreservation, and this viability is approximately equal to, or more similar to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of equivalent cells (e.g., parent cells, or enucleated cells described herein that have not been cryopreserved or cryopreserved) after the same period of cryopreservation or cryopreservation. In some embodiments, the post-cryohibernation viability exhibited by enucleated cells, measured 24 hours after cryopreservation, is greater than or equal to the viability of equivalent enucleated cells that have not been cryopreserved. In some embodiments, the post-cryohibernation viability exhibited by enucleated cells, measured 24 hours after cryopreservation, is greater than or equal to the viability of equivalent enucleated cells that have not been cryopreserved. Viability in this context may be measured by the trypan blue dye exclusion method described herein. In some embodiments, trypan blue dye exclusion is carried out by (a) centrifuging aliquots of nucleateless cells in a suspension to create a cell pellet; (b) resuspending the cell pellet in serum-free medium to produce a serum-free cell suspension; (c) mixing one part trypan blue dye with one part serum-free cell suspension; and (d) counting the nucleateless cells from (c) within 3-5 minutes, so that at least a portion of the nucleateless cells are not stained with trypan blue dye. This indicates viability. In some embodiments, viability is measured using annexin V cell surface staining. In some embodiments, viability is measured by the expression of exogenous polypeptides. For example, the viability of enucleated cells can be determined by the expression of exogenous antibodies or single-domain antibodies expressed by the enucleated cells.In some embodiments, viability is measured by the expression of any one of the cell surface markers described herein, examples of which include CD105, CD90, CD45, CXCR4, PSGL-1, or CCR2. In some embodiments, viability is measured by the cellular activity of enucleated cells. In some embodiments, viability is measured by the homing ability of enucleated cells, determined by chemosensing or chemokine homing activity described herein.

[0041] In one embodiment, the enucleated cells of this disclosure are cryopreserved. In some embodiments, the enucleated cells are cryopreserved for about 24 hours to about 5 years. In some embodiments, the enucleated cells are cryopreserved for about 12 hours to about 24 hours, about 24 hours to about 48 hours, about 48 hours to about 72 hours, about 72 hours to about 96 hours, about 96 hours to about 120 hours, about 120 hours to about 144 hours, about 144 hours to about 168 hours, 7 days to about 14 days, about 14 days to about 21 days, about 21 days to about 28 days, about 28 days to about 30 days, about 30 days to about 31 days, about 1 month to about 2 months, about 2 months to about 3 months, about 3 months to about It is frozen for 4 months, approximately 4 to 5 months, approximately 5 to 6 months, approximately 6 to 7 months, approximately 7 to 8 months, approximately 8 to 9 months, approximately 9 to 10 months, approximately 10 to 11 months, approximately 11 to 12 months, approximately 1 to 1.5 years, approximately 1.5 to 2 years, approximately 2 to 2.5 years, approximately 2.5 to 4 years, approximately 4 to 4.5 years, approximately 4.5 to 5 years, or longer.

[0042] In some embodiments, enucleated cells are cryopreserved for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, at least about 120 hours, at least about 132 hours, at least about 144 hours, at least about 156 hours, at least about 168 hours, at least about 14 days, at least about 21 days, at least about 28 hours, at least about 30 days, at least about 31 days, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1.5 years, at least about 2 years, at least about 2.5 years, at least about 3 years, at least about 3.5 years, at least about 4 years, at least about 4.5 years, at least about 5 years or longer. In some embodiments, the enucleated cells are cryopreserved for at least about 24 hours. In some embodiments, the enucleated cells are cryopreserved for at least about 7 days. In some embodiments, the enucleated cells are cryopreserved for at least about 1 month. In some embodiments, the enucleated cells are cryopreserved for at least about 1 year.

[0043] In some embodiments, enucleated cells are cryopreserved for approximately 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, 14 days, 21 days, 28 hours, 30 days, 31 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years.

[0044] In some embodiments, enucleated cells are cryopreserved at temperatures below at least about -70°C. In some embodiments, enucleated cells are cryopreserved between about -70°C and about -80°C, about -80°C and about -90°C, about -90°C and about -100°C, about -100°C and about -110°C, about -110°C and about -120°C, about -120°C and about -130°C, about -130°C and about -140°C, about -140°C and about -150°C, about -150°C and about -160°C, about -160°C and about -170°C, about -170°C and about -180°C, about -180°C and about -190°C, or -190°C and about -200°C.

[0045] In some ways, enucleated cells are cryopreserved at temperatures of at least approximately -70°C, at least approximately -80°C, at least approximately -90°C, at least approximately -100°C, at least approximately -110°C, at least approximately -120°C, at least approximately -130°C, at least approximately -140°C, at least approximately -150°C, at least approximately -160°C, at least approximately -170°C, at least approximately -180°C, at least approximately -190°C, at least approximately -200°C, or higher. In some ways, enucleated cells are cryopreserved at temperatures of approximately -70°C, approximately -80°C, approximately -90°C, approximately -100°C, approximately -110°C, approximately -120°C, approximately -130°C, approximately -140°C, approximately -150°C, approximately -160°C, approximately -170°C, approximately -180°C, approximately -190°C, approximately -200°C, or higher.

[0046] In some embodiments, enucleated cells are cryopreserved in liquid nitrogen. In some embodiments, the cryopreserved composition is cryopreserved in liquid nitrogen. In some embodiments, enucleated cells are cryopreserved in dry ice. In some embodiments, enucleated cells are cryopreserved in a freezer. In some embodiments, the freezer is set to any temperature in this disclosure.

[0047] In some embodiments, the cryopreserved composition further comprises a cryopreserved medium. In some embodiments, the cryopreserved medium is a serum cryopreserved medium. In some embodiments, the serum cryopreserved medium has fetal bovine serum. In some embodiments, the cryopreserved medium is a serum-free medium. In some embodiments, the cryopreserved medium contains dimethyl sulfoxide (DMSO). In some embodiments, the cryopreserved medium contains about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% DMSO. In some embodiments, the cryopreserved medium contains glycerol. In some embodiments, the cryopreserved medium is a xeno-free medium. Non-limiting examples of xeno-free mediums include xeno-free X-VIVO medium, xeno-free mesenchymal stem cell medium, and StemPro® MSC SFM xeno-free medium. In some embodiments, the xenofree medium is supplemented with human serum, platelet lysate, holotransferrin, or insulin. In some embodiments, the xenofree medium is supplemented with human serum. In some embodiments, the xenofree medium is supplemented with platelet lysate. In some embodiments, the xenofree medium is supplemented with holotransferrin. In some embodiments, the xenofree medium is supplemented with insulin. In some embodiments, the freezing medium is CryoStor® medium. Non-limiting examples of CryoStor® medium include CS10, CS5, CS2, and CSB. In some embodiments, CryoStor® is CryoStor® CS10 medium.

[0048] In some embodiments, the frozen composition further comprises a non-pyrogenic solution. Non-limiting examples of non-pyrogenic solutions include Ringer's lactate solution, cupric chloride solution, mannitol solution, phosphate buffer (PBS), sodium chloride solution, and sodium lactate solution. In some embodiments, the non-pyrogenic solution is PBS. In some embodiments, the non-pyrogenic solution is sodium lactate solution. In some embodiments, the non-pyrogenic solution is sterilized.

[0049] In some embodiments, cryopreserved enucleated cells further comprise one or more therapeutic agents.

[0050] In some embodiments, cryopreserved enucleated cells release one or more therapeutic agents. In some embodiments, cryopreserved enucleated cells release one or more therapeutic agents in amounts approximately equal to or greater than those released by otherwise identical, non-cryopreserved enucleated cells and otherwise identical target cells. In some embodiments, cryopreserved enucleated cells release about 1 to about 50 times more of one or more therapeutic agents than other equivalent enucleated cells. In some embodiments, cryopreserved enucleated cells release about 1 to 2 times, 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times more than the amount of 1 or more therapeutic agent released by otherwise identical enucleated cells. In some embodiments, cryopreserved enucleated cells release about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, or more amounts of one or more therapeutic agents compared to the amount of one or more therapeutic agents released by otherwise identical enucleated cells. In some embodiments, the amount of one or more therapeutic agents released by cryopreserved enucleated cells is equal to the amount of one or more therapeutic agents released by otherwise identical enucleated cells.

[0051] In some embodiments, the amount of one or more released therapeutic agents is measured in vitro. In some embodiments, the amount of one or more released therapeutic agents is measured in vivo. In some embodiments, the amount of one or more released therapeutic agents is measured ex vivo.

[0052] In some embodiments, the therapeutic agent is a virus. In some embodiments, the amount of therapeutic agent released is a measure of viral titer in target cells. In some embodiments, viral titer is measured in plaque-forming units (PFUs). In some embodiments, PFUs are measured as PFUs per milliliter (mL). In some embodiments, PFUs are measured as PFUs per gram. In some embodiments, target cells are a biological sample. Non-limiting examples of biological samples include isolated cells, cell supernatants, tissue biopsies, tumor biopsies, cell lines, cell cultures, and biological fluids (e.g., saliva, blood, plasma, serum, urine, feces, lymph, cerebrospinal fluid). In some embodiments, target cells are lung cells. In some embodiments, target cells are hepatocytes. In some embodiments, target cells are one or more cells from a subject. In some embodiments, the subject has a disease. In some embodiments, target cells are diseased cells. In some embodiments, target cells are cancer cells. In some embodiments, target cells are solid tumor cells.

[0053] In some embodiments, cryopreserved enucleated cells release approximately the same amount of virus to target cells as, or a greater amount of virus to, otherwise identical, non-cryopreserved enucleated cells. In some embodiments, cryopreserved enucleated cells release approximately the same amount of virus to target cells as, otherwise identical, non-cryopreserved enucleated cells. In some embodiments, cryopreserved enucleated cells release approximately 1 to 50 times the amount of virus to target cells as, otherwise equivalent, enucleated cells. In some embodiments, cryopreserved enucleated cells release to target cells approximately 1 to 2 times, 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times more virus than would be released by otherwise identical enucleated cells to otherwise identical target cells. In some embodiments, cryopreserved enucleated cells release to target cells about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, or more amounts of virus compared to the amount of virus released to target cells by otherwise identical enucleated cells. In some embodiments, the amount of virus released to target cells by cryopreserved enucleated cells is equal to the amount of virus released to target cells by otherwise identical enucleated cells.

[0054] In some embodiments, the therapeutic agent is a cytokine or its cytokine receptor-binding fragment. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured by the amount of soluble cytokine or cytokine receptor-binding fragment present. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in micrograms (μg) per mL. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in nanograms (ng) per mL. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in picograms (pg) per mL. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in the supernatant of cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in the supernatant of otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in the supernatant of cryopreserved enucleated cells and the supernatant of otherwise identical enucleated cells.

[0055] In some embodiments, cryopreserved nucleated cells release larger amounts of cytokines or cytokine receptor-binding fragments compared to otherwise identical enucleated cells. In some embodiments, cryopreserved enucleated cells release about 1 to 50 times more amounts of cytokines or cytokine receptor-binding fragments compared to otherwise equivalent enucleated cells. In some embodiments, cryopreserved enucleated cells release about 1 to 2 times, 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times more than the amount of cytokines or cytokine receptor-binding fragments released by otherwise identical enucleated cells. In some embodiments, cryopreserved enucleated cells release about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, or more amounts of cytokines or cytokine receptor-binding fragments released by otherwise identical enucleated cells. In some embodiments, the amount of cytokines or cytokine receptor-binding fragments released by cryopreserved enucleated cells is equal to the amount of cytokines or cytokine receptor-binding fragments released by otherwise identical enucleated cells.

[0056] In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in target cells introduced into cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in target cells introduced into cryopreserved nucleated cells and otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment is measured as the amount of mRNA of the cytokine or cytokine receptor-binding fragment. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells, and the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells.

[0057] In some embodiments, target cells introduced into cryopreserved enucleated cells have a higher amount of cytokine or cytokine receptor-binding fragment mRNA compared to otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells have approximately 1 to 50 times more amount of cytokine or cytokine receptor-binding fragment mRNA compared to otherwise identical target cells introduced into equivalent enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells have about 1 to 2 times, about 2 to 3 times, about 3 to 4 times, about 4 to 5 times, about 5 to 10 times, about 10 to 15 times, about 15 to 20 times, about 20 to 25 times, about 25 to 30 times, about 30 to 35 times, about 35 to 40 times, about 40 to 45 times, or about 45 to 50 times compared to the amount of mRNA of cytokines or cytokine receptor binding fragments present in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells contain approximately 1 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 25 times, approximately 30 times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times, or more amounts of mRNA of cytokines or cytokine receptor-binding fragments compared to target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of mRNA of cytokines or cytokine receptor-binding fragments present in target cells introduced into cryopreserved enucleated cells is equal to the amount of mRNA of cytokines or cytokine receptor-binding fragments present in otherwise identical target cells introduced into otherwise identical enucleated cells.

[0058] In some embodiments, the amount of cytokine or cytokine receptor-bound fragment released is measured through the release of another cytokine or cytokine receptor-bound fragment by target cells introduced into cryopreserved enucleated cells. In some embodiments, the other cytokine or cytokine receptor-bound fragment is a soluble cytokine or cytokine receptor-bound fragment. In some embodiments, the other cytokine or cytokine receptor-bound fragment is any cytokine or cytokine receptor-bound fragment of this disclosure. In some embodiments, the other cytokine or cytokine receptor-bound fragment is IFN-γ. In some embodiments, the other cytokine or cytokine receptor-bound fragment is TNF-α. In some embodiments, the other cytokine or cytokine receptor-bound fragment is IL-6.

[0059] In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured in target cells introduced into cryopreserved nucleated cells and in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment is measured as the amount of mRNA of the cytokine or cytokine receptor-binding fragment. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment released is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells, and the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells.

[0060] In some embodiments, target cells introduced into cryopreserved enucleated cells release a greater amount of another cytokine or its cytokine receptor-binding fragment compared to otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells release about 1 to 50 times more of another cytokine or its cytokine receptor-binding fragment compared to otherwise identical target cells introduced into otherwise equivalent enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells release about 1 to 2 times, 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times more than the amount of another cytokine or cytokine receptor binding fragment released by other target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells release about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, or more amounts of another cytokine or cytokine receptor-binding fragment compared to other identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of another cytokine or cytokine receptor-binding fragment released by target cells introduced into cryopreserved enucleated cells is equal to the amount of another cytokine or cytokine receptor-binding fragment released by other identical target cells introduced into otherwise identical enucleated cells.

[0061] In some embodiments, the amount of a cytokine or its cytokine receptor-binding fragment is measured as the amount of mRNA of another cytokine or its cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells. In some embodiments, the amount of a cytokine or its cytokine receptor-binding fragment is measured as the amount of mRNA of another cytokine or its cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of mRNA of a cytokine or its cytokine receptor-binding fragment is measured as the amount of mRNA of another cytokine or its cytokine receptor-binding fragment present in targets introduced into cryopreserved enucleated cells, and the amount of mRNA of another cytokine or its cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells.

[0062] In some embodiments, target cells introduced into cryopreserved enucleated cells have a large amount of mRNA of another cytokine or its cytokine receptor-binding fragment compared to otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells have about 1 to 50 times more mRNA of another cytokine or its cytokine receptor-binding fragment compared to otherwise identical target cells introduced into otherwise equivalent enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells have about 1 to 2 times, about 2 to 3 times, about 3 to 4 times, about 4 to 5 times, about 5 to 10 times, about 10 to 15 times, about 15 to 20 times, about 20 to 25 times, about 25 to 30 times, about 30 to 35 times, about 35 to 40 times, about 40 to 45 times, or about 45 to 50 times compared to the amount of mRNA of another cytokine or cytokine receptor binding fragment present in other target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells contain approximately 1 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 25 times, approximately 30 times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times, or more amounts of mRNA of another cytokine or cytokine receptor-binding fragment compared to other target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of mRNA of another cytokine or cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells is equal to the amount of mRNA of another cytokine or cytokine receptor-binding fragment present in other target cells introduced into otherwise identical enucleated cells. In some embodiments, the cryopreserved composition further comprises an intracellular organelle. In some embodiments, an intracellular organelle for the synthesis or secretion of a therapeutic agent. In some embodiments, the intracellular organelle synthesizes or secretes a therapeutic agent in the absence of a nucleus.In some embodiments, the intracellular organelle is the Golgi apparatus, the endoplasmic reticulum, or a combination thereof.

[0063] In some embodiments, enucleated cells, or compositions containing enucleated cells, are stable at 4°C for approximately 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, the compositions are stable at room temperature for approximately 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, the compositions are stable at 37°C for approximately 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, enucleated cells, or compositions comprising enucleated cells, may continue to survive after being administered to a subject requiring it to treat a disease or condition described herein. In some embodiments, enucleated cells, or compositions comprising enucleated cells, may continue to survive for approximately equal to or longer than 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or longer after being administered to a subject.

[0064] In some embodiments, enucleated cells may be obtained from the parent cells of the subject requiring treatment with enucleated cells as described herein. In some embodiments, enucleated cells may be obtained from allogeneic parent cells of the subject requiring treatment with enucleated cells as described herein.

[0065] Enucleated cells may be smaller than their nucleated counterparts (e.g., nucleated parent cells), and for this reason, they may be better able to migrate through vascular structures and small openings in tissue parenchyma. Furthermore, removing a large, dense nucleus reduces the major physical barrier, allowing cells to move freely through blood vessels and small openings in tissue parenchyma. Thus, enucleated cells have improved in vivo distribution within the body and migration to target tissues. In some embodiments, enucleated cells have a diameter of at least 1 micrometer (μm). In some embodiments, the diameter of enucleated cells is greater than 1 μm. In some embodiments, the diameter of enucleated cells is 1-100 μm (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, 5-90 μm, 5-80 μm, 5-70 μm, 5-60 μm, 5-50 μm, 5-40 μm, 5-30 μm, 5- The diameters are 20 μm, 5-10 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, 10-15 μm, 15-90 μm, 15-80 μm, 15-70 μm, 15-60 μm, 15-50 μm, 15-40 μm, 15-30 μm, and 15-20 μm. In some embodiments, the diameter of enucleated cells is 10-30 μm. In some embodiments, the diameter of the enucleated cells is 5–25 μm (e.g., 5–20 μm, 5–15 μm, 5–10 μm, 10–25 μm, 10–20 μm, 10–15 μm, 15–25 μm, 15–20 μm, or 20–25 μm). In some embodiments, the enucleated cells have a diameter of approximately 8 μm. In some embodiments, it may be advantageous for some enucleated cells to be small enough to allow for better homing or delivery to the target site. For example, the enucleated cells described herein may be able to pass through narrow passages in lung tissue or lung structures (such as alveolar ducts or microcapillaries) that most cells (such as parent cells) cannot pass through.

[0066] In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% or less of the average diameter of the nucleated parent cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 50% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 60% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 70% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 80% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 90% or less of the average diameter of the nucleated cells.

[0067] In some embodiments, enucleated cells have significant therapeutic value because they may continue to survive, not differentiate into other cell types, secrete bioactive molecules, physically migrate / homing within approximately 5 days or less, be extensively enucleated ex vivo to perform specific therapeutic functions, and fuse with the same or other cell types to transfer desired innate or enucleated products. Thus, enucleated cells have broad utility as cell vehicles for delivering therapeutically important biomolecules and disease-targeting cargo (such as genes, viruses, bacteria, mRNA, shRNA, siRNA, polypeptides (including antibody and antigen-binding fragments), plasmids, gene-editing mechanisms, or nanoparticles). This disclosure enables the creation of safe (e.g., no unwanted DNA is transferred to the target) and controllable (e.g., cell death occurs within approximately 5 days) cell-based carriers. These carriers may be genetically enucleated to deliver cargo to humans that combat specific diseases and promote health. In some embodiments, enucleated cells continue to survive, migrate, or homing for approximately 12, 24, 36, 48, 60, 72, 84, 96, 108 hours, 5, 6, 7, 8, 9 days, or longer, or more, after being administered to a target requiring administration.

[0068] In some embodiments, enucleated cells are manipulated to express at least one of the following: an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous protein, a gene editing mechanism, or a combination thereof. In some embodiments, the exogenous DNA molecule is single-stranded DNA, double-stranded DNA, oligonucleotides, plasmids, bacterial DNA molecules, DNA viruses, or a combination thereof. In some embodiments, the exogenous RNA molecule is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), RNA viruses, or a combination thereof. In some embodiments, the exogenous protein is a cytokine, growth factor, hormone, antibody or its antigen-binding fragment, enzyme, antigen, immunomodulatory protein, or a combination thereof. In some embodiments, the exogenous protein includes a functional fragment of a full-length protein. In some embodiments, the antibody is a single-domain antibody or its antigen-binding fragment. In some embodiments, the parental cell (e.g., a nucleated cell) is genetically enucleated before enucleation (e.g., pre-enucleation). In some embodiments, parental cells are genetically enucleated after enucleation (e.g., post-enucleation). For example, the enucleated cells disclosed herein may be engineered to express interleukin-12 (IL-12). In another example, the enucleated cells disclosed herein may be engineered to contain a virus such as vesicular stomatitis virus (VSV). In some embodiments, VSV may encode an activator such as interferon-beta (IFN-β).

[0069] (b) Transmembrane part In some embodiments, enucleated cells comprising at least one transmembrane moiety, or compositions comprising enucleated cells, are described herein. In some embodiments, the enucleated cells comprise an exogenous polypeptide. The exogenous polypeptide may be covalently fused to the transmembrane moiety. In some embodiments, the exogenous polypeptide is complexed with the transmembrane moiety. In some embodiments, the transmembrane moiety comprises a full-length protein or a variant thereof or a fragment thereof. In some embodiments, the transmembrane moiety is endogenous to the parental cell to be enucleated in order to obtain the enucleated cell. In some embodiments, the transmembrane moiety may be an exogenous transmembrane moiety to the parental cell or the enucleated cell. In some embodiments, the transmembrane moiety is selected from transmembrane proteins (bitopic transmembrane proteins) comprising a single transmembrane α-helix. The transmembrane moiety comprises a polytopic transmembrane α-helix protein. In some embodiments, the transmembrane moiety comprises a polytopic transmembrane β-sheet protein. In some embodiments, the transmembrane moiety comprises a type I, type II, type III, or type IV' transmembrane protein. Non-limiting examples of transmembrane proteins may include any combination of CD4, CD14, glycophorin a (GPA), or integrins.

[0070] In some embodiments, the transmembrane portion is added to the exogenous polypeptide by modification. For example, the transmembrane portion may be added to the N-terminus or C-terminus of the exogenous polypeptide to insert it into the cell membrane of the enucleated cell described herein. Non-limiting examples of modifications made to the exogenous polypeptide to add a transmembrane portion include adding glycosylphosphatidylinositol, farnesyl, palmitic acid, myristic acid, or a combination thereof to the exogenous polypeptide.

[0071] In some embodiments, the transmembrane portion is genetically modified to fuse with or complex with at least one exogenous therapeutic agent described herein. In some embodiments, the enucleated cell includes an immune evasion portion. In some embodiments, the immune evasion includes a "don't eat me" signaling peptide, e.g., CD47 (e.g., NCBI gene ID 961), programmed cell death ligand 1 (PD-L1, e.g., NCBI gene ID 29126), major histocompatibility complex, class I, E (HLA-E, e.g., NCBI gene ID 3133), major histocompatibility complex, class I, G (HLA-I, e.g., NCBI gene ID 3135), fragments thereof, or combinations thereof.

[0072] (c) Targeting moiety In some embodiments, enucleated cells containing a targeting moiety are described herein. The targeting moiety described herein is designed to guide the enucleated cells to target cells or a target environment (e.g., tissue) within a target after delivery to the target (e.g., systemic delivery). In some embodiments, the targeting moiety is expressed on the surface of the enucleated cell. In some embodiments, the targeting moiety is complexed with a transmembrane moiety described herein. In some embodiments, the targeting moiety is secreted by the enucleated cell. In some embodiments, enucleated cells containing a targeting moiety localize to target cells or a target environment with a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold increase compared to the localization of equivalent enucleated cells lacking the targeting moiety. In some embodiments, enucleated cells containing the targeting region localize to target cells or target environments with an increase of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to equivalent enucleated cells lacking the targeting region. In some embodiments, the target cells or target environment are in vivo. In some embodiments, the target cells or target environment are ex vivo.

[0073] In some embodiments, the targeting portion includes an exogenous antibody or exogenous antigen-binding fragment for targeting a biomarker described herein. In some embodiments, the targeting portion includes an exogenous antibody or exogenous antigen-binding fragment for targeting a chemokine receptor or chemokine ligand involved in chemokine signaling, or a portion thereof. In some embodiments, the exogenous antibody is an exogenous single-domain antibody or a fragment thereof.

[0074] In some embodiments, the targeted portion targets a biomarker, which is expressed by or associated with the target cell or microenvironment. In some embodiments, the biomarker may be released by the target cell. The biomarker may indicate the presence of a disease or pathological condition. In some embodiments, the biomarker is expressed by immune cells in response to the target cell or microenvironment associated with the disease or pathological condition. In some embodiments, the biomarker may be an epitope or an antigen. In some embodiments, the biomarker, including an epitope, may be conjugated by an antibody different from the antibody or its antigen-binding fragment (e.g., a therapeutic agent) that confers therapeutic properties.

[0075] In some embodiments, the targeting moiety targets biomarkers expressed or released by lung cells or lung cancer cells. Non-limiting examples of cancer cell biomarkers include carbonic anhydrase 9 (CA9, e.g., NCBI gene ID 768), carbonic anhydrase 12 (CA12, e.g., NCBI gene ID 771), cancer / testicular antigen 83 (CXorf61, e.g., NCBI gene ID 203413), desmoglein 3 (DSG3, e.g., NCBI gene ID 1830), FAT atypical cadherin 2 (FAT2, e.g., NCBI gene ID 2196), G protein-coupled receptor 87 (GPR87, e.g., NCBI gene ID 53836), KISS1 receptor (KISS1R, e.g., NCBI gene ID 84634), LY6 / PLAUR domain-containing 3 (LYPD3, e.g., NCBI gene ID 27076), and lysinus. Quality carrier family 7 members 11 (SLC7A11, e.g., NCBI gene ID 23657), TMPRSS4 (e.g., NCBI gene ID 56649), transmembrane serine protease 4 (TFPI, e.g., NCBI gene ID 7035), midkine (MDK, e.g., NCBI gene ID 4192), secretory phosphorylated protein 1 (OPN, e.g., NCBI gene ID 6696), matrix metallopeptidase 2 (MMP2, e.g., NCBI gene ID 4313), TIMP metallopeptidase inhibitor 1 (TIMP1, e.g., NCBI gene ID 7076), cell adhesion molecule 5 (CEA, e.g., NCBI gene ID 1048), cytokeratin 19 fragments (CYFRA 21-1, e.g., NCBI gene ID 3880), Serpin family B member 3 (SCC, e.g., NCBI gene ID 6317), Advanced Glycation End Product organism-specific receptor (AGER, e.g., NCBI gene ID 177), Adipogenesis regulator (C10orf116, e.g., NCBI gene ID 10974), Adusin 2 (ADD2, e.g., NCBI gene ID 119), Periaxin (PRX, e.g., NCBI gene ID 57716), Laminin subunit beta 3 (LAMB3, e.g., NCBI gene ID 3914), Synemin (SYNM, e.g., NCBI gene ID 23336), Spectrin alpha, Red blood cell 1 (SPTA1, e.g., NCBI gene ID 6708),Ankyrin 1 (ANK1, e.g., NCBI gene ID 286), hemoglobin subunit epsilon 1 (HBE1, e.g., NCBI gene ID 3046), hemoglobin subunit gamma 1 (HBG1, e.g., NCBI gene ID 3047), carbonic anhydrase 1 (CA1, e.g., NCBI gene ID 759), tenascin XB (TNXB, e.g., NCBI gene ID 7148), multimelin 2 (MMRN2, e.g., NCBI gene ID 7 9812), hemoglobin subunit alpha-1 (HBA1, e.g., NCBI gene ID 3039), caveolin-1 (CAV1, e.g., NCBI gene ID 857), hemoglobin subunit beta (HBB, e.g., NCBI gene ID 3043), collagen type VI alpha-6 chain (COL6A6, e.g., NCBI gene ID 131873), chromosome 1 open reading frame 198 (C1orf198, e.g., NCBI gene ID84886), Chloride intracellular channel 2 (CLIC2, e.g., NCBI gene ID1193), SdpC synthetic operon transcription regulator (ArsR family) (SDPR, e.g., NCBI gene ID8436), EH domain-containing 2 (EHD2, e.g., NCBI gene ID30846), Apolipoprotein A2 (APOA2, e.g., NCBI gene ID336), NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7, e.g., NCBI gene ID4713), Protein kinase C delta-binding protein (PRKCDBP, e.g., NCBI gene ID112464), Laminin subunit alpha 3 (LAMA3, e.g., NCBI gene ID), EvC ciliary complex subunit 2 (LBN, e.g., NCBI gene ID132884), Serpin family A member 3 (ACT, e.g., NCBI gene ID12), Insulin-like growth factor-binding protein 3 (3 IGFBP3 (e.g., NCBI gene ID 3486), prostaglandin D2 synthase (L-PGDS, e.g., NCBI gene ID 5730), retinoic acid receptor beta (HAP, e.g., NCBI gene ID 5915), hepatocyte growth factor (HGF, e.g., NCBI gene ID 3082), eukaryotic translation initiation factor 4 gamma-2 (AAG1 / 2, e.g., NCBI gene ID 1982),Clusterin (CLU, e.g., NCBI gene ID 1191), Streptococcal superantigen SSA (SSA, e.g., NCBI gene ID 6737), Tetanic (TTA, e.g., NCBI gene ID 100189453), Apolipoprotein A4 (APOA4, e.g., NCBI gene ID 337), Fibrinogen-like protein A (FIBA, e.g., NCBI gene ID 105209070), Serum amyloid A cluster (SAA, e.g., NCBI gene ID 6288), Ceruloplasmin (CP, e.g., NCBI gene ID 1356) ), haptoglobin (HP, e.g., NCBI gene ID 3240), transthyretin (TTR, e.g., NCBI gene ID 7276), keratin 2 (KRT2A, e.g., NCBI gene ID 3849), glutamate transporter (GLT1B, e.g., NCBI gene ID 6506), casein kinase 1 (CK1, e.g., NCBI gene ID 1452), AKT serine / threonine kinase 1 (AKT, e.g., NCBI gene ID 207), mannose-binding lectin 2 (MBL2, e.g., NCBI gene ID 4153), fibril Norgen alpha chain (FGA, e.g., NCBI gene ID 2243), gelsolin (GSN, e.g., NCBI gene ID 2934), haptoglobin (HP, e.g., NCBI gene ID 3240), phycolin 3 (FCN3, e.g., NCBI gene ID 8547), carnosine dipeptidase 1 (CNDP1, e.g., NCBI gene ID 84735), calcitonin-related polypeptide alpha (CALCA, e.g., NCBI gene ID 796), carbamoyl phosphate synthase 1 (CPS1, e.g., NCBI gene ID 1373), clo Mogranin B (CHGB, e.g., NCBI gene ID 1114), Involucrin (IVL, e.g., NCBI gene ID 3713), Forward Gradient 2 (AGR2, e.g., NCBI gene ID 10551), Nuclear Autoantigen Sperm Protein (NASP, e.g., NCBI gene ID 4678), Phosphofructokinase, Platelet (PFKP, e.g., NCBI gene ID 5214), Thrombospondin 2 (THBS2, e.g., NCBI gene ID 7058), Thioredoxin Domain-containing 17 (TXNDC17, e.g., NCBI gene ID 84817),Proprotein convertase subtilisin / kexin type 1 (PCSK1, e.g., NCBI gene ID 5122), cellular retinoic acid-binding protein 2 (CRABP2, e.g., NCBI gene ID 1382), acyl-CoA binding domain-containing protein 3 (ACBD3, e.g., NCBI gene ID 64746), desmoglein 2 (DSG2, e.g., NCBI gene ID 1829), LPS-responsive beige-like anchor protein (LRBA, e.g., NCBI gene ID 987), serine / threonine kinase receptor-related protein (STRAP, e.g., NCBI gene ID 11171), VGF nerve growth factor-inducible (VGF, e.g., NCBI Gene ID 7425), NOP2 nucleolar protein (NOP2, e.g., NCBI gene ID 4839), lipocalin 2 (LCN2, e.g., NCBI gene ID 3934), creatine kinase, mitochondrial 1B (CKMT1B, e.g., NCBI gene ID 1159), aldo-keto reductase family 1 member B10 (AKR1B10, e.g., NCBI gene ID 57016), carboxypeptidase D (CPD, e.g., NCBI gene ID 1362), proteasome activator subunit 3 (PSME3, e.g., NCBI gene ID 10197), virin 1 (VIL1, e.g., NCBI gene ID 7429), Serpin family B member 5 (SERPINB5, e.g., NCBI gene ID 5268), ribosomal protein L5 (RPL5, e.g., NCBI gene ID 6125), placophyllin 1 (PKP1, e.g., NCBI gene ID 5317), ribosomal protein L10 (RPL10, e.g., NCBI gene ID 6134), aldo-keto reductase family 1 member B10 (AKR1B10, e.g., NCBI gene ID 57016), aldo-keto reductase family 1 member C1 (AKR1C1, e.g., NCBI gene ID 1645), proliferating cell nuclear antigen (PCNA, e.g., NCBI gene ID 5111), ribosomal protein S2 (RPS2, e.g., NCBI gene ID 5111) Gene ID 6187), Aldo-keto reductase family member 1 C3 (AKR1C3, e.g., NCBI gene ID 8644), Acyl-CoA binding domain-containing 3 (ACBD3, e.g., NCBI gene ID 64746), Bicinin-like 1 (VSNL1, e.g., NCBI gene ID 7447), Adenosyl homocysteinase (AHCY, e.g., NCBI gene ID 191), IMMP10, Activation kinase 2 (PAK2, e.g., NCBI gene ID 5062), Involucrin (IVL, e.g., NCBI gene ID 3713), Isoleucine tRNA synthetase (IARS, e.g., NCBI gene ID 3376), Proteasome 26S subunit ubiquitin receptor, Non-ATPase2 (PSMD2, e.g., NCBI gene ID 5708), guanylate-binding protein 5 (GBP5, e.g., NCBI gene ID 115362), minichromosome maintenance complex component 6 (MCM6, e.g., NCBI gene ID 4175), N-myc downstream regulator 1 (NDRG1, e.g., NCBI gene ID 10397), NOP58 ribonucleoprotein (NOP58, e.g., NCBI gene ID 51602), S100 calcium-binding protein A2 (S100A2, e.g., NCBI gene ID 6273), Neuregulin 1 (NRG1, e.g., NCBI gene ID 3084), Neuregulin 2 (NRG2, e.g., NCBI gene ID 9542), Carnosine dipeptidase 1 (CNDP1, e.g., NCBI gene ID 84735), Ubiquitin cross-reactive protein (UCRP, e.g., NCBI gene ID 9636), Kramer (CER, e.g., NCBI gene ID 8110), Plasminogen activator (UPA, e.g., NCBI gene (ID5328), Matrix metallopeptidase 14 (MT1-MMP, e.g., NCBI gene ID4323), Stratifin (SFN, e.g., NCBI gene ID2810), Transferrin (TF, e.g., NCBI gene ID7018), Albumin (ALB, e.g., NCBI gene ID213), S100 calcium-binding protein A9 (S100A9, e.g., NCBI gene ID6280), Stasmin 1 (STMN, e.g., NCBI gene ID392) 5) It includes enolase (ENO), plasminogen activator (PLAU, e.g., NCBI gene ID 5328), insulin-like growth factor binding protein 7 (IGFBP7, e.g., NCBI gene ID 3490), matrix metallopeptidase 14 (MMP14, e.g., NCBI gene ID 4323), thrombospondin 1 (THBS1, e.g., NCBI gene ID 7057), or thrombospondin 2 (THBS2, e.g., NCBI gene ID 7058).

[0076] In some embodiments, the targeting portion targets biomarkers expressed or released by metastatic cancer cells. For example, cancer cells may originate in one tissue and then metastasize to another location. In some embodiments, metastatic cancer cells express a non-limiting example of cancer biomarkers described herein. In some embodiments, metastatic cancer cells express cancer biomarkers, including melanoma-associated antigen (MAGE family member A3 (MAGE-A3, e.g., NCBI gene ID 4102)), membrane-associated glycoprotein (MUC-1, e.g., NCBI gene ID 4582), glycoprotein epithelial cell adhesion molecule (EpCAM, e.g., NCBI gene ID 4072), KRAS proto-oncogene (KRAS, e.g., NCBI gene ID 3845), anaplastic lymphoma kinase (ALK, e.g., NCBI gene ID 238), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, e.g., NCBI gene ID 1493), programmed cell death protein 1 (PD-1, e.g., NCBI gene ID 513). 3) Epidermal growth factor (EGF, e.g., NCBI gene ID 1950), serine protease ester (EA, e.g., NCBI gene ID 5328), telomerase reverse transcriptase (TERT, e.g., NCBI gene ID 7015), PRAME nuclear receptor transcription regulator (PRAME, e.g., NCBI gene ID 23532), receptor tyrosine protein kinase erbB-2 (HER, e.g., NCBI gene ID 2064), or vascular endothelial growth factor (VEGF, e.g., NCBI gene ID 7422), carcinoembryonic antigen (CEA, e.g., NCBI gene ID 1048), MAGE family member A1 (MAGE-A1, e.g., NCBI gene ID 4100), MAGE family member A1 This includes MAGE-A4 (e.g., NCBI gene ID 4103), Survivin, six-transmembrane epithelial antigen of the prostate gland 1 (STEAP1, e.g., NCBI gene ID 26872), SRY (sex-determining region Y) box 2 (SOX2, e.g., NCBI gene ID 6657), or cancer / testis / antigen 1 (CTAG1B, e.g., NCBI gene ID 1485).

[0077] In some embodiments, the targeting moiety targets a biomarker expressed or released by endothelial cells. In some embodiments, endothelial cells are vascular cells. In some embodiments, endothelial cells are lymphatic cells. In some embodiments, the biomarker is expressed or released by vascular cells. In some embodiments, the biomarker is expressed or released by lymphatic cells. Non-limiting examples of endothelial cell biomarkers include angiotensin I-converting enzyme (ACE / CD143, e.g., NCBI gene ID 1636), CD93 molecule (C1qR1 / CD93, e.g., NCBI gene ID 22918), cadherin 5 (VE-cadherin, e.g., NCBI gene ID 1003), D6 protein (CC chemokine receptor D6, e.g., NCBI gene ID 1238), platelet and endothelial cell adhesion molecule 1 (CD31 / PECAM-1). (e.g., NCBI gene ID 5175), CD34 molecule (CD34, e.g., NCBI gene ID 947), CD36 molecule (CD36 / SR-B3, e.g., NCBI gene ID 948), CD151 molecule (CD151, e.g., NCBI gene ID 977), CD160 molecule (CD160, e.g., NCBI gene ID 11126), CD300 molecule-like family member g (CD300 g / nepmucin, e.g., NCBI gene ID 146894) ), CDC-like kinase 1 (CL-K1 / COLEC11, e.g., NCBI gene ID 78989), cleavage factor polyribonucleotide kinase subunit 1 (CL-P1 / COLEC12, e.g., NCBI gene ID 81035), coagulation factor III / tissue factor (e.g., NCBI gene ID 2152), C-type lectin domain family 4 member M (DC-SIGNR / CD299, e.g., NCBI gene ID 10332), discoidin, CUB and LCCL domain-containing 2 (DCBLD2 / ESDN, e.g., NCBI gene ID 131566), endothelial cell surface expression chemotactic and apoptosis regulator (ECSCR, e.g., NCBI gene ID 641700), basidine (Ok blood type) (EMMPRIN / CD147, e.g., NCBI gene ID 682), endoglin / CD105 (e.g., NCBI gene ID 5077), endomucin (e.g., NCBI gene ID 2022),Endosialin / CD248 (e.g., NCBI gene ID 57124), Protein C receptor (EPCR, e.g., NCBI gene ID 10544), Erythropoietin R (e.g., NCBI gene ID 2056), Endothelial cell adhesion molecule (ESAM, e.g., NCBI gene ID 90952), Fatty acid binding protein 5 (FABP5 / E-FABP, e.g., NCBI gene ID 2171), Fatty acid binding protein 6 (FABP6, e.g., NCBI gene ID 2172), Intercellular adhesion molecule 1 (ICAM-1 / CD54, e.g., NCBI gene ID 3383), Intercellular adhesion molecule 2 (ICAM-2 / CD102, e.g., NCBI gene ID 3384), Interleukin 1 receptor (IL-1 RI, e.g., NCBI gene ID 3553), Interleukin 13 receptor alpha-1 (IL-13 R-alpha1 (e.g., NCBI gene ID 3597), integrin alpha4 / CD49d (e.g., NCBI gene ID 3676), integrin α4β1 (e.g., NCBI gene ID 3688), integrin α4β7 / LPAM-1 (e.g., NCBI gene ID 3676), integrin β 2 / CD18 (e.g., NCBI gene ID 3689), KLF transcription factor 4 (KLF4, e.g., NCBI gene ID 9314), lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1, e.g., NCBI gene ID 10894), melanoma cell adhesion molecule (MCAM / CD146, e.g., NCBI gene ID 4162), nectin cell adhesion molecule 2 (nectin-2 / CD112, e.g., NCBI gene ID 5819), PD-ECGF / thymidine phosphorylase (e.g., NCBI gene ID 1890), podocalyxin (e.g., For example, NCBI gene ID 5420), podoplanin (e.g., NCBI gene ID 10630), sphingosine-1-phosphate receptor 1 (S1P1 / EDG-1, e.g., NCBI gene ID 1901), sphingosine-1-phosphate receptor 2 (S1P2 / EDG-5, e.g., NCBI gene ID 9294), sphingosine-1-phosphate receptor 3 (S1P3 / EDG-3, e.g., NCBI gene ID 1903), sphingosine-1-phosphate receptor 4 (S1P4 / EDG-6, e.g., NCBI gene ID 8698),Sphingosine-1-phosphate receptor 5 (S1P5 / EDG-8, e.g., NCBI gene ID 53637), E-selectin / CD62E (e.g., NCBI gene ID 6401), P-selectin / CD62P (e.g., NCBI gene ID 6403), Molasses-like slow (SLAM / CD150, e.g., NCBI gene ID 6504), Stabilin-1 (e.g., NCBI gene ID 23166), Stabilin-2 (e.g., NCBI gene ID 55576), Plexin domain-containing 1 (TEM7 / PLXDC1, e.g., NCBI gene ID 5 7125), ANTXR cell adhesion molecule 1 (TEM8 / ANTXR1, e.g., NCBI gene ID 84168), thrombomodulin / BDCA-3 (e.g., NCBI gene ID thrombomodulin), thrombospondin type 1 domain-containing 1 (THSD1, e.g., NCBI gene ID 55901), thrombospondin type 1 domain-containing 7A (THSD7A, e.g., NCBI gene ID 221981), TEK receptor tyrosine kinase (Tie-2, e.g., NCBI gene ID 7010), TNF receptor superfamily member 1A (TNF RI / TNFRSF1A (e.g., NCBI gene ID 7132), TNF receptor superfamily member 1B (TNF RII / TNFRSF1B, e.g., NCBI gene ID 7133), basidine (Ok blood type) (TRA-1-85 / CD147, e.g., NCBI gene ID 682), TNF receptor superfamily member 10b (TRAIL R2 / TNFRSF10B (e.g., NCBI gene ID 8795), TNF receptor superfamily member 10a (TRAILR1 / TNFRSF10A, e.g., NCBI gene ID 8797), vascular cell adhesion molecule 1 (VCAM-1 / CD106, e.g., NCBI gene ID 7412), EGF-like domain multiple 7 (VE-Statin, e.g., NCBI gene ID: 51162), fms-related receptor tyrosine kinase 1 (VEGFR1 / Flt-1, e.g., NCBI gene ID 2321), kinase insertion domain receptor (VEGFR2 / KDR / Flk-1, e.g., NCBI gene ID 3791), fms-related receptor tyrosine kinase 4 (VEGFR3 / Flt-4, e.g., NCBI gene ID 2324),It contains an angiogenic factor with a G patch and FHA domain 1 (VG5Q, e.g., NCBI gene ID 55109), or a von Willebrand factor domain 2 (vWF-A2, e.g., NCBI gene ID 7450).

[0078] In some embodiments, the targeting moiety includes a chemokine receptor or chemokine ligand involved in chemokine signaling, or a moiety thereof (e.g., SDF-1α / CXCR4, CCL2 / CCR2, etc.), or an adhesion molecule (e.g., PSGL-1, etc.). As shown herein, enucleated cells may be enucleated to express functional CXCR4, CCR2, or even glycosylated PSGL-1, which may significantly enhance the specific targeting of enucleated cells. In some embodiments, the targeting moiety (e.g., CXCR4, CCR2, or PSGL-1) may be expressed on the surface of enucleated cells. Non-limiting examples of cell surface proteins that can be expressed on the cell surface of enucleated cells as the targeting moiety include chemokines, such as CXCR4, CCR2, CCR1, CCR5, CXCR7, CXCR2, and CXCR1. In some embodiments, enucleated cells can be enucleated to secrete targeted moieties or tethered to the extracellular matrix (e.g., SDF1α or CCL2). Non-limiting examples of targeted moieties that may be secreted by enucleated cells include SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11, and CXCL12. In some embodiments, enucleated cells include cell matrix receptors, and intercellular adhesion molecules include integrins, cadherins, glycoproteins, and heparin sulfate proteoglycans.

[0079] In some embodiments, enucleated cells may further include surface markers that help evade the target immune system (e.g., by manipulation or from cells from which enucleated cells were obtained). For example, in some embodiments, enucleated cells may include CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof. Without being bound by any particular theory, CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof are thought to help prevent enucleated cells from being phagocytosed by macrophages. Non-limiting examples of cell matrix receptors and cell adhesion molecules include integrins, cadherins, glycoproteins, or heparin sulfate proteoglycans. In some embodiments, the cell matrix receptor or cell adhesion molecule includes PD-L1, HLA-E, or HLA-G. Non-limiting examples of therapeutic molecules include tumor antigens and immunomodulatory peptides, polyamines, and ATP. In some embodiments, the therapeutic molecule can be recognized by immune cells and can induce an immune response. For example, the therapeutic molecule may be either 4-1BB or one of the cytokines described herein for inducing an immune response.

[0080] (d) Therapeutic agents In some embodiments, the enucleated cells of the Disclosure comprise at least one therapeutic agent. In some embodiments, the enucleated cells of the Disclosure comprise at least two, three, four, five, six, seven, eight, nine, ten, or more therapeutic agents. In some embodiments, the therapeutic agent comprises an activator. In some embodiments, the therapeutic agent is exogenous to the enucleated cell or its parent cell. The activator comprises at least one of the following: DNA molecules, RNA molecules, proteins (e.g., enzymes, antibodies, antigens, toxins, cytokines, protein hormones, growth factors, cell surface receptors, or vaccines), peptides (e.g., peptide hormones or antigens), small molecules (e.g., steroids, polyketides, alkaloids, toxins, antibiotics, antivirals, colchicine, taxol, mitomycin, or emtansine), exogenous gene editing systems, nanoparticles, or other activators (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses, exosomes, lipids, or ions). In some embodiments, the enucleated cells are manipulated to produce (e.g., express, and optionally release or secrete) the therapeutic agent. In some embodiments, the parent may be manipulated to produce a therapeutic agent before enucleation for the production of enucleated cells.

[0081] In some embodiments, the therapeutic agent is a virus. In some embodiments, the virus is a replication component virus. In some embodiments, the virus is a replication failure virus. In some embodiments, the virus is an adeno-associated virus (AAV). Non-limiting examples of oncolytic viruses include AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9. In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is human T-lymphotropic virus (HTLV). In some embodiments, the retrovirus is human foam virus. In some embodiments, the retrovirus is a gamma-retrovirus. In some embodiments, the gamma-retrovirus is murine leukemia virus (MLV). In some embodiments, the gamma-retrovirus is Moloney's murine sarcoma virus. In some embodiments, the retrovirus is an endogenous retrovirus. In some embodiments, the virus is a lentivirus. In some embodiments, the lentivirus is human immunodeficiency virus type 1 (HIV-1). In some embodiments, the lentivirus is HIV-2. In some embodiments, the virus is a poxvirus. In some embodiments, the poxvirus is vaccinia virus. In some embodiments, the poxvirus is monkeypox virus. In some embodiments, the poxvirus is smallpox virus. In some embodiments, the virus is parvovirus. In some embodiments, the parvovirus is parvovirus B19. In some embodiments, the virus is baculovirus. In some embodiments, the baculovirus is nuclear polyhedron virus (NPV). In some embodiments, the virus is herpesvirus. Non-exclusive examples of herpesviruses include varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, Kaposi's sarcoma-associated herpesvirus, B virus, herpes simplex virus type 1 (HSV-1), HSV-2, and varisserovirus.

[0082] In some embodiments, the virus is an oncolytic virus. Non-limiting examples of oncolytic viruses include tarimodine laherpalepbec, ONYX-015, GL-ONC1, CV706, Voyager-V1, HSV-1716, vaccinia virus, adenovirus, HSV-1, HSV-2, B19PV, H1PV, monkeypox virus, Sindbis virus, Zika virus, measles virus, Newcastle disease virus, coxsackievirus A21, poliovirus, Seneca Valley virus, reovirus, Maraba virus, varicella stomatitis virus, poliovirus, reovirus, Seneca virus, ECHO-7, and Semryqui Forest virus. In some embodiments, the oncolytic virus is a coxsackievirus. In some embodiments, the oncolytic virus is varicella stomatitis virus (VSV). In some embodiments, the oncolytic virus is an adenovirus. In some embodiments, the oncolytic virus is a retrovirus.

[0083] In some embodiments, at least one therapeutic agent comprises an engineered oncolytic moiety. In some embodiments, the engineered oncolytic moiety comprises an oncolytic virus. In some embodiments, the oncolytic virus comprises adenovirus, vaccinia virus (e.g., Copenhagen strain, Western Reserve strain, or YS strain), reovirus, herpes simplex virus, Newcastle disease virus, poxvirus, myxoma virus, picornavirus, influenza virus, coxsackievirus, parvovirus or rhabdovirus (e.g., vesicular stomatitis virus), or variants thereof. In some embodiments, the engineered oncolytic moiety comprises an adenovirus moiety or an adenovirus (e.g., Delta-24 strain or ONYX-015 strain). In some embodiments, the engineered oncolytic moiety comprises modifications to the oncolytic virus genome encoding the engineered oncolytic virus. In some embodiments, the engineered oncolytic virus is engineered to enhance its selectivity for infecting cancer cells. In some embodiments, the engineered oncolytic virus comprises a genomic mutation, deletion, cleavage, substitution, or combination thereof of the oncolytic virus. In some embodiments, the viral genome of the engineered oncolytic virus comprises an expression cassette. For example, the expression cassette may comprise an exogenous gene encoding an exogenous protein and a promoter for driving the expression of the exogenous protein. In some embodiments, the oncolytic virus genome of the engineered oncolytic virus comprises replacing one or more genes within the oncolytic virus genome with an expression cassette disclosed herein. In some embodiments, the exogenous protein encoded by the expression cassette may exert an additive or synergistic effect on the therapeutic effect of the oncolytic virus (e.g., targeting and killing cancer cells). For example, the expression cassette may encode an exogenous cytokine or an immune checkpoint inhibitor.

[0084] In some embodiments, the oncolytic moiety comprises one or more polynucleotides encoding one or more components of the oncolytic virus or engineered oncolytic virus described herein. In some embodiments, one or more polynucleotides are engineered to produce the engineered oncolytic virus or engineered oncolytic virus described herein.

[0085] Adenovirus In some embodiments, the oncolytic viruses described herein are adenoviruses or engineered adenoviruses, or include them. In some embodiments, engineered adenoviruses include viral genome regions of adenoviruses that are deleted (complete or partial deletion), defunctionalized, modified to have reduced function, or substituted with other sequences, or any combination thereof. In some embodiments, engineered adenoviruses can enhance tumor selectivity, for example, by weakening the ability of the virus to replicate in normal quiescent cells without affecting the ability of the virus to replicate in tumor cells. In some embodiments, engineered adenoviruses have improved properties for use in cancer treatment, such as selective replication in cancer cells, attenuation of viral pathogenicity, enhanced lytic activity, modification of antiviral immune responses that may lead to rapid clearance of the adenovirus, or modification of virus-induced systemic antitumor immunity, or any combination thereof.

[0086] Generally, the adenovirus infection cycle proceeds in two stages. In the initial stage, the initiation of adenovirus genome replication takes place, enabling the production of regulatory proteins and proteins involved in viral DNA replication and transcription. In the later stage, the synthesis of structural proteins is triggered. The genes in the initial stage are distributed in four regions: E1, E2, E3, and E4 (E stands for "initial"). The three regions E1, E2, and E4 are essential for viral replication. E1 is located at the 5' end of the adenovirus genome and contains two viral transcription units (E1A and E1B). In particular, the E1A transcription unit encodes a protein that activates the transcription of other viral genes and induces transcription from the promoters of the E1B, E2A, E2B, E3, and E4 regions and later genes.

[0087] In some embodiments, the adenovirus includes the Delta-24 strain, Delta-24-RGD strain, ICOVIR-5 strain, ICOVIR-7 strain, ONYX-015 strain, ColoAd1 strain, H101 strain, or AD5 / 3-D24-GMCSF strain, or variants thereof. In some embodiments, the Delta-24 strain includes a deletion of 24 nucleotides (SEQ ID NO: 3) in the CR2 portion of the E1A gene (SEQ ID NO: 2), which contains the region (nucleotides 923-946) responsible for binding the retinoblastoma (Rb) protein, corresponding to amino acids 122-129 of the encoded E1A protein. In some embodiments, the deletion in the CR2 portion of E1A (SEQ ID NO: 1) includes residues 122-129 of the E1A amino acid sequence (SEQ ID NO: 4). E1A binds the Rb protein and releases E2F. This E2F functions as a transcription factor for genes that promote progression to the S phase of the cell cycle and enhance viral replication. In some embodiments, deletion of E1A enhances the selectivity of the virus against cancer cells. Because mE1A cannot bind the Rb protein and release E2F, the delta-24 adenovirus cannot replicate in normal cells. However, in some embodiments, in cancer cells, Rb deficiency indicates the presence of a free E2F protein that drives the cell to the S state, thus enhancing viral replication. In some embodiments, this approach forms the backbone of delta-24 adenovirus-based oncolytic virus therapy, mediating the selectivity of delta-24 adenovirus against cancer cells. In some embodiments, the E1A region includes a nucleic acid sequence that shares at least 80%, 85%, 90%, 95%, or 99% homology with SEQ ID NO: 2. In some embodiments, the E1A region includes a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2. In some embodiments, the E1A region includes a nucleic acid sequence containing SEQ ID NO: 2. In some embodiments, the delta-24 adenovirus is derived from any adenovirus serotype. In some embodiments, the delta-24 adenovirus is derived from adenovirus serotype 5 (Ad-5, SEQ ID NO: 6).

[0088] In some embodiments, the delta-24 strain contains one or more nucleotide substitutions in 24 nucleotides. For example, the 24 nucleotides can be substituted with nucleotides encoding RGD to generate a modified adenovirus strain of delta-24-RGD. In some embodiments, delta-24-RGD contains peptide substitutions within the adenovirus genome. In some embodiments, the peptide enables the delta-24-RGD virus to attach to and infect cells via integrins. These integrins are membrane receptors that are very abundant in tumor cells, such as glioblastoma cells. In some embodiments, delta-24-RGD is an adenovirus containing a deletion of 24 bases (bases 923-946) in the E1A gene and an insertion of an amino acid sequence (RGD-4C peptide) that is at least 80% identical to or shares at least 80% homology with SEQ ID NO: 5. In some embodiments, delta-24-RGD is an adenovirus containing a 24-base (bases 923-946) deletion in the E1A gene and insertion of the amino acid sequence of SEQ ID NO: 5, an integrin-binding motif that strongly binds to ανβ3 and ανβ5 integrins, into the HI loop of a fiber knob protein. In some embodiments, the E1A deletion enhances the virus's selectivity for cancer cells, and the RGD-4C sequence enhances the virus's infectivity to tumors such as gliomas that express low levels of the adenovirus receptor. In some embodiments, delta-24-RGD has a potent antitumor mechanism by replicating within human tumors, inducing tumor necrosis, and triggering an immune response.

[0089] In some embodiments, the ONYX-015 strain is a hybrid of the Ad2 and Ad5 serotypes of the virus, having deletions in the E1B-55K and E3B regions to enhance cancer selectivity. In some embodiments, H101 is a modified version of ONYX-015. In some embodiments, ICOVIR-5 and ICOVIR-7 include a deletion of the retinoblastoma (Rb) binding site of E1A and substitution of the E1A promoter with the E2F promoter. In some embodiments, ColoAd1 is a chimeric Ad-dllp / Ad3 serotype. In some embodiments, AD5 / 3-D24-GMCSF (CGTG-102) is a capsid-modified adenovirus of the Ad5 and Ad3 serotypes encoding GM-CSF (the capsid protein knob of the Ad5 serotype is replaced with a knob domain from the Ad3 serotype).

[0090] In some embodiments, the enucleated cells described herein include one or more polynucleotides encoding one or more components of the Delta-24 virus or its variants disclosed herein. In some embodiments, one or more polynucleotides include a nucleic acid sequence that shares at least 80%, 85%, 90%, 95%, or 99% homology with SEQ ID NO: 6 (Human Adenovirus 5, NCBI reference sequence: AC_000008.1). In some embodiments, one or more polynucleotides include a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6.

[0091] The therapeutic agent may be or may contain the targeted moiety described herein. Non-limiting examples of targeted moieties that may be produced by or contained in enucleated cells include chemokine receptors, adhesion molecules, and antigens. In some embodiments, the therapeutic agent may be or may contain the transmembrane moiety described herein.

[0092] In some embodiments, the therapeutic agent is recombinantly expressed by enucleated cells or their parent cells. In some embodiments, the parent cells from which the enucleated cells are derived or obtained are manipulated to produce or express the therapeutic agent. In some embodiments, the expression of the therapeutic agent is stable (e.g., persistent). In some embodiments, the expression of the therapeutic agent by the parent cells is transient (e.g., non-persistent). In some embodiments, the parent cells are enucleated before manipulating the enucleated cells to recombinantly express the therapeutic agent.

[0093] In some embodiments, the therapeutic agent is not spontaneously expressed in the cells from which the enucleated cells originate or are obtained (e.g., without manipulation) (e.g., the therapeutic agent is exogenous to the parent cells). In some embodiments, the therapeutic agent is not spontaneously expressed in the target (e.g., the therapeutic agent is exogenous to the target). In some embodiments, the therapeutic agent is not spontaneously expressed in the target site of treatment (e.g., a tumor, or a specific tissue, such as the brain, intestines, lungs, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is exogenous to the target site). In some embodiments, levels of the therapeutic agent are not naturally present in the enucleated cells of the parent cells.

[0094] In some embodiments, the therapeutic agent is spontaneously expressed (e.g., without manipulation) in the cells from which enucleated cells originate or are obtained (e.g., the therapeutic agent is endogenous to enucleated cells). In some embodiments, the therapeutic agent is spontaneously expressed in the target (e.g., the therapeutic agent is endogenous to the target). In some embodiments, the therapeutic agent is spontaneously expressed in the target site of treatment (e.g., a tumor, or a specific tissue, such as the brain, intestines, lungs, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is endogenous to the target site of treatment).

[0095] In some embodiments, the therapeutic agent is derived from synthetic cells and loaded into enucleated cells. For example, the therapeutic agent may be taken up into cells. Alternatively, the therapeutic agent may be synthesized by cells and then delivered to target cells.

[0096] In some embodiments, compared to the original cells from which enucleated cells are derived or obtained, the therapeutic agent includes modified, cleaved, or non-mutated versions and / or copies of DNA molecules, RNA molecules, proteins, peptides, small molecule activators, and / or gene editing factors. For example, the therapeutic agent can modify mutated p53 or EGFR in target cells as part of the treatment of lung cancer.

[0097] In some embodiments, the therapeutic agent comprises any combination of at least two (e.g., at least two, three, four, five, or more) different therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule activators, or therapeutic gene editing factors. For example, in some embodiments, the therapeutic agent comprises a therapeutic DNA molecule and a small molecule activator. For example, in some embodiments, the therapeutic agent comprises two different small molecule activators. For example, in some embodiments, the therapeutic agent comprises a chemokine receptor (e.g., for targeting) and a small molecule activator.

[0098] In some embodiments, the therapeutic agent comprises RNA molecules, including messenger RNA (mRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA, long non-coding RNA (lncRNA), or RNA viruses. In some embodiments, the therapeutic agent comprises DNA molecules, such as single-stranded DNA, double-stranded DNA, oligonucleotides, plasmids, bacterial DNA molecules, or DNA viruses. In some embodiments, the therapeutic agent comprises a protein or a portion thereof. In some embodiments, the protein is a cytokine, growth factor, hormone, antibody or its antigen-binding fragment, small peptide-based drug, or enzyme. In some embodiments, enucleated cells transiently express the therapeutic agent. In some embodiments, the expression of the therapeutic agent is inducible. In some embodiments, the expression of the therapeutic agent is persistent.

[0099] In some embodiments, the therapeutic agent comprises an exogenous factor. In some embodiments, the exogenous factor is an exogenous polypeptide. In some embodiments, the exogenous polypeptide is encoded by an exogenous polynucleotide delivered to a parent cell or an enucleated cell. In some embodiments, the exogenous polypeptide is synthesized or released by at least one intracellular organelle of the enucleated cell. In some embodiments, the exogenous polypeptide is released by the enucleated cell. In some embodiments, the exogenous polypeptide is expressed on the cell surface or on the enucleated cell. In some embodiments, the enucleated cell delivers the exogenous polypeptide to a target cell. In some embodiments, the target cell is a cancer cell expressing any cancer biomarker described herein. In some embodiments, the target cell is an endothelial cell expressing an endothelial biomarker described herein. In some embodiments, the endothelial cell is an angiocyte. In some embodiments, the endothelial cell is a lymphatic cell.

[0100] In some embodiments, the exogenous polypeptide comprises any one cytokine from among the cytokines described herein or their cytokine receptor-binding fragments. Non-limiting examples of cytokines include interleukin (IL) 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13, IL-14, IL-15, IFN-α, IFN-β, IFN-γ, IL-17, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the exogenous polypeptide comprises a soluble cytokine. For example, the exogenous polypeptide may include the extracellular domain or fragment of a cytokine. In some embodiments, the exogenous polypeptide includes solubility determined by a turbidimetric solubility assay or a thermodynamic solubility assay by dissolving the exogenous polypeptide in a solvent, examples of which solvents include organic solvents (including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc.) or inorganic solvents (including water or phosphate-buffered saline (PBS)). In some embodiments, the exogenous polypeptide contains solubility of at least 0.0001 mg / ml, 0.0005 mg / ml, 0.001 mg / ml, 0.005 mg / ml, 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 5.0 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 500 mg / ml, 1,000 mg / ml, 5,000 mg / ml, 10,000 mg / ml, 50,000 mg / ml, or 100,000 mg / ml.

[0101] Figure 4A shows the workflow for incorporating the target agent (IL-12p70 in this case). Figure 4B shows the results following the workflow in Figure 4A. Here, the supernatant values ​​for IL-12p70 secretion are similar between fresh cells and cells cryopreserved in two ways. In Figure 5A, the initiation protocol is similar, but the sample type is different. In Figure 4A, the sample type evaluated is supernatant. In Figure 5A, different tissue types and markers were analyzed. Details are shown superimposed on the right side of Figure 5A. Following the protocol in Figure 5A, the graph in Figure 5B shows a comparison of IL-12 supernatant measurements between fresh cells and cryopreserved cells (similar to Figure 4B). Figure 5C analyzes the amount of IL-12p70 in the plasma of mice on days 1, 2, and 3 after injection for mice without enucleated cells, mice with fresh enucleated cells containing IL-12, and mice with cryopreserved enucleated cells containing IL-12. Figure 5D analyzes the amount of IFN-γ in the plasma of mice on days 1, 2, and 3 after injection for mice without enucleated cells, mice containing fresh enucleated cells containing IL-12, and mice containing cryopreserved enucleated cells containing IL-12. Figure 5E analyzes the amount of IL-12 mRNA in the lung tissue of mice on days 1, 2, and 3 after injection for mice without enucleated cells, mice containing fresh enucleated cells containing IL-12, and mice containing cryopreserved enucleated cells containing IL-12. Figure 5F analyzes the amount of IFN-γ mRNA in the lung tissue of mice on days 1, 2, and 3 after injection for mice without enucleated cells, mice containing fresh enucleated cells containing IL-12, and mice containing cryopreserved enucleated cells containing IL-12. Figure 5G shows the amount of IL-12 mRNA in the liver tissue of mice on days 1, 2, and 3 after injection, for mice without enucleated cells, mice containing fresh enucleated cells containing IL-12, and mice containing cryopreserved enucleated cells containing IL-12.Figure 5H shows the amount of IFN-γ mRNA in the liver tissue of mice on days 1, 2, and 3 after injection, for mice without enucleated cells, mice containing fresh enucleated cells containing IL-12, and mice containing cryopreserved enucleated cells containing IL-12. Figure 5I shows the total DiD+ cells in the lung tissue of mice on days 1, 2, and 3 after injection, for mice without enucleated cells, mice containing fresh enucleated cells containing IL-12, and mice containing cryopreserved enucleated cells containing IL-12.

[0102] As shown in Figure 6A, enucleated cells containing mlIFN-β-containing oncolytic virus were injected into the tail vein of mice, and the viral titer was quantitatively expressed after 16 hours, as shown in Figure 6B. By comparing enucleated cells before and after freezing, additional data on the PFU / mL of the enucleated cell inoculum were generated (Figure 6C).

[0103] In some embodiments, the exogenous polypeptide comprises a member of the tumor necrosis factor (TNF) superfamily or a catalytically active fragment thereof. Non-limiting examples of TNF superfamily members include lymphotoxin α (TNFβ), tumor necrosis factor (TNFα), lymphotoxin β (TNFγ), OX40 ligand (CD252, Gp34 or CD134L), CD40 ligand (CD154, TRAP, Gp39 or T-BAM), Fas ligand (CD178, APTL or CD95L), CD27 ligand (CD70), CD30 ligand (CD153), CD137 ligand (4-1 This includes BBL, TNF-related apoptosis-inducing ligands (CD253 or APO-2L), nuclear factor-κB receptor activator ligands (CD254, OPGL, TRANCE or ODF), TNF-related weak apoptosis-inducing factors (APO-3L or DR3L), proliferation-inducing ligands (CD256, tumor marker genes (TALL-2 or TRDL1), B cell activators (CD257, BlyS, TALL-1 or TNFSF20), LIGHT (CD258 or HVEML), vascular endothelial growth inhibitors (TL1 or TL-1A), TNF superfamily member 18 (GITRL, AITRL or TL-6), or ectodysprasin A (ED1-A1 or ED1-A2).

[0104] In some embodiments, the therapeutic agent comprises any one of the immune checkpoint proteins described herein, or an immune checkpoint inhibitor for inhibiting any one of the immune checkpoint proteins described herein. Non-limiting examples of immune checkpoint proteins include PD-1, PD-L1, CTLA-4, VISTA, B7-H3 (also known as CD276), A2AR, CD27, LAG3, TIM-3, Ig and T cell immune receptors (TIGIT) containing the ITIM domain, CD73, NKG2A, PVRIG, PVRL2, CEACAM1, CEACAM5, CEACAM6, FAK, CCR-2, CCL-2, LIF, CD47, SIRPα, M-CSF, CSF-1R, IL-3, IL-1RAP, IL-8, SEMA4D, angiopoietin-2, CLEVER-1, Axl, phosphatidylserine, or fragments thereof.

[0105] In some embodiments, the therapeutic agent is an exogenous gene editing system. In some embodiments, the exogenous gene editing system includes at least one nucleic acid contact moiety. In some embodiments, the exogenous gene editing system modulates the expression of a target gene in a target cell. For example, an enucleated cell can deliver the exogenous gene editing system to a target cell, where the exogenous gene editing system modulates the expression of a target gene in the target cell. In some embodiments, the exogenous gene editing system includes at least one guide nucleic acid. In some embodiments, the at least one guide nucleic acid is encoded by an enucleated cell. In some embodiments, the at least one guide nucleic acid is encoded from an exogenous nucleic acid as part of the exogenous gene editing system. In some embodiments, the at least one guide nucleic acid can be complexed with at least one nucleic acid contact moiety in a nanoparticle. For example, the exogenous gene editing system may include a nucleic acid contact moiety containing a Cas RNP, where the nucleic acid contact moiety is Cas9 or a fragment thereof, and Cas9 complexes with the guide nucleic acid to form a Cas9 RNP.

[0106] In some embodiments, a nucleic acid contact moiety can be complexed with at least one guide nucleic acid and recruited to a target gene to regulate its expression. In some embodiments, the nucleic acid contact moiety can regulate the expression of a target gene by directly contacting and cleaving the target gene or its transcript. For example, the nucleic acid contact moiety may include Cas9 or Cas12. Here, the nucleic acid contact moiety can be complexed with and recruited by a guide nucleic acid that is at least partially complementary to the nucleic acid sequence of the target gene. Upon contact with the nucleic acid sequence of the target gene, the nucleic acid contact moiety can consequently cleave the target gene. In some embodiments, the nucleic acid contact moiety can regulate the expression of a target gene by cleaving the transcript of the target gene. For example, the nucleic acid contact moiety may include Cas13. Here, the nucleic acid contact moiety can be complexed with and recruited by a guide nucleic acid that is at least partially complementary to the transcript of the target gene. Upon contact with the transcript of the target gene, the nucleic acid contact moiety can consequently cleave the transcript and reduce the expression of the target gene.

[0107] In some embodiments, the nucleic acid contact moiety can be inactivated with respect to its enzymatic cleavage activity (e.g., inactivated Cas9 or dCas9). In such scenarios, the nucleic acid contact moiety can be operably bound to a transcription regulator, such as a transcription activator or transcription repressor. For example, the nucleic acid contact moiety can be covalently bound to a transcription activator or transcription repressor, which can be recruited to the target gene by the nucleic acid contact moiety, which is compounded with at least one guide nucleic acid and the nucleic acid sequence of the target gene. Recruitment of the transcription activator or transcription repressor to the target gene may result in transcriptional activation or repression of the target gene.

[0108] In some embodiments, the nucleic acid contact moiety comprises a ribonucleic acid interference (RNAi) nucleic acid. For example, the nucleic acid contact moiety may be a target gene or an siRNA or antisense oligonucleotide for binding to a transcript of the target gene. In some embodiments, the nucleic acid contact moiety comprises a nuclease, such as an endonuclease (e.g., a heterologous endonuclease). In some embodiments, the nuclease may be a restriction enzyme. Suitable nucleases include, but are not limited to, CRISPR-related (Cas) proteins or Cas nucleases comprising type I CRISPR-related (Cas) polypeptides, type II CRISPR-related (Cas) polypeptides, type III CRISPR-related (Cas) polypeptides, type IV CRISPR-related (Cas) polypeptides, type V CRISPR-related (Cas) polypeptides, and type VI CRISPR-related (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-related RNA-binding proteins; recombinases; flippases; transposases; Argonaut (Ago) proteins (e.g., prokaryotic Argonaut (pAgo), archaeal Argonaut (aAgo), and eukaryotic Argonaut (eAgo)); any derivatives thereof; any variants thereof and any fragments thereof.

[0109] In some embodiments, the nucleic acid contact moiety includes a DNA nuclease, an example of which is a nuclease-deficient, engineered (e.g., programmable or targetable) DNA nuclease. In some embodiments, the nucleic acid contact moiety includes a nuclease-null DNA-binding protein derived from a DNA nuclease that does not induce transcriptional activation or repression of a target DNA sequence, unless it is present in the complex with one or more heterogeneous effectors of the Disclosure. In some embodiments, the nucleic acid contact moiety includes a nuclease-null DNA-binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence (e.g., which may be altered or enhanced by the presence of heterogeneous effectors of the Disclosure).

[0110] In some embodiments, the nucleic acid contact moiety includes an RNA nuclease, an example of which is an engineered (e.g., programmable or targetable) RNA nuclease. In some embodiments, the nucleic acid contact moiety includes a nuclease-null RNA-binding protein derived from an RNA nuclease that does not induce transcriptional activation or repression of a target RNA sequence, unless it is present in the complex with one or more heterogeneous effectors of this disclosure. In some embodiments, the nucleic acid contact moiety includes a nuclease-null RNA-binding protein derived from an RNA nuclease that can induce transcriptional activation or repression of a target RNA sequence (e.g., which may be altered or enhanced by the presence of heterogeneous effectors of this disclosure).

[0111] In some embodiments, the nucleic acid contact portion includes a nucleic acid-guided targeting system. In some embodiments, the nucleic acid contact portion includes a DNA-guided targeting system. In some embodiments, the nucleic acid contact portion includes an RNA-guided targeting system. The nucleic acid-guided targeting system may include, and can utilize, at least one guide nucleic acid described herein that facilitates the specific binding of a CRISPR-Cas system (e.g., its nuclease-deficient forms, such as dCas9 or dCas14) to a target gene (e.g., a target endogenous gene) or a target gene regulatory sequence. Binding specificity can be determined by the use of a guide nucleic acid, such as a single guide RNA (sgRNA) or a portion thereof. In some embodiments, by using different sgRNAs, the compositions and methods of this disclosure can be used with (e.g., as targets to) different target genes (e.g., target endogenous genes) or target gene regulatory sequences.

[0112] Prokaryotic CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associates) systems, such as class II CRISPR-Cas systems (Cas9 and Cpfl, etc.), can be reused in the compositions and methods of this disclosure as tools for gene expression, epigenome editing, and chromatin loop regulation. Nuclease-inactivated Cas (dCas) proteins conjugated with heterologous gene effectors can enable the regulation of the expression of target genes (such as target endogenous genes) adjacent to the dCas-binding site.

[0113] In some embodiments, the nucleic acid contact moiety comprises a CRISPR-associated (Cas) protein or Cas nuclease that functions in a non-natural CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) system. In bacteria, this system can provide adaptive immunity against foreign DNA.

[0114] In a wide variety of organisms, including diverse mammals, animals, plants, microorganisms, and yeasts, the CRISPR / Cas system (e.g., modified and / or unmodified) can be used as a genome manipulation tool, or modified to guide the manipulated protein to specifically bind to a target gene locus, as disclosed herein. The CRISPR / Cas system may include guide nucleic acids as described herein, examples of which include guide RNA (gRNA) compounded with a Cas protein for targeted regulation of gene expression and / or activity or nucleic acid binding. RNA-guided Cas proteins (e.g., Cas nucleases such as Cas9 nuclease) can specifically bind to a target polynucleotide (e.g., DNA) in a sequence-dependent manner. If the Cas protein has nuclease activity, it can cleave DNA.

[0115] In some cases, Cas proteins may mutate and / or modify, producing nuclease-deficient proteins or proteins with reduced nuclease activity compared to wild-type Cas proteins. Nuclease-deficient proteins may retain their ability to bind DNA, but may lack or have reduced nucleic acid cleavage activity.

[0116] In some embodiments, the nucleic acid contact moiety includes a Cas protein that forms a complex with a guide nucleic acid (such as a guide RNA or a portion thereof). In some embodiments, the nucleic acid contact moiety includes a Cas protein that forms a complex with a single guide nucleic acid (such as a single guide RNA (sgRNA)). In some embodiments, the nucleic acid contact moiety includes an RNA-binding protein (RBP) optionally complexed with a guide nucleic acid (such as a guide RNA (e.g., sgRNA) that can form a complex with a Cas protein). In some embodiments, the nucleic acid contact moiety includes a nuclease-null DNA-binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the nucleic acid contact moiety includes an RNA-derived nuclease-null RNA-binding protein.

[0117] Any suitable CRISPR / Cas system may be used herein. CRISPR / Cas systems can be referred to using various nomenclature systems. A CRISPR / Cas system may be a Type I, Type II, Type III, Type IV, Type V, Type VI system, or any other suitable CRISPR / Cas system. The CRISPR / Cas systems used herein may be Class 1, Class 2, or any other appropriately classified CRISPR / Cas system. The determination of Class 1 or Class 2 can be based on the gene encoding the effector module. Class 1 systems generally have a crRNA-effector complex of multiple subunits, while Class 2 systems generally have a single protein (e.g., Cas9, Cpfl, C2c1, C2c2, C2c3) or a crRNA-effector complex. Class 1 CRISPR / Cas systems can utilize a complex of multiple Cas proteins to enable regulation. Class 1 CRISPR / Cas systems can include, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, IIIC, IIID), and type IV (e.g., IV, IVA, IVB) CRISPR / Cas types. Class 2 CRISPR / Cas systems can enable regulation using a single large Cas protein. Class 2 CRISPR / Cas systems can include, for example, type II (e.g., II, IIA, IIB) and type V CRISPR / Cas types. CRISPR systems may be complementary to each other and / or functional units may be trans-lent to facilitate targeting of CRISPR loci.

[0118] If the nucleic acid contact region can contain a Cas protein or a derivative thereof, the Cas protein or its derivative may be a Class 1 or Class 2 Cas protein. A Cas protein can be a Type I, Type II, Type III, Type IV, Type V Cas protein, or Type VI Cas protein. A Cas protein may contain one or more domains. Non-limiting examples of domains include guide nucleic acid recognition and / or binding domains, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domains, RNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. Guide nucleic acid recognition and / or binding domains may interact with the guide nucleic acid. Nuclease domains may contain catalytic activity for nucleic acid cleavage. Nuclease domains may lack catalytic activity to prevent nucleic acid cleavage. A Cas protein may be a chimeric Cas protein or a fragment thereof fused to another protein or polypeptide. A Cas protein may be a chimera of various Cas proteins, for example, it may contain domains of different Cas proteins.

[0119] Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e(CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas 9(Csnl or Csx12), Cas10, CaslOd, Cas10, CaslOd, CasF, CasG, CasH, Cpfl, Csyl, Csy2, Csy3, Csel(CasA), Cse2(CasB), Cse3(CasE), Cse4(CasC), Cscl, Csc2, Cs This includes a5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csfl, Csf2, Csf3, Csf4, Cul966, Cas13a, Cas13b, Cas13c, Cas13d, Cas13X, Cas13Y, Cas14 (for example, Cas14 variants, such as Cas14a, Cas14b, Cas14c, etc.), and their homologs or modified versions.

[0120] The Cas protein or its fragments or derivatives may originate from any suitable organism. Non-limiting examples include: Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus species, Staphylococcus aureus, Nocardiopsis dasonvillei, Streptomyces pristinea espiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocardarius, Pseudomycoides, Bacillus serenityredens, Exigova Cterium sibiricum, Lactobacillus delbrücki, Lactobacillus salivarius, Microsilla marina, Burkholderia bacterium, Polaromonas naphthalenivorance, Polaromonas species, Crocosphaera watsonyi, Cyanoseis species, Microcystis erginosa, Pseudomonas erginosa, Synechococcus species, Acethalobium arabicicum, Ammonifex degensii, Caldicerulosylpter vesii, Candidetus desalhordis, Clostridium botulinum, Clostridium difficile, Finegordia magna, Natroanaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus cardus, Acidithiobacillus ferrooxydance, Allochromatium binosum, Malinobacter species, Nitrosococcus halophilus, Nitrosococcus watsonyi, Pseudoalteromonas haloplanc This includes *Tystis*, *Ctedonobacter racemifer*, *Methanohalobium ebestigatum*, *Anabaena variabilis*, *Nodularia spumigena*, *Nostoc*, *Arthrospira maxima*, *Arthrospira pratensis*, *Arthrospira*, *Ringbia*, *Microcoleus xonoplastes*, *Oschilatoria*, *Petrotoga mobilis*, *Thermosispho africanus*, *Acaryochloris marina*, *Leptototricia shahii*, and *Francisella novicida*. In some embodiments, this organism is *Streptococcus pyogenes*. In some embodiments, this organism is *Staphylococcus aureus*.In some aspects, this organism is *Streptococcus thermophilus*.

[0121] Cas proteins can originate from various bacterial species, including Veillonella atypical, Fusobacterium nucleatum, Philifacter allosis, Solobacterium moolei, Coprococcus catus, Treponema denticola, Peptoniphyllus dueldenii, Catenibacterium mitsokai, Streptococcus mutans, Listeria inocure, Staphylococcus pseudointermedius, Acidaminococcus intestin, Orsenella uli, Oenococcus kitaharae, and Bifidobacterium Bacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegordia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma obipneumoniae, Mycoplasma canis, Mycoplasma sinobie, Eubacterium rectale, Thermophyllus streptococcus, Eubacterium dolichum, Lactobacillus coliniformis subspecies torucens, Iliobacter polytropus, Ruminococcus albus, Ackermansia muciniphylla, Acidothermus ceruloricicus Bifidobacterium longum, Bifidobacterium denthium, Corynebacterium diphtheriae, Elusimicrobium minutum, Nitratifractor sarsuginis, Spheroketa clobus, Fibrobacter succinogenes subspecies succinogenes, Bacteroides fragilis, Capnositophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella luminicola, Flavobacterium columnare, Aminomonas pausivorans, Rhodospirillum labrum, Candidatus puniseispirina Mu marinum, Verminephrobacter eiseniae, Ralstonia sizgii, Dinoroseobacter sibae, Azospirillum, Nitrobacter hamburgensis, Brasirhizobium, Worinera succinogenes, Campylobacter jejuni subspecies jejuni, Helicobacter mustelae, Bacillus cereus, Acidoborax ebreus, Clostridium perfringens, Parvibacrum labmentivorance, Rosebria intestinalis, Meningococcus, Pasteurella multofida subspecies multofida, Stella wazwartensis,This includes, but is not limited to, Proteobacterium, Legionella pneumophila, Parasterella exclementihominis, Vorineella succinogenes, and Francisella novicida.

[0122] The Cas proteins used herein may be wild-type or modified forms of the Cas protein. A Cas protein may be an active variant, an inactive variant, or a fragment of a wild-type or modified Cas protein. Compared to the wild-type version of the Cas protein (e.g., the wild-type version of Cas14), a Cas protein may contain amino acid changes, including deletions, insertions, substitutions, mutations, variants, fusions, chimeras, or any combination thereof. A Cas protein may be a polypeptide having at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity with the wild-type Cas protein. The Cas protein may be a polypeptide having up to approximately 5%, up to approximately 10%, up to approximately 20%, up to approximately 30%, up to approximately 40%, up to approximately 50%, up to approximately 60%, up to approximately 70%, up to approximately 80%, up to approximately 90%, or up to approximately 100% sequence identity and / or similarity with the wild-type exemplary Cas protein. The variant or fragment may contain at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity or similarity with the wild-type or modified Cas protein or its portion. A variant or fragment can target a nucleic acid locus when combined with a guide nucleic acid, but on the other hand, it lacks nucleic acid cleavage activity.

[0123] Cas proteins can contain one or more nuclease domains, such as a DNase domain. For example, the Cas9 protein can contain a RuvC-like nuclease domain and / or an HNH-like 20 nuclease domain. In the nuclease-active form of Cas9, the RuvC and HNH domains can each cleave different double-stranded DNA, causing double-strand breaks in the DNA. Cas proteins can contain only one nuclease domain (for example, Cpfl contains a RuvC domain but lacks an HNH domain). In some embodiments, no nuclease domains are present. In some embodiments, nuclease domains are present but inactive, or their activity is reduced or minimal. In some embodiments, nuclease domains are present and active.

[0124] Deleting or mutating one or more nuclease domains (e.g., RuvC, HNH) of a Cas protein may render the nuclease domain non-functional or reduce its nuclease activity. For example, in a Cas protein containing at least two nuclease domains (e.g., Cas9), deletion or mutation of one of the nuclease domains may result in the resulting Cas protein (known as nickase) being able to produce single-strand breaks at the CRISPR RNA (crRNA) recognition sequence in double-stranded DNA, but not double-strand breaks. Such a nickase may be able to cleave either the complementary or non-complementary strand, but not both. Deleting or mutating all nuclease domains of a Cas protein (e.g., both the RuvC and HNH nuclease domains of the Cas9 protein, and the RuvC nuclease domain of the Cpfl protein) may result in the resulting Cas protein having a reduced or complete inability to cleave both strands of double-stranded DNA. An example of a mutation that can convert the Cas9 protein to nickase is the D10A mutation in the RuvC domain of Cas9 from Streptococcus pyogenes (where aspartic acid at position 10 of Cas9 is changed to alanine). H939A (where histidine at amino acid position 839 is changed to alanine) or H840A (where histidine at amino acid position 840 is changed to alanine) in the HNH domain of Cas9 from Streptococcus pyogenes can convert Cas9 to nickase. Examples of mutations that can convert the Cas9 protein to inactive Cas9 are the D10A mutation in the RuvC domain (where aspartic acid at position 10 of Cas9 is changed to alanine), and the H939A mutation (where histidine at amino acid position 839 is changed to alanine) or H840A mutation (where histidine at amino acid position 840 is changed to alanine) in the HNH domain of Cas9 from Streptococcus pyogenes.

[0125] Nuclease-inactive Cas proteins may contain one or more mutations compared to the wild-type version of the protein. Mutations may result in the nucleic acid cleavage activity of one or more of the nucleic acid cleavage domains of the wild-type Cas protein being reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% or less. Mutations may result in one or more of the nucleic acid cleavage domains retaining the ability to cleave the complementary strand of the target nucleic acid while reducing their ability to cleave the non-complementary strand. Mutations may result in one or more of the nucleic acid cleavage domains retaining the ability to cleave the non-complementary strand of the target nucleic acid while reducing their ability to cleave the complementary strand. Mutations may result in one or more of the nucleic acid cleavage domains lacking the ability to cleave both the complementary and non-complementary strands of the target nucleic acid. The residues to be mutated within the nuclease domain can correspond to one or more catalytic residues of the nuclease. For example, mutating residues in the wild-type exemplary Streptococcus pyogenes Cas9 polypeptide (such as Asp10, His840, Asn854, and Asn856) can inactivate one or more of the nucleic acid cleavage domains (e.g., nuclease domains). The residues to be mutated within the nuclease domain of the Cas protein may correspond to Asp10, His840, Asn854, and Asn856 in the wild-type Streptococcus pyogenes Cas9 polypeptide, as determined, for example, by sequence and / or structural alignment.

[0126] The Cas protein may contain amino acid sequences that have at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity with the nuclease domain (e.g., RuvC domain, HNH domain) of the wild-type Cas protein.

[0127] Cas proteins, their variants, or derivatives can be modified, for example, as part of a complex disclosed herein, to enhance the regulation of gene expression by the compositions and methods of this disclosure. Cas proteins can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, enzyme activity, and / or binding to other factors (such as heterodimerizing or oligomerizing domains) to induce ligands. Cas proteins can also be modified to alter any other activity or property of the protein (such as stability). For example, one or more nuclease domains of a Cas protein can be modified, deleted, or inactivated, or a Cas protein can be cleaved to remove domains that are not essential for the desired function of the protein or complex. Cas proteins can be modified to modulate (e.g., enhance or decrease) the activity of Cas proteins that regulate gene expression by a complex of this disclosure including heterogeneous gene effectors.

[0128] For example, Cas proteins can be bound (e.g., by fusion, covalent, or non-covalent) to heterogeneous gene effectors (e.g., epigenetic modification domains, transcriptional activation domains, and / or transcriptional repressor domains). Cas proteins can be bound (e.g., by fusion, covalent, or non-covalent) to oligomerization or dimerization domains (e.g., heterodimerization domains) disclosed herein. Cas proteins can be bound (e.g., by fusion, covalent, or non-covalent) to heterogeneous polypeptides that improve or decrease their stability. Cas proteins can be bound (e.g., by fusion, covalent, or non-covalent) to sequences (e.g., degrons) that can promote the degradation of Cas proteins or complexes containing Cas proteins, examples of which include inducible degrons (e.g., auxin-inducible).

[0129] The Cas protein can be a fusion protein (for example, a fusion protein comprising the Cas protein and one or more of the partners disclosed herein). The fusion domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or internally within the Cas protein.

[0130] A partner of the Cas protein (e.g., covalently or noncovalently bound to the dCas protein as disclosed herein) can be a transcription effector (e.g., a transcription activator or transcription repressor). As provided herein, the transcription effector can be heterogeneous to the cell.

[0131] In some embodiments, the transcription effector may be a histone epigenetic modifier (or histone modifier). In some cases, a histone epigenetic modifier can modulate histones through methylation (e.g., histone methylation modifiers, amino acid methyltransferases, e.g., KRAB). In some cases, a histone epigenetic modifier can modulate histones through acetylation. In some cases, a histone epigenetic modifier can modulate histones through phosphorylation. In some cases, a histone epigenetic modifier can modulate histones through ADP-ribosylation. In some cases, a histone epigenetic modifier can modulate histones through glycosylation. In some cases, a histone epigenetic modifier can modulate histones through SUMOylation. In some cases, a histone epigenetic modifier can modulate histones through ubiquitination. In some cases, a histone epigenetic modifier can modulate histones by remodeling the histone structure, for example, via ATP hydrolysis-dependent treatment.

[0132] In some embodiments, transcription effectors can be gene epigenetic modifiers (or gene modifiers). In some cases, gene modifiers can regulate genes through methylation (e.g., gene methylation modifiers, such as DNA methyltransferases or DNMTs). In some cases, gene modifiers can regulate genes through acetylation.

[0133] In some embodiments, the transcription effector is derived from the family of the relevant histone acetyltransferase. Non-limiting examples of histone acetyltransferases include the GNAT subfamily, MYST subfamily, p300 / CBP subfamily, HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0134] In some embodiments, the transcription effector is derived from histone lysine methyltransferase. Non-limiting examples of histone lysine methyltransferase include the EZH subfamily, non-SET subfamily, other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, and PRDM1 Includes 2, PRDM13, PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4 and SYMD5.

[0135] Examples of proteins (or fragments thereof) that can be used as fusion partners to increase transcription include transcription activators (examples include VP16, VP64, VP48, VP160, p65 subdomains (e.g., derived from NFκB), and the activation domain and / or TAL activation domain of EDLL (e.g., for plant activity)); histone lysine methyltransferases (examples include SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, etc.); histone lysine demethylases (examples include JHDM2a / b, UTX, JMJD3, etc.); histone acetyltransferases (examples include GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI) 60, CLOCK, etc.; and DNA demethylases (examples include, but are not limited to, 10-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, or combinations thereof).

[0136] Examples of proteins (or fragments thereof) that can be used as fusion partners to reduce transcription include transcriptional repressors (examples include Kruppel association boxes (KRAB or SKD)); KOX1 repressor domains; Mad mSIN3 interaction domains (SID); ERF repressor domains (ERD), SRDX repressor domains (e.g., for repression in plants); histone lysine methyltransferases (examples include Pr-SET7 / 8, SUV4-20H1, RIZ1, etc.); histone lysine demethylases (examples include JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD1A / RBP2, JARID1B / PLU-1, JARID This includes, but is not limited to, 1C / SMCX, JARIDID / SMCY, histone lysine deacetylases (examples include HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.), DNA methylases (examples include Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), etc.), and peripheral recruitment elements (examples include lamin A, lamin B, or combinations thereof).

[0137] The Cas protein can be provided in any form. For example, the Cas protein can be provided in protein form (e.g., as the Cas protein alone, or in combination with a guide nucleic acid as a ribonucleoprotein). The Cas protein can be provided in a complex, for example, with a guide nucleic acid and / or one or more heterologous gene effectors of this disclosure. The Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA. The nucleic acid encoding the Cas protein can be codon-optimized for efficient translation into the protein in a particular cell or organism.

[0138] In some embodiments, the Cas protein, its variant, or derivative is a nuclease-inactive Cas (dCas) protein. An inactive Cas protein can be a protein lacking nucleic acid cleavage activity.

[0139] Cas proteins can include modified forms of wild-type Cas proteins. Modified forms of wild-type Cas proteins can include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the Cas protein. For example, modified forms of Cas proteins may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of wild-type Cas proteins (e.g., Cas9 from Streptococcus pyogenes). Modified forms of Cas proteins may have virtually no nucleic acid cleavage activity. When a Cas protein is a modified form that has virtually no nucleic acid cleavage activity, it is sometimes called enzymatically inactive, "inactivated," and / or "inactive form" (abbreviated as "d"). Inactive Cas proteins (e.g., dCas, dCas9, dCas14) can bind to target polynucleotides but may not cleave the target polynucleotides at all, or only cleave them minimally. In some embodiments, the inactive Cas protein is the inactive Cas14 protein.

[0140] A dCas polypeptide (e.g., dCas14 polypeptide), in combination with, for example, heterogeneous gene effectors (may be multiple) disclosed herein, can associate with a single guide RNA (sgRNA) to activate or repress the transcription of a target gene (e.g., a target endogenous gene). The sgRNA can be introduced into cells expressing Cas or a variant thereof, as provided herein. In some cases, such cells may contain one or more different sgRNAs targeting the same target gene (e.g., a target endogenous gene) or a target gene regulatory sequence. In other cases, the sgRNAs target different nucleic acids within the cell (e.g., different target genes, different target gene regulatory sequences, or different sequences within the same target gene or target gene regulatory sequence).

[0141] Enzymatically inactive may refer to a nuclease that can bind to a nucleic acid sequence within a polynucleotide in a sequence-specific manner but does not cleave the target polynucleotide, or cleaves it at a significantly lower frequency. The enzymatically inactive guide region may contain an enzymatically inactive domain (e.g., a nuclease domain). Enzymatically inactive may refer to a complete lack of activity. Enzymatically inactive may refer to substantially no activity. Enzymatically inactive may refer to essentially no activity. Enzymatically inactive may refer to activity of less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% compared to the activity of an equivalent wild-type enzyme.

[0142] In some embodiments, the nucleic acid contact moieties disclosed herein do not include a nucleic acid-guided targeting system. For example, the nucleic acid contact moieties may include proteins that bind to a target gene (e.g., a target endogenous gene) or a target gene regulatory sequence based on protein structural features (such as certain nucleases disclosed herein).

[0143] In some embodiments, the enucleated cells include additional therapeutic agents, examples of which are disclosed herein. In some embodiments, the composition containing the enucleated cells is formulated for administration to a subject disclosed herein together with the additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject sequentially, simultaneously, substantially sequentially, or substantially simultaneously.

[0144] (e) Pharmaceutical preparations In some embodiments, pharmaceutical formulations and / or compositions comprising the compositions disclosed herein and pharmaceutically acceptable carriers, excipients, diluents, or spray inhalants are disclosed herein. In some embodiments, the pharmaceutical formulations are in unit dose form. In some embodiments, the compositions disclosed herein comprise one or more activators or therapeutic agents.

[0145] In some embodiments, the composition comprises two or more activators or two or more therapeutic agents disclosed herein. In some embodiments, the two or more activators are contained in a single dosing unit, for example, when the enucleated cells contain two or more therapeutic agents. In embodiments, the two or more activators are contained in separate dosing units, for example, when the enucleated cells are administered separately from additional therapeutic agents or adjuvants. In some embodiments, the pharmaceutical composition described herein comprises at least one additional activator other than the enucleated cells described herein. In some embodiments, the at least one additional activator is a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormone agent, an anti-angiogenic agent, a cardioprotective agent, and / or a checkpoint inhibitor.Non-restrictive checkpoint inhibitors include IMP321 / eftilagimod alfa (Imtech), relatrimab BMS-986016, ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), semiplimab (Ributayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), ipilimumab (Yervoy), LAG525, MK-4280, irinotecan, oxaliplatin, REGN3767, TSR-033, and BI. 754111, Sym022, FS118 (bispecific anti-LAG3 / PD-L1 antagonist mAb), MGD013 (bispecific anti-LAG3 / PD-1 antagonist mAb), TSR-022, niraparib, bevacizumab, MBG453, decitabine, spartalizumab, Sym023, INCAGN2390, LY3321367, ramucirumab, abemaciclib, merestinib, BMS-986258, SHR-1702, camrelizumab, MK-7684, etigirimab / OMP-313 M32, Tiragolumab / MTIG7192A / RG-6058, BMS-986207, AB-154, ASP-8374, JNJ-61610588, CA-170d, Enobrituzumab / MGA271, MGD009, I-8H9 / Ombrutamab, Trastuzumab, MGD013 (Anti-PD-1, Anti-LAG-3 Dual Checkpoint Inhibitor), BGB-A1217, CM-24 (MK-6018), BMS This includes 986178, MEDI6469, PF-04518600, GSK3174998, MOXR0916, utomirimab (PF-05082566), urerumab (BMS-663513)ES101, BMS-986156, TRX-518, AMG 228, JTX-2011, GSK3359609, BMS-986226, MEDI-570, or valrirumab (CDX-1127). Such compounds or drugs may be present in combination in amounts effective for the intended purpose.Further non-limiting examples of additional therapeutic agents include CPI-006 (to inhibit CD73 and enable T cell and APC activation); monalizumab (to inhibit NKG2A); COM701 (to inhibit PVRIG / PVRL2 and enable T cell activation); CM24 (to inhibit CEACAM1 and enable T cell and NK cell activation); NEO-201 (to inhibit CEACAM5 and CEACAM6, enabling T cell activation while inhibiting tumor cell growth); and defactinib (FAK (To inhibit and prevent tumor growth); PF-04136309 (To inhibit CCR-2 and CCL-2, enabling T cell recruitment and activation); MSC-1 (To inhibit LIF, enabling T cell and APC activation while preventing cancer growth); Hu5F9-G4 (5F9), ALX148, TTI-662 and RRx-001 (To inhibit CD47 or SIRPα, enabling T cell and APC activation); Lacunotuzumab (MCS-110), LY3022855, SNDX-6 352, emactuzumab (RG7155) and pexidartinib (PLX3397) (to inhibit M-CSF or CSF-1R and enable APC activation); CAN04 and canakinumab (ACZ885) (to inhibit IL-3 or IL-1RAP and enable T cell and APC activation); BMS-986253 (to inhibit IL-8 and reduce the immunosuppressive tumor microenvironment while hindering tumor growth); pepinemab (VX15 / 2503) (to inhibit SEMA4D and hinder tumor growth) This includes: reducing the immunosuppressive tumor microenvironment; trevananib (to inhibit angiopoietin-2 and enable APC activation while hindering cancer growth); FP-1305 (to inhibit CLEVER-1 and enable APC activation); enapotamab vedotin (EnaV) (to inhibit Axl and enable APC activation while hindering cancer growth); or bavituximab (to inhibit phosphatidylserine and enable T cell and APC activation while hindering cancer growth).

[0146] The composition may contain at least an exogenous therapeutic agent as an active ingredient in the form of a free acid or free base, or a pharmaceutically acceptable salt. Furthermore, the methods and compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds having the same type of activity. In some embodiments, the therapeutic agent exists in a non-solvated form or in a solvated form with a pharmaceutically acceptable solvent (such as water or ethanol). The solvated form of the therapeutic agent is also considered to be disclosed herein.

[0147] In certain embodiments, the compositions provided herein include one or more preservatives for inhibiting microbial activity. Suitable preservatives include mercury-containing substances (such as melfen and thimerosal), stabilized chlorine dioxide, and quaternary ammonium compounds (such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride).

[0148] In some embodiments, the compositions described herein benefit from antioxidants, metal chelating agents, thiol-containing compounds, and other generally stabilizing agents. Examples of such stabilizers include, but are not limited to, (a) glycerol at approximately 0.5% to 2% w / v, (b) methionine at approximately 0.1% to 1% w / v, (c) monothioglycerol at approximately 0.1% to 2% w / v, (d) EDTA at approximately 1 mM to 10 mM, (e) ascorbic acid at approximately 0.01% to 2% w / v, (f) polysorbate 80 at approximately 0.003% to 0.02% w / v, (g) polysorbate 20 at approximately 0.001% to 0.05% w / v, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparin analogs, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0149] In some embodiments, the pharmaceutical formulation is formulated for administration to a target by intrathecal, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or any combination thereof. In some embodiments, the pharmaceutical formulation is formulated for intravenous administration. In some embodiments, the pharmaceutical formulation further comprises at least one additional activator. In some embodiments, the at least one additional activator comprises cytokines, growth factors, hormones, enzymes, small molecules, compounds, or any combination thereof.

[0150] (f) Preparations for administration Formulations are disclosed herein. In some embodiments, the formulation comprises a plurality of enucleated cells formulated from a cryopreserved composition, the cryopreserved composition comprising a plurality of cryopreserved enucleated cells, at least a subset of the plurality of enucleated cells comprising (i) a therapeutic agent and (ii) intracellular organelles sufficient to release the therapeutic agent in vivo or ex vivo in an amount substantially equal to or greater than the amount of therapeutic agent released by otherwise identical, non-cryopreserved enucleated cells.

[0151] In some embodiments, multiple enucleated cells include a diameter that is approximately 70% or less of the average diameter of the nucleated parent cell. In some embodiments, multiple enucleated cells include a diameter that is approximately 1 micrometer (μm) to approximately 100 μm. In some embodiments, multiple enucleated cells include a diameter that is approximately 5 μm to approximately 25 μm. In some embodiments, multiple enucleated cells include a diameter that is approximately 8 μm.

[0152] In some embodiments, one or more structural features include one or more tunnel nanotubes. In some embodiments, the intracellular organelle includes the Golgi apparatus, the endoplasmic reticulum, or any combination thereof.

[0153] The compositions and / or formulations described herein may be formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled-release formulations, rapidly dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated tablets, capsules, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations, and mixed formulations of immediate-release and controlled-release. In one embodiment, the therapeutic agent discussed herein (e.g., therapeutic agent) may be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one embodiment, a formulation suitable for intramuscular, subcutaneous, or intravenous injection comprises a physiologically acceptable sterile aqueous or non-aqueous solution, a dispersion, a suspension or emulsion, and a sterile powder for rehydrating into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, and cremophor), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate fluidity may be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, or by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives, such as preservatives, humectants, emulsifiers, and distributors. Prevention of microbial growth may be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. In some cases, it is desirable to include isotonic agents such as sugars and sodium chloride. Absorption may be prolonged by the use of agents that delay the absorption of the injectable drug form (such as aluminum monostearate and gelatin).

[0154] For intravenous injection, infusion, or infusion, the compositions described herein are formulated in aqueous solutions, preferably physiologically compatible buffers (such as Hanks' solution, Ringer's solution, or saline buffer). For transmucosal administration, appropriate penetrating agents are used in the formulation to penetrate the barrier. Such penetrating agents are generally known in the art. For other parenteral injections, suitable formulations preferably comprise aqueous or non-aqueous solutions containing physiologically compatible buffers or excipients. Such excipients are known.

[0155] Parenteral injection may include bolus injection or continuous infusion. The injectable composition may be supplied in unit dosage forms, such as ampoules or multi-dose containers, with added preservatives. The compositions described herein may be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulation agents (such as suspending agents, stabilizers and / or dispersants). In one embodiment, the active ingredient is in powder form for preparation with a suitable vehicle (such as sterile pyrogen-free water) before use.

[0156] For inhalation administration, the therapeutic agent is formulated for use as an aerosol, mist, or powder. The pharmaceutical compositions described herein are conveniently delivered in the form of aerosol spray dispensing from a pressurized pack or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of pressurized aerosols, the dosage unit may be determined by providing a valve for dispensing a measured amount. Capsules and cartridges (e.g., gelatin, to be an example) for use in inhalers or blowers may be formulated containing a powder mixture of the therapeutic agent described herein and a suitable powder base (e.g., lactose or starch). Formulations containing the compositions are prepared as a solution in saline by employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizers or dispersants known in the art. Preferably, these compositions and formulations are prepared using suitable non-toxic and pharmaceutically acceptable ingredients. The selection of a suitable carrier depends on the exact properties of the desired nasal dosage form (e.g., solution, suspension, ointment, or gel). Generally, nasal dosage forms contain a large amount of water in addition to the active ingredient. Other components (such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents, or buffers, as well as other stabilizers and solubilizers) may be present in trace amounts, at their discretion. Preferably, the nasal dosage form needs to be isotonic with nasal secretions.

[0157] Oral pharmaceutical preparations are obtained by mixing one or more solid excipients with one or more of the compositions described herein, optionally grinding the resulting mixture, adding a suitable adjuvant if desired, and then processing the granular mixture to obtain tablets or sugar-coated tablet cores. Suitable excipients include, for example, fillers (such as sugars, including lactose, sucrose, mannitol, or sorbitol); cellulose preparations (e.g., corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.); or other materials (such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate). Optionally, disintegrants may be added, examples of which include cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or its salts (such as sodium alginate). In some embodiments, dyes or pigments are added to the tablet or sugar-coated tablet coating to identify or characterize different combinations of active therapeutic agent doses.

[0158] In some embodiments, the composition of the exogenous therapeutic agent is in the form of a capsule, which includes a push-in capsule made of gelatin, and a soft, sealed capsule made of gelatin and a plasticizer (such as glycerol or sorbitol). The push-in capsule contains the active ingredient mixed with a filler (such as lactose), a binder (such as starch), and / or a lubricant (such as talc or magnesium stearate), and optionally a stabilizer. In the soft capsule, the active therapeutic agent is dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, a stabilizer is added. For example, the capsule may be prepared by placing a bulk blend of the therapeutic formulation inside the capsule. In some embodiments, the formulation (non-aqueous suspension and solution) is placed in a soft gelatin capsule. In other embodiments, the formulation is placed in a standard gelatin capsule or a non-gelatin capsule (such as a capsule containing HPMC). In other embodiments, the formulation is placed in a dispensing capsule, which is either swallowed whole or the capsule is opened and the contents are dispensed onto food before consumption.

[0159] Compositions for oral administration are in a state of dosage suitable for such administration. In one embodiment, a solid oral dosage form is prepared by mixing the composition with one or more of the following: antioxidants, flavoring agents, and carrier materials (such as binders, suspensions, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents). In some embodiments, the solid dosage forms disclosed herein are in the form of tablets (including suspension tablets, fast-dissolving tablets, chewable disintegrating tablets, rapid disintegrating tablets, effervescent tablets, or caplets), pills, powders, capsules, solid dispersions, solid solutions, biodegradable dosage forms, controlled-release formulations, pulse-release formulations, multi-particle dosage forms, beads, pellets, and granules. In other embodiments, the composition is in the form of a powder. Compressed tablets are solid dosage forms prepared by compressing a bulk blend of the above formulations. In various embodiments, the tablets contain one or more flavoring agents. In other embodiments, the tablets contain a film surrounding the finally compressed tablet. In some embodiments, the film coating may provide delayed release of the therapeutic agent from the formulation. In other embodiments, the film coating assists patient compliance. The film coating may range from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms (e.g., tablets, effervescent tablets, and capsules) are prepared by mixing particles of the therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into unit dosage forms (tablets, pills, and capsules, etc.) with equivalent effects. In some embodiments, each unit dose includes the film coating.

[0160] In another embodiment, the dosage form includes microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulated material. Non-limiting examples of materials include pH adjusters, erosion accelerators, defoamers, antioxidants, flavoring agents, and carrier materials (such as binders, suspenders, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents).

[0161] The dosage form of an orally administered liquid formulation is optionally an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form optionally contains additives, examples of which include (a) disintegrants, (b) dispersants, (c) wetting agents, (d) at least one preservative, (e) viscosity enhancers, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion further contains a crystal formation inhibitor.

[0162] In some embodiments, the compositions described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of immiscible phases in another phase, usually existing in the form of droplets. Generally, emulsions are produced by strong mechanical dispersion. SEDDS, in contrast to emulsions or microemulsifies, spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to disperse the droplets throughout the solution. Furthermore, the stability of unstable or hydrophobic active ingredients is ensured by optionally adding water or an aqueous phase immediately before administration. Thus, SEDDS provide an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS result in improved bioavailability of hydrophobic active ingredients.

[0163] The compositions described herein (e.g., pharmaceutical compositions) may be formulated for administration to a subject via a route of administration, including, but not limited to, intravenous, intra-arterial, oral, parenteral, oral, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal routes of administration. The compositions described herein may include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly dissolving formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations, and mixed formulations of immediate-release and controlled-release.

[0164] Oral formulations are administered using a variety of formulations known in the art. Furthermore, the oral dosage forms described herein may further include a biodegradable (hydrolyzable) polymer carrier that also serves to adhere the dosage form to the buccal mucosa. For oral or sublingual administration, the composition may take the form of a tablet, lozenge, or gel formulated in a preferred manner.

[0165] For intravenous injection, the composition is optionally formulated as an aqueous solution, preferably as a physiologically compatible buffer (such as Hanks' solution, Ringer's solution, or saline buffer). For transmucosal administration, an appropriate penetrating agent is used in the formulation to penetrate the barrier. For other parenteral injections, the appropriate formulation preferably comprises an aqueous or non-aqueous solution containing a physiologically compatible buffer or excipient.

[0166] Parenteral injection may optionally involve bolus injection or continuous infusion. The injectable formulation may optionally be supplied in unit dosage forms, such as ampoules or multi-dose containers, with the addition of preservatives. In some embodiments, the compositions described herein are suitable for parenteral injection as sterile suspensions, solutions, or emulsions in oily or aqueous vehicles and contain formulation agents (such as suspending agents, stabilizers, and / or dispersants). The parenteral administration composition comprises an aqueous solution of an agent that modulates carotid body activity in an aqueous form. Furthermore, a suspension of the agent that modulates carotid body activity may optionally be prepared as needed (e.g., an oily injection suspension).

[0167] Suitable formulation techniques include, for example, one or a combination of the following methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., Ulster coating), tangential coating, top spray, tableting, extrusion molding, etc.

[0168] In some embodiments, compositions are provided for oral administration to a subject, comprising a therapeutic agent particle and at least one dispersant or suspension agent. The formulation may be a powder and / or granule for suspension, which, when mixed with water, yields a substantially homogeneous suspension.

[0169] Furthermore, the composition optionally includes one or more pH adjusters or buffers (including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid); bases (such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane); and buffers (such as citrate / dextrose, sodium bicarbonate, and ammonium chloride). Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0170] Furthermore, the composition optionally includes one or more salts in amounts necessary to bring the osmotic pressure of the composition within an acceptable range. Such salts include those having a sodium, potassium, or ammonium cation and an anion of chloride, citric acid, ascorbic acid, boric acid, phosphate, bicarbonate, sulfuric acid, thiosulfate, or bisulfite, and preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0171] Other compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances (such as melfen and thimerosal), stabilized chlorine dioxide, and quaternary ammonium compounds (such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride).

[0172] In one embodiment, the aqueous suspensions and dispersions described herein remain homogeneous for at least 4 hours. In one embodiment, the aqueous suspension is resuspended into a homogeneous suspension by physical stirring for less than 1 minute. In yet another embodiment, stirring is not required to maintain a homogeneous aqueous dispersion.

[0173] Nasal aerosol formulations are generally aqueous solutions designed to be administered into the nasal cavity as drops or sprays. Nasal solutions are generally isotonic and may be similar to nasal secretions in that they are slightly buffered to maintain a pH of approximately 5.5 to 6.5, although additionally, pH values ​​outside this range may be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0174] Aerosol formulations for inhalation and inhalation agents may be designed so that the agent, or combination of agents, is delivered to the target respiratory system when administered via the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer. Inhalations or blown-inhalations containing fine powder or liquid drugs may be delivered to the respiratory system as a pharmaceutical aerosol of the agent, or a solution or suspension of a combination of agents in a propellant, for example, to assist in dispersal. The propellant may be a liquefied gas, which may include halocarbons, such as fluorocarbons (fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons and hydrochlorocarbons, etc.), as well as hydrocarbons and hydrocarbon ethers.

[0175] Aerosol formulations may also contain other components (e.g., ethanol, isopropanol, propylene glycol), and even surfactants or other components (such as oils and detergents). These components may help stabilize the formulation and / or lubricate valve components.

[0176] Aerosol formulations may be packaged under pressure, and may be formulated as aerosols using solutions, suspensions, emulsions, powders, and semi-solid preparations. For example, a solution aerosol formulation comprises a solution of an agent (such as a transporter, carrier, or ion channel inhibitor) in a (substantially) pure propellant, or as a mixture of a propellant and a solvent. The solvent may be used to dissolve the agent and / or to slow the evaporation of the propellant. The solvent may include, for example, water, ethanol, and glycol. Any combination of suitable solvents may be used in optional combinations with preservatives, antioxidants, and / or other aerosol components.

[0177] Aerosol formulations may be dispersions or suspensions. A suspension aerosol formulation comprises a suspension of an agent, or a combination of agents (e.g., a transporter, a carrier, or an ion channel inhibitor), and a dispersant. The dispersant may include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin, and corn oil. A suspension aerosol formulation may also contain lubricants, preservatives, antioxidants, and / or other aerosol components.

[0178] Aerosol formulations may also be formulated as emulsions. Emulsified aerosol formulations may include, for example, an alcohol such as ethanol, a surfactant, water, and a propellant, or a combination of agents (e.g., a transporter, carrier, or ion channel). The surfactant used may be nonionic, anionic, or cationic. An example of an emulsion aerosol formulation includes, for example, ethanol, a surfactant, water, and a propellant. Another example of an emulsion aerosol formulation includes, for example, vegetable oil, glyceryl monostearate, and propane.

[0179] method In some embodiments, methods for producing or using compositions disclosed herein are disclosed herein. In some embodiments, the method includes a high-throughput technique for enucleated cells for producing compositions containing enucleated cells for biomedical applications with minimal residual nucleated parent cells. In some embodiments, the method is for cryopreserving enucleated cells. In some embodiments, cryopreserved enucleated cells release a certain amount or more of therapeutic agent compared to otherwise identical, non-cryopreserved enucleated cells.

[0180] In some embodiments, the method involves inducing the expression of a suicide gene under conditions suitable for killing residual nucleated parental cells in the composition. The methods disclosed herein also provide ways to use enucleated cells as fusion partners (e.g., fusion with other cells in vivo or ex vivo), or as therapeutic agent delivery vehicles, or a combination thereof.

[0181] In some embodiments, methods for producing enucleated cells described herein are disclosed, comprising enucleating nucleated parent cells. In some embodiments, parent cells may be treated with exogenous molecules to soften the cytoskeleton of the parent cells. For example, parent cells may be treated with cytochalasin to soften the cortical actin cytoskeleton. In some embodiments, the nuclei are physically extracted from the cell bodies by centrifugation to produce enucleated cells. In some embodiments, centrifugation involves the use of a density gradient, in which enucleated cells and intact nucleated cells precipitate into different layers, so that the enucleated cells are at least isolated. In some embodiments, centrifugation involves continuous flow centrifugation. Example 3 shows an exemplary continuous flow centrifugation experiment for obtaining enucleated cells from nucleated cells. In some embodiments, continuous flow centrifugation is fixed-angle centrifugation. In some embodiments, the use of continuous flow centrifugation increases the volume that can be centrifuged. For example, using continuous flow centrifugation increases the volume that can be centrifuged compared to swing-bucket centrifugation (to produce an equivalent density gradient). In some embodiments, centrifugation involves zone centrifugation, in which enucleated cells are separated from nucleated cells based on differences in size, mass, or a combination thereof. Example 2 illustrates a method for generating enucleated cells by zone centrifugation. In some embodiments, the method includes inducing cell death in nucleated cells after centrifugation or enucleation. For example, nucleated cells can be manipulated to have heterologous polynucleotides encoding heterologous gene products described herein, and the expression of the heterologous gene product induces cell death in at least one nucleated cell.

[0182] In some embodiments, a composition containing tens of millions of enucleated cells ("enucleated cell fraction") is obtained by the methods disclosed herein. In some embodiments, the composition also contains residual nucleated cells ("nucleated cell fraction"). In some embodiments, the composition is further processed to purify the enucleated cell fraction from the nucleated cell fraction. In some embodiments, the enucleated cell fraction is formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier, excipient, or diluent.

[0183] In some embodiments, the method for producing enucleated cells does not consist of, and does not involve, the differentiation of parental cells. For example, enucleated cells are not obtained by differentiating nucleated erythrocyte progenitor cells into differentiated enucleated erythrocytes. In some embodiments, enucleated cells are not terminally differentiated cells. In some embodiments, enucleated cells are not platelets. In some embodiments, enucleated cells are not obtained from platelet-derived cells. In some embodiments, enucleated cells are not erythrocytes. In some embodiments, enucleated cells are not obtained from erythrocyte-derived cells.

[0184] In some embodiments, a nucleus-containing parental cell is manipulated to express at least one of the therapeutic agents, transmembrane portions, immune-evading portions, or targeting portions described herein, and then the nucleus of the parental cell is removed. In some embodiments, a nucleus-containing parental cell is enucleated, and the enucleated cell is manipulated to express the therapeutic agent, transmembrane portion, immune-evading portion, or targeting portion described herein. In some embodiments, the parental cell is manipulated to express one or more of the above biomolecules (e.g., immune-evading portions and / or targeting portions), and the resulting enucleated cell (e.g., already expressing immune-evading portions and / or targeting portions) is further manipulated to express a second of the above biomolecules (e.g., a therapeutic agent). In this form, the enucleated cells of the Disclosure can be extensively manipulated before enucleation, can be stored for extended periods as needed (e.g., by lyophilization, cryopreservation, or freezing), and can be rapidly manipulated to express the therapeutic agent when the time is approaching.

[0185] In some embodiments, the composition has a volume containing about 10 milliliters (mL) or more to about 10,000 mL. In some embodiments, the composition has volumes of about 10 mL or more to about 100 mL, about 10 mL or more to about 1,000 mL, about 10 mL or more to about 2,000 mL, about 10 mL or more to about 3,000 mL, about 10 mL or more to about 4,000 mL, about 10 mL or more to about 5,000 mL, about 10 mL or more to about 6,000 mL, about 10 mL or more to about 7,000 mL, about 10 mL or more to about 8,000 mL, about 10 mL or more to about 9,000 mL, about 10 mL or more to about 10,000 mL, about 100 mL or more to about 1,000 mL, and about 100 mL or more to about 2,000 mL. 0mL, about 100mL or more - about 3,000mL, about 100mL or more - about 4,000mL, about 100mL or more - about 5,000mL, about 100mL or more - about 6,000mL, about 100mL or more - about 7,000mL, about 100mL or more - about 8,000mL, about 100m L or more ~ ​​approx. 9,000 mL, approx. 100 mL or more ~ ​​approx. 10,000 mL, approx. 1,000 mL or more ~ ​​approx. 2,000 mL, approx. mL or more ~ ​​approximately 6,000mL, approximately 1,000mL or more ~ ​​approximately 7,000mL, approximately 1,000mL or more ~ ​​approximately 8,000mL, approximately 1,000mL or more ~ ​​approximately 9,000mL, approximately 1,000mL or more ~ ​​approximately 10,000mL, approximately 2,000mL or more ~ ​​approximately 3,000mL, approximately 2 ,000mL or more to approx. 4,000mL, approx. 2,000mL or more to approx. 5,000mL, approx. 2,000mL or more to approx. 6,000mL, approx. 2,000mL or more to approx. 7,000mL, approx. Approximately 2,000mL or more ~ ​​approx. 10,000mL, approx. 3,000mL or more ~ ​​approx. 4,000mL, approx. 3,000mL or more ~ ​​approx. 5,000mL, approx. 3,000mL or more ~ ​​approx. 6,000mL, approx. 3,000mL or more ~ ​​approx. 7,000mL, approx. 3,000mL or more ~ ​​approx. mL, approximately 3,000 mL or more ~ ​​approximately 9,000 mL, approximately 3,000 mL or more ~ ​​approximately 10,000 mL, approximately 4,000 mL or more ~ ​​approximately 5,000 mL, approximately 4,000 mL or more ~ ​​approximately 6,000 mL, approximately 4,000 mL or more ~ ​​approximately 7,000 mL, approximately 4,000 mL or more ~ ​​approximately 8,000mL, approx. 4,000mL or more ~ ​​approx. 9,000mL, approx. 4,000mL or more ~ ​​approx. 10,000mL, approx. 5,000mL or more ~ ​​approx. 6,000mL, approx. 00mL or more ~ ​​approximately 8,000mL, approximately 5,000mL or more ~ ​​approximately 9,000mL, approximately 5,000mL or more ~ ​​approximately 10,000mL, approximately 6,000mL or more ~ ​​approximately 7,000mL, approximately 6,000mL or more ~ ​​approximately 8, Having a volume containing 000 mL, approximately 6,000 mL or more to approximately 9,000 mL, approximately 6,000 mL or more to approximately 10,000 mL, approximately 7,000 mL or more to approximately 8,000 mL, approximately 7,000 mL or more to approximately 9,000 mL, approximately 7,000 mL or more to approximately 10,000 mL, approximately 8,000 mL or more to approximately 9,000 mL, approximately 8,000 mL or more to approximately 10,000 mL, or approximately 9,000 mL or more to approximately 10,000 mL. In some embodiments, the composition has a volume containing about 10 mL or more, about 100 mL or more, about 1,000 mL or more, about 2,000 mL or more, about 3,000 mL or more, about 4,000 mL or more, about 5,000 mL or more, about 6,000 mL or more, about 7,000 mL or more, about 8,000 mL or more, about 9,000 mL or more, or about 10,000 mL or more. In some embodiments, the composition has a volume containing at least about 10 mL or more, about 100 mL or more, about 1,000 mL or more, about 2,000 mL or more, about 3,000 mL or more, about 4,000 mL or more, about 5,000 mL or more, about 6,000 mL or more, about 7,000 mL or more, about 8,000 mL or more, or about 9,000 mL or more. In some embodiments, the composition has a volume including at most about 100 mL or more, about 1,000 mL or more, about 2,000 mL or more, about 3,000 mL or more, about 4,000 mL or more, about 5,000 mL or more, about 6,000 mL or more, about 7,000 mL or more, about 8,000 mL or more, about 9,000 mL or more, or about 10,000 mL or more.

[0186] In some embodiments, the composition has a volume containing about 10 mL to about 10,000 mL. In some embodiments, the composition has volumes of about 10 mL to about 100 mL, about 10 mL to about 1,000 mL, about 10 mL to about 2,000 mL, about 10 mL to about 3,000 mL, about 10 mL to about 4,000 mL, about 10 mL to about 5,000 mL, about 10 mL to about 6,000 mL, about 10 mL to about 7,000 mL, about 10 mL to about 8,000 mL, about 10 mL to about 9,000 mL, about 10 mL to about 10,000 mL, about 100 mL to about 1,000 mL, about 100 mL to about 2,000 mL, about 100 mL to about 3,000 mL, and about 100 mL to about 4 ,000mL, about 100mL to about 5,000mL, about 100mL to about 6,000mL, about 100mL to about 7,000mL, about 100mL to about 8,000mL, about 100mL to about 9,000mL, about 100mL to about 10,000mL, about 1,000mL to about 2,000 mL, approximately 1,000mL to approximately 3,000mL, approximately 1,000mL to approximately 4,000mL, approximately 1,000mL to approximately 5,000mL, approximately 1,000mL to approximately 6,000mL, approximately 1,000mL to approximately 7,000mL, approximately 1,000mL to approximately 8,000mL, approximately 1,000mL ~9,000mL, 1,000mL~10,000mL, 2,000mL~3,000mL, 2,000mL~4,000mL, 2,000mL~5,000mL, 2,000mL~6,000mL, 2,000mL~7,000mL, 2,000mL to 8,000mL, 2,000mL to 9,000mL, 2,000mL to 10,000mL, 3,000mL to 4,000mL, 3,000mL to 5,000mL, 3,000mL to 6,000mL, 3,000mL to 6,000mL 7,000mL, approximately 3,000mL to approximately 8,000mL, approximately 3,000mL to approximately 9,000mL, approximately 3,000mL to approximately 10,000mL, approximately 4,000mL to approximately 5,000mL, approximately 4,000mL to approximately 6,000mL, approximately 4,000mL to approximately 7,000mL, approximately 4 ,000mL~about 8,000mL, about 4,000mL~about 9,000mL, about 4,000mL~about 10,000mL, about 5,000mL~about 6,000mL, about 5,000mL~about 7,000mL, about 5,000mL~about 8,000mL, about 5,000mL~about 9,Having a volume containing 000 mL, approximately 5,000 mL to approximately 10,000 mL, approximately 6,000 mL to approximately 7,000 mL, approximately 6,000 mL to approximately 8,000 mL, approximately 6,000 mL to approximately 9,000 mL, approximately 6,000 mL to approximately 10,000 mL, approximately 7,000 mL to approximately 8,000 mL, approximately 7,000 mL to approximately 9,000 mL, approximately 7,000 mL to approximately 10,000 mL, approximately 8,000 mL to approximately 9,000 mL, approximately 8,000 mL to approximately 10,000 mL, or approximately 9,000 mL to approximately 10,000 mL. In some embodiments, the composition has a volume containing about 10 mL, about 100 mL, about 1,000 mL, about 2,000 mL, about 3,000 mL, about 4,000 mL, about 5,000 mL, about 6,000 mL, about 7,000 mL, about 8,000 mL, about 9,000 mL, or about 10,000 mL. In some embodiments, the composition has a volume containing at least about 10 mL, about 100 mL, about 1,000 mL, about 2,000 mL, about 3,000 mL, about 4,000 mL, about 5,000 mL, about 6,000 mL, about 7,000 mL, about 8,000 mL, or about 9,000 mL. In some embodiments, the composition has a volume containing up to about 100 mL, about 1,000 mL, about 2,000 mL, about 3,000 mL, about 4,000 mL, about 5,000 mL, about 6,000 mL, about 7,000 mL, about 8,000 mL, about 9,000 mL, or about 10,000 mL.

[0187] In some embodiments, a cell processing method is described herein in which a portion of nucleated cells (parent cells) is denucleated using continuous flow centrifugation to produce an enucleated cell fraction. Here, continuous flow centrifugation is fixed-angle centrifugation. In some embodiments, continuous flow centrifugation is swing bucket centrifugation.

[0188] In some embodiments, the resulting composition includes an enucleated cell fraction, which may constitute 100% of the composition. In other embodiments, there may be a nucleated cell fraction of the composition consisting of nucleated parent cells that were not enucleated. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 30% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 35% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 40% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 45% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 50% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 55% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 60% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 65% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 70% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 75% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 80% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 85% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 90% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 95% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 96% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 97% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 98% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 99% of the composition.

[0189] In some embodiments, cell isolation, cell segregation, or cell sorting is a process of isolating one or more specific cell populations from a heterogeneous mixture of cells. In some embodiments, the cell enucleation methods disclosed herein are performed on an isolated population of homogeneous cells. In some embodiments, the cell enucleation methods disclosed herein are performed on a heterogeneous mixture of cells. In some embodiments, the methods disclosed herein involve isolating a homogeneous population of cells from a heterogeneous mixture of cells using a preferred cell separation technique, which includes, but is not limited to, immunomagnetic cell separation, fluorescence-activated cell sorting, density gradient centrifugation, immunodensity cell isolation, microfluidic cell sorting, buoyancy-activated cell sorting, aptamer-based cell isolation, complement depletion, or any combination thereof.

[0190] During centrifugation, as the sample rotates, denser particles can move to the outer edges of the mixture, while less dense objects accumulate closer to the center. Biological samples can be centrifuged until cell types are isolated into layers. During centrifugation, each cell type can settle to its isodensity point, which is the point in the medium gradient where the density of the cells and the medium are equal. Examples of density gradient media include Lymphoprep®, Lympholyte®, Ficoll-Paque®, Percoll®, OptiPrep®, Cell Separation with Accuspin®, Aystem-Histopaque® medium, Histopaque® medium, Histopaque® iodation gradient medium, inorganic salts, nonionic iodized density gradient media, polyhydric alcohols, and polysaccharides. For example, Lymphoprep®, Lympholyte®, and Ficoll-Paque® are composed of sugars and sodium diatrizoate and can be used to isolate mononuclear cells from peripheral blood, umbilical cord blood, and bone marrow. Percoll® consists of colloidal silica particles coated with polyvinylpyrrolidone and is widely used to isolate cells, organelles, viruses, and other intracellular particles. OptiPrep® is a culture medium consisting of iodixanol in water and is used to isolate viruses, organelles, macromolecules, and cells.

[0191] In some embodiments, a cell processing method is disclosed herein for producing an enucleated cell fraction by using continuous flow centrifugation to enucleate a portion of nucleated cells. In some embodiments, a cell processing method is disclosed herein for producing an enucleated cell fraction by using zone centrifugation to enucleate a portion of nucleated cells. In some embodiments, continuous flow centrifugation is fixed-angle centrifugation. In some embodiments, continuous flow centrifugation is swing-bucket centrifugation. In some embodiments, a density gradient is generated by continuous flow centrifugation. In some embodiments, the density gradient separates the enucleated cell fraction from nucleated cells in the composition. In some embodiments, the density gradient includes a polysaccharide density gradient. In some embodiments, the polysaccharide density gradient includes a ficol density gradient. In some embodiments, the method further includes generating a ficol gradient by polymerizing sucrose molecules with epichlorohydrin to obtain osmotically inert polysaccharides.

[0192] In some embodiments, the gradient includes a range of 2 to 20 of the density gradient. In some embodiments, the gradient is a density gradient of ranges 2-3, 2-4, 2-5, 2-6, 2-8, 2-10, 2-12, 2-14, 2-16, 2-18, 2-20, 3-4, 3-5, 3-6, 3-8, 3-10, 3-12, 3-14, 3-16, 3-18, 3-20, 4-5, 4-6, 4-8, 4-10, 4-12, 4-14, 4-16, 4-18, 4-20, 5-6, 5-8, 5-10, 5-12 Includes ranges, ranges 5-14, ranges 5-16, ranges 5-18, ranges 5-20, ranges 6-8, ranges 6-10, ranges 6-12, ranges 6-14, ranges 6-16, ranges 6-18, ranges 6-20, ranges 8-10, ranges 8-12, ranges 8-14, ranges 8-16, ranges 8-18, ranges 8-20, ranges 10-12, ranges 10-14, ranges 10-16, ranges 10-18, ranges 10-20, ranges 12-14, ranges 12-16, ranges 12-18, ranges 12-20, ranges 14-16, ranges 14-18, ranges 14-20, ranges 16-18, ranges 16-20, or ranges 18-20. In some embodiments, the gradient includes 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 ranges of density gradient. In some embodiments, the gradient includes at least 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, or 18 ranges of density gradient. In some embodiments, the gradient includes up to 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 ranges of density gradient.In some embodiments, the gradient includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 ranges of the density gradient. In some embodiments, the gradient includes at least 7 ranges of the density gradient. In some embodiments, the gradient includes at least 5 ranges of the density gradient. In some embodiments, the gradient includes at least 3 ranges of the density gradient. In some embodiments, the gradient includes 7 ranges of the density gradient. In some embodiments, the gradient includes 5 ranges of the density gradient. In some embodiments, the gradient includes 3 ranges of the density gradient.

[0193] In some embodiments, the gradient is approximately 7.5% to 30% density gradient, 7.5% to 10% density gradient, 7.5% to 12.5% ​​density gradient, 7.5% to 15% density gradient, 7.5% to 16% density gradient, 7.5% to 17% density gradient, 7.5% to 18% density gradient, 7.5% to 19% density gradient, and 7.5% to 20% density gradient. Medium, approximately 7.5% density gradient medium to approximately 25% density gradient medium, approximately 7.5% density gradient medium to approximately 27.5% density gradient medium, approximately 7.5% density gradient medium to approximately 30% density gradient medium, approximately 10% density gradient medium to approximately 12.5% ​​density gradient medium, approximately 10% density gradient medium to approximately 15% density gradient medium, approximately 10% density gradient medium to approximately 16% density gradient medium, approximately 10% density gradient medium to approximately 17% density gradient medium, approximately 10% density gradient medium to approximately 18% density gradient medium, approximately 10% density gradient medium to approximately 19% density gradient medium, approximately 10% density gradient medium to approximately 2 0% density gradient medium, approximately 10% density gradient medium to approximately 25% density gradient medium, approximately 10% density gradient medium to approximately 27.5% density gradient medium, approximately 10% density gradient medium to approximately 30% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 15% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 16% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 17% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 18% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 19% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 20% density gradient medium, Approximately 12.5% ​​density gradient medium to approximately 25% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 27.5% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 30% density gradient medium, approximately 15% density gradient medium to approximately 16% density gradient medium, approximately 15% density gradient medium to approximately 17% density gradient medium, approximately 15% density gradient medium to approximately 18% density gradient medium, approximately 15% density gradient medium to approximately 19% density gradient medium, approximately 15% density gradient medium to approximately 20% density gradient medium, approximately 15% density gradient medium to approximately 25% density gradient medium, approximately 15% density gradient medium to approximately 27%.5% density gradient medium, approximately 15% density gradient medium ~ approximately 30% density gradient medium, approximately 16% density gradient medium ~ approximately 17% density gradient medium, approximately 16% density gradient medium ~ approximately 18% density gradient medium, approximately 16% density gradient medium ~ approximately 19% density gradient medium, approximately 16% density gradient medium ~ approximately 20% density gradient medium, approximately 16% density gradient medium ~ approximately 25% density gradient medium, approximately 16% density gradient medium ~ approximately 27.5% density gradient medium, approximately 16% density gradient medium ~ approximately 30% density gradient medium, approximately 17% density gradient medium ~ approximately 18% density gradient medium, approximately 17% density gradient medium ~ approximately 19% density gradient medium, approximately 17% density gradient medium ~ approximately 20% density gradient medium, approximately 17% density gradient medium ~ approximately 25% density gradient medium, approximately 17% density gradient medium ~ approximately 27.5% density gradient medium, approximately 17% density gradient medium ~ approximately 30% density gradient medium, approximately 18% density gradient medium ~ approximately Includes 19% density gradient media, approximately 18% to approximately 20% density gradient media, approximately 18% to approximately 25% density gradient media, approximately 18% to approximately 27.5% density gradient media, approximately 18% to approximately 30% density gradient media, approximately 19% to approximately 20% density gradient media, approximately 19% to approximately 25% density gradient media, approximately 19% to approximately 27.5% density gradient media, approximately 19% to approximately 30% density gradient media, approximately 20% to approximately 25% density gradient media, approximately 20% to approximately 27.5% density gradient media, approximately 20% to approximately 30% density gradient media, approximately 25% to approximately 27.5% density gradient media, approximately 25% to approximately 30% density gradient media, or approximately 27.5% to approximately 30% density gradient media. .

[0194] In some embodiments, the gradient is a ficol gradient. In some embodiments, the ficol gradient includes a range of 2 to 20 of the density ficol gradient. In some embodiments, the Ficol gradient is a density Ficol gradient of ranges 2-3, 2-4, 2-5, 2-6, 2-8, 2-10, 2-12, 2-14, 2-16, 2-18, 2-20, 3-4, 3-5, 3-6, 3-8, 3-10, 3-12, 3-14, 3-16, 3-18, 3-20, 4-5, 4-6, 4-8, 4-10, 4-12, 4-14, 4-16, 4-18, 4-20, 5-6, 5-8, 5-10, 5 Range ~12 range, Range 5 ~14 range, Range 5 ~16 range, Range 5 ~18 range, Range 5 ~20 range, Range 6 ~8 range, Range 6 ~10 range, Range 6 ~12 range, Range 6 ~14 range, Range 6 ~16 range, Range 6 ~18 range, Range 6 ~20 range, Range 8 ~10 range, Range 8 ~12 range, Range 8 ~14 range, Range 8 ~16 range, Range 8 ~18 range, Range 8 ~20 range The range includes ranges 10-12, 10-14, 10-16, 10-18, 10-20, 12-14, 12-16, 12-18, 12-20, 14-16, 14-18, 14-20, 16-18, 16-20, or 18-20. In some embodiments, the Ficol gradient includes ranges 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, the Ficol gradient includes at least ranges 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, or 18 of the density Ficol gradient. In some embodiments, the Ficol gradient includes up to 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 ranges of density Ficol gradient.In some embodiments, the ficol gradient includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes at least 7 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes at least 5 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes at least 3 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes 7 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes 5 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes 3 ranges of the density ficol gradient.

[0195] In some embodiments, the Ficol density gradient is approximately 7.5% to 10% Ficol, approximately 7.5% to 12.5% ​​Ficol, approximately 7.5% to 15% Ficol, approximately 7.5% to 16% Ficol, approximately 7.5% to 17% Ficol, approximately 7.5% to 18% Ficol, approximately 7.5% to 19% Ficol, approximately 7.5% to 20% Ficol, approximately 7.5% to 25% Ficol, and approximately 7.5% to 27.5% Ficol. , approximately 7.5% Ficol to approximately 30% Ficol, approximately 10% Ficol to approximately 12.5% ​​Ficol, approximately 10% Ficol to approximately 15% Ficol, approximately 10% Ficol to approximately 16% Ficol, approximately 10% Ficol to approximately 17% Ficol, approximately 10% Ficol to approximately 18% Ficol, approximately 10% Ficol to approximately 19% Ficol, approximately 10% Ficol to approximately 20% Ficol, approximately 10% Ficol to approximately 25% Ficol, approximately 10% Ficol to approximately 27.5% Ficol, approximately 10% Ficol to approximately 30% Ficol, approximately 12.5% ​​Ficol Fikol ~ approx. 15% Fikol, approx. 12.5% ​​Fikol ~ approx. 16% Fikol, approx. 12.5% ​​Fikol ~ approx. 17% Fikol, approx. 12.5% ​​Fikol ~ approx. 18% Fikol, approx. 12.5% ​​Fikol ~ approx. 19% Fikol, approx. 12.5% ​​Fikol ~ approx. 20% Fikol, approx. 12.5% ​​Fikol ~ approx. 25% Fikol, approx. 12.5% ​​Fikol ~ approx. 27.5% Fikol, approx. 12.5% ​​Fikol ~ approx. 30% Fikol, approx. 15% Fikol ~ approx. 16% Fikol, approx. 15% Fikol ~ approx. 17% Fikol, approx. 15% Ficol to approximately 18% Ficol, approximately 15% Ficol to approximately 19% Ficol, approximately 15% Ficol to approximately 20% Ficol, approximately 15% Ficol to approximately 25% Ficol, approximately 15% Ficol to approximately 27.5% Ficol, approximately 15% Ficol to approximately 30% Ficol, approximately 16% Ficol to approximately 17% Ficol, approximately 16% Ficol to approximately 18% Ficol, approximately 16% Ficol to approximately 19% Ficol, approximately 16% Ficol to approximately 20% Ficol, approximately 16% Ficol to approximately 25% Ficol, approximately 16% Ficol to approximately 27% Ficol.5% Ficol, approximately 16% to approximately 30% Ficol, approximately 17% to approximately 18% Ficol, approximately 17% to approximately 19% Ficol, approximately 17% to approximately 20% Ficol, approximately 17% to approximately 25% Ficol, approximately 17% to approximately 27.5% Ficol, approximately 17% to approximately 30% Ficol, approximately 18% to approximately 19% Ficol, approximately 18% to approximately 20% Ficol, approximately 18% to approximately 25% Ficol, approximately 18% to approximately 27.5% Ficol, approximately Includes 18% to approximately 30% Ficol, approximately 19% to approximately 20% Ficol, approximately 19% to approximately 25% Ficol, approximately 19% to approximately 27.5% Ficol, approximately 19% to approximately 30% Ficol, approximately 20% to approximately 25% Ficol, approximately 20% to approximately 27.5% Ficol, approximately 20% to approximately 30% Ficol, approximately 25% to approximately 27.5% Ficol, or approximately 27.5% to approximately 30% Ficol.

[0196] In some embodiments, the ficol density gradient includes about 7.5% ficol, about 10% ficol, about 12.5% ​​ficol, about 15% ficol, about 16% ficol, about 17% ficol, about 18% ficol, about 19% ficol, about 20% ficol, about 25% ficol, about 27.5% ficol, or about 30% ficol. In some embodiments, the ficol density gradient includes at least about 7.5% ficol, about 10% ficol, about 12.5% ​​ficol, about 15% ficol, about 16% ficol, about 17% ficol, about 18% ficol, about 19% ficol, about 20% ficol, about 25% ficol, or about 27.5% ficol. In some embodiments, the Ficol density gradient includes up to about 10% Ficol, about 12.5% ​​Ficol, about 15% Ficol, about 16% Ficol, about 17% Ficol, about 18% Ficol, about 19% Ficol, about 20% Ficol, about 25% Ficol, about 27.5% Ficol, or about 30% Ficol. In some embodiments, the Ficol density gradient includes about 25% Ficol, about 17% Ficol, about 16% Ficol, about 15% Ficol, or about 12.5% ​​Ficol. In some embodiments, the Ficol density gradient includes about 25% Ficol. In some embodiments, the Ficol density gradient includes about 17% Ficol. In some embodiments, the Ficol density gradient includes about 16% Ficol. In some embodiments, the Ficol density gradient includes about 15% Ficol. In some embodiments, the Ficol density gradient includes about 12.5% ​​Ficol.

[0197] In some embodiments, the cell processing method disclosed herein comprises using continuous flow centrifugation to denucleate a portion of nucleated cells to produce an enucleated cell fraction, wherein the portion of nucleated cells to be denucleated is approximately equal to or greater than 10% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 20% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 25% of the nucleated cells. In some embodiments, the proportion of nucleated cells is approximately equal to or greater than 30% of the nucleated cells. In some embodiments, the proportion of nucleated cells is approximately equal to or greater than 35% of the nucleated cells. In some embodiments, the proportion of nucleated cells is approximately equal to or greater than 40% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 45% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 50% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 55% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 60% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 65% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 70% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 75% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 80% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 85% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 90% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 95% of the nucleated cells.

[0198] In some embodiments, the enucleated cell fraction produced by the method disclosed herein is approximately 1 × 10⁶ 5 Enucleated cells, 1 x 10 6 Enucleated cells, approximately 1 x 107 Enucleated cells, 3×10 5 Enucleated cells, 5×10 5 Enucleated cells, 7×10 7 of enucleated cells, 8×10 7 of enucleated cells, 9×10 7 of enucleated cells, 10×10 7 of enucleated cells, 15×10 7 of enucleated cells, 20×10 7 of enucleated cells, 50×10 7 of enucleated cells, 70×10 7 Enucleated cells, 90×10 7 Enucleated cells, 100×10 7 of enucleated cells, 150×10 7 of enucleated cells, 200×10 7 [[ID=,29]]of enucleated cells, 250×10 7 Enucleated cells, 300×10 7 Enucleated cells or 500×1E 7 including enucleated cells of 500×10 or more.

[0199] In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 50% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 60% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 70% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 80% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 90% or less of the average diameter of the nucleated cells.

[0200] In some embodiments, the diameter of enucleated cells in the enucleated cell fraction includes approximately 5 μm or more, approximately 10 μm or more, approximately 20 μm or more, approximately 30 μm or more, approximately 40 μm or more, approximately 50 μm or more, approximately 60 μm or more, approximately 70 μm or more, approximately 80 μm or more, or approximately 90 μm or more. In some embodiments, the diameter of enucleated cells in the enucleated cell fraction is in the range of approximately 1 μm to approximately 10 μm. In some embodiments, the diameter of enucleated cells in the enucleated cell fraction is approximately 1 μm to 2 μm, 1 μm to 3 μm, 1 μm to 4 μm, 1 μm to 5 μm, 1 μm to 6 μm, 1 μm to 7 μm, 1 μm to 8 μm, 1 μm to 9 μm, 1 μm to 10 μm, 2 μm to 3 μm, 2 μm to 4 μm, 2 μm to 5 μm, 2 μm to 6 μm, 2 μm to 7 μm, 2 μm to 8 μm, 2 μm to 9 μm, 2 μm to 10 μm, 3 μm to 4 μm, 3 μm to 5 μm, 3 μm to 6 μm, 3 μm to 7 μm, 3 μm to 8 μm m is in the range of approximately 3μm to 9μm, approximately 3μm to 10μm, approximately 4μm to 5μm, approximately 4μm to 6μm, approximately 4μm to 7μm, approximately 4μm to 8μm, approximately 4μm to 9μm, approximately 4μm to 10μm, approximately 5μm to 6μm, approximately 5μm to 7μm, approximately 5μm to 8μm, approximately 5μm to 9μm, approximately 5μm to 10μm, approximately 6μm to 7μm, approximately 6μm to 8μm, approximately 6μm to 9μm, approximately 6μm to 10μm, approximately 7μm to 8μm, approximately 7μm to 9μm, approximately 7μm to 10μm, approximately 8μm to 9μm, approximately 8μm to 10μm, or approximately 9μm to 10μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is in the range of approximately 1 μm, approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, approximately 9 μm, or approximately 10 μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is in the range of at least approximately 1 μm, approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, or approximately 9 μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is in the range of at most approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, approximately 9 μm, or approximately 10 μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is approximately 8 μm.

[0201] In some embodiments, the cell processing method further includes generating a density gradient by centrifuging a density gradient medium at an acceleration for at least about 1 minute (min), at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some embodiments, the cell processing method further includes generating a density gradient by centrifuging polysaccharides at an acceleration for at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some embodiments, the cell processing method further includes generating a density gradient by centrifuging the polysaccharides at an accelerated rate for at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, or at least about 50 minutes. In some embodiments, the cell processing method further includes generating a density gradient by centrifuging the polysaccharides at an accelerated rate for at least about 30 minutes.

[0202] In some embodiments, a method for using continuous flow centrifugation for cell processing to enucleate a portion of nucleated cells and produce an enucleated cell fraction further includes generating a density gradient by centrifugating polysaccharides with minimal deceleration. In some embodiments, the density gradient includes centrifugating polysaccharides with a maximum centrifugal force of about 20,000 relative centrifugal force (RCF) to about 250,000 RCF. In the step embodiment, the density gradient is approximately 20,000 RCF to approximately 30,000 RCF, approximately 20,000 RCF to approximately 40,000 RCF, approximately 20,000 RCF to approximately 50,000 RCF, approximately 20,000 RCF to approximately 60,000 RCF, approximately 20,000 RCF to approximately 70,000 RCF, approximately 20,000 RCF to approximately 80,000 RCF, approximately 20,000 RCF to approximately 100,000 RCF, approximately 20,000 RCF to approximately 120,000 RCF, and approximately 20,000 RCF. F ~ approx. 150,000 RCF, approx. 20,000 RCF ~ approx. 200,000 RCF, approx. 20,000 RCF ~ approx. 250,000 RCF, approx. 30,000 RCF ~ approx. 40,000 RCF, approx. F, about 30,000RCF to about 60,000RCF, about 30,000RCF to about 70,000RCF, about 30,000RCF to about 80,000RCF, about 30,000RCF to about 100,000RCF, about 30,000RCF to about 120,000RCF, about 30,000RCF to about 150,000RCF, about 30,000RCF to about 200,000RCF, about 30,000RCF to about 250,000RCF, about 40,000RCF to about 50,000RCF, Approx. 40,000RCF~Approx. 60,000RCF, Approx. 40,000RCF~Approx. 70,000RCF, Approx. 40,000RCF~Approx. 80,000RCF, Approx. 40,000RCF~Approx. 100,000RCF, Approx. 40,000RCF~Approx. 12 0,000RCF, about 40,000RCF to about 150,000RCF, about 40,000RCF to about 200,000RCF, about 40,000RCF to about 250,000RCF, about 50,000RCF to about 60,000RCF, about 50 ,000RCF~Approx. 70,000RCF, Approx. 50,000RCF~Approx. 80,000RCF, Approx. 50,000RCF~Approx. 100,000RCF, Approx. 50,000RCF~Approx. 120,000RCF, Approx. 50,000RCF~Approx. 150,000RCF, about 50,000RCF to about 200,000RCF, about 50,000RCF to about 250,000RCF, about 60,000RCF to about 70,000RCF, about 60,000RCF to about 80,0 00RCF, about 60,000RCF to about 100,000RCF, about 60,000RCF to about 120,000RCF, about 60,000RCF to about 150,000RCF, about 60,000RCF to about 200, 000RCF, about 60,000RCF to about 250,000RCF, about 70,000RCF to about 80,000RCF, about 70,000RCF to about 100,000RCF, about 70,000RCF to about 120, 000RCF, about 70,000RCF to about 150,000RCF, about 70,000RCF to about 200,000RCF, about 70,000RCF to about 250,000RCF, about 80,000RCF to about 100 ,000RCF, about 80,000RCF to about 120,000RCF, about 80,000RCF to about 150,000RCF, about 80,000RCF to about 200,000RCF, about 80,000RCF to about 25 0,000RCF, about 100,000RCF~about 120,000RCF, about 100,000RCF~about 150,000RCF, about 100,000RCF~about 200,000RCF, about 100,000RCF This includes centrifuging polysaccharides with a maximum centrifugal force of F ~ approximately 250,000 RCF, approximately 120,000 RCF ~ approximately 150,000 RCF, approximately 120,000 RCF ~ approximately 200,000 RCF, approximately 120,000 RCF ~ approximately 250,000 RCF, approximately 150,000 RCF ~ approximately 200,000 RCF, approximately 150,000 RCF ~ approximately 250,000 RCF, or approximately 200,000 RCF ~ approximately 250,000 RCF. In some embodiments, the density gradient involves centrifuging the polysaccharides at a minimum centrifugal force of about 20,000 RCF, about 30,000 RCF, about 40,000 RCF, about 50,000 RCF, about 60,000 RCF, about 70,000 RCF, about 80,000 RCF, about 100,000 RCF, about 120,000 RCF, about 150,000 RCF, about 200,000 RCF, or about 250,000 RCF.This includes centrifuging polysaccharides with maximum centrifugal forces of approximately 000 RCF, approximately 100,000 RCF, approximately 120,000 RCF, approximately 150,000 RCF, or approximately 200,000 RCF. In some embodiments, the density gradient includes centrifuging polysaccharides with maximum centrifugal forces of approximately 30,000 RCF, approximately 40,000 RCF, approximately 50,000 RCF, approximately 60,000 RCF, approximately 70,000 RCF, approximately 80,000 RCF, approximately 100,000 RCF, approximately 120,000 RCF, approximately 150,000 RCF, approximately 200,000 RCF, or approximately 250,000 RCF.

[0203] (a) Modification and / or storage of disclosed cells Nucleated ("parent") cells may be engineered to express one or more exogenous factors before or after enucleation, or in combination thereof. In some embodiments, one or more exogenous biomolecules include a targeting moiety, a transmembrane moiety, a biomolecular suicide switch, or a therapeutic agent, or a combination thereof. In some embodiments, the targeting moiety includes an adhesion molecule, a chemokine, or a retention receptor, or both. In some embodiments, the targeting moiety is engineered to target a target tissue, cell, or environment disclosed herein (e.g., lymphoid tissue of the subject). Additionally or alternatively, the resulting enucleated cells are engineered to express and, optionally, secrete the therapeutic agent. In some embodiments, the therapeutic agent includes an antibody or its antigen-binding fragment (e.g., a single-domain antibody). In some embodiments, the enucleated cells may be administered to a subject in need to treat a disease or condition in the subject.

[0204] Various methods may be used to introduce biomolecules (e.g., therapeutic agents, transmembrane portions, immune-evading portions, and / or targeting portions) into parental or enucleated cells as described herein. Non-limiting examples of methods that may be used to introduce biomolecules into parental or enucleated cells include liposome-mediated transfer, adenovirus, adeno-associated virus, herpesvirus, retrovirus-based vector, lentiviral vector, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalement, hydrodynamic delivery, magnetofection, nanoparticle transfection, or combinations thereof. In some embodiments of any of the compositions and methods provided herein, therapeutic agents, viruses, antibodies, or nanoparticles may be introduced into enucleated cells.

[0205] In some embodiments, biomolecules are introduced into parental or enucleated cells via mRNA transfection. In mRNA transduction, mRNA can be incubated with a transfection reagent (e.g., lipofectamine). The mRNA transfection reagent mixture can then be added to a parental or enucleated cell suspension or cell culture. The mRNA transfection reagent and cell suspension or cell culture can then be incubated to enable transfection of mRNA into parental or enucleated cells. In some embodiments, biomolecules are introduced into parental or enucleated cells via siRNA transfection. In siRNA transduction, siRNA can be incubated with a transfection reagent (e.g., lipofectamine). The siRNA transfection reagent mixture can then be added to a parental or enucleated cell suspension or cell culture. The siRNA transfection reagent and cell suspension or cell culture can then be incubated to enable transfection of siRNA into parental or enucleated cells. In some embodiments, biomolecules are introduced into parental cells by oncolytic virus-mediated infection. In oncolytic virus-mediated infection, parent cells are incubated with an oncolytic virus (such as an adenovirus) up to a multiple of infection (MOI) to enable infection of the parent cells. In some embodiments, the oncolytic virus carries a therapeutic agent. Following incubation, the supernatant of the parent cells may be removed to eliminate any free viruses remaining in the parent cell culture. In some embodiments, biomolecules are introduced into parent cells or enucleated cells via lentivirus overexpression. In lentivirus overexpression, parent cells or enucleated cells are incubated with a lentivirus (such as HIV) at MOI to enable infection of the parent cells or enucleated cells. In some embodiments, the lentivirus carries a selective gene (e.g., an antibiotic resistance gene). After incubation, the supernatant of the parent cells or enucleated cells may be removed to eliminate any free viruses remaining in the parent cell culture or enucleated cell culture.Infected parental cell cultures or infected enucleated cell cultures can then be cultured with a selective agent (such as an antibiotic). Surviving infected parental cells or enucleated cells can be isolated and further cultured and propagated. In some embodiments, biomolecules are introduced into the parental or enucleated cells via peptide loading. In peptide loading, the parental or enucleated cells can be seeded onto a surface (such as a glass slide). In some embodiments, the parental or enucleated cells adhere to the surface. The parental or enucleated cells can be incubated with biomolecules (such as peptides) and Arg9. Arg9 can be a cell-permeable peptide that can pass through the membrane of the parental or enucleated cells.

[0206] (i) Cryopreservation / cryohibernation In some embodiments, enucleated cells are preserved by cryopreservation. Cryopreservation includes freezing enucleated cells, and cryopreservation includes storing enucleated cells at a temperature below room temperature but not freezing. In some embodiments, enucleated cells are preserved by cryopreservation, cryopreservation, or freeze-drying. Cryopreservation includes freezing enucleated cells, and cryopreservation includes storing enucleated cells at a temperature below room temperature but not freezing. In some embodiments, cryopreservation includes storing enucleated cells at about 4°C. In some embodiments, cryopreservation includes storing enucleated cells at a maximum of about 4°C. In some embodiments, cryopreservation includes storing enucleated cells for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 1 month, at least about 1 month, or at least about 1 year. In some embodiments, cryo-hibernation involves contacting or storing enucleated cells in a culture medium (such as a cell culture medium). In some embodiments, the cell culture medium includes xeno-free medium. In some embodiments, the cell culture medium contains about 5% to about 20% serum.

[0207] In some embodiments, the enucleated cells described herein can be cryopreserved. In some embodiments, the cryopreserved enucleated cells, after thawing, are as viable as otherwise equivalent, uncryopreserved enucleated cells.

[0208] This disclosure also provides a method for delivering one or more therapeutic agents. In some embodiments, one or more therapeutic agents are viruses. In some embodiments, one or more therapeutic agents are cytokines or cytokine receptor-binding fragments thereof. In some embodiments, one or more therapeutic agents are viruses or cytokine receptor-binding fragments thereof.

[0209] In some embodiments, one or more therapeutic agents are present in a plurality of enucleated cells of the Disclosure. In some embodiments, the plurality of enucleated cells are cryopreserved. In some embodiments, at least a subset of the plurality of cryopreserved enucleated cells contains one or more therapeutic agents.

[0210] In some embodiments, the method further includes preparing a fluid preparation. In some embodiments, the fluid preparation comprises cryopreserved enucleated cells of the Disclosure. In some embodiments, the fluid preparation further comprises a non-pyrogenic solution.

[0211] In some embodiments, at least a subset of multiple cryopreserved enucleated cells containing one or more therapeutic agents represents about 5% to about 100% of the multiple cryopreserved cells. In some embodiments, at least subsets of multiple cryopreserved enucleated cells comprising one or more therapeutic agents are approximately 5% to approximately 10%, approximately 10% to approximately 15%, approximately 15% to approximately 20%, approximately 20% to approximately 25%, approximately 25% to approximately 30%, approximately 30% to approximately 35%, approximately 35% to approximately 40%, approximately 40% to approximately 45%, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, or approximately 95% to approximately 100%.

[0212] In some embodiments, at least a subset of multiple cryopreserved enucleated cells comprising one or more therapeutic agents is at least about 10%, at least about 15%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100%.

[0213] In some embodiments, at least a subset of multiple cryopreserved enucleated cells comprising one or more therapeutic agents is about 10%, about 15%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% of the multiple cryopreserved enucleated cells.

[0214] In some embodiments, the method further includes introducing a fluid formulation into the sample. In some embodiments, the sample is a biopsy, tissue sample, blood sample, serum sample, cell line, or isolated cells. In some embodiments, the sample is a subject sample. In some embodiments, the sample is a subject. In some embodiments, the subject has a disease. Non-limiting examples of diseases include cancer, autoimmune diseases, fibrotic diseases, and inflammatory diseases.

[0215] In some embodiments, the introduction of the fluid formation is carried out under conditions sufficient to deliver one or more therapeutic agents. In some embodiments, conditions sufficient to deliver one or more therapeutic agents are incubation of the fluid formation with the sample. In some embodiments, the fluid formation is incubated with the sample for about 1 hour to about 14 days. In some embodiments, the fluid formation is incubated with the sample for about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 7 hours, about 7 to 8 hours, about 8 to 9 hours, about 9 to 10 hours, about 10 to 11 hours, about 11 to 12 hours, about 12 to 15 hours, about 15 to 18 hours, about 18 to 21 hours, about 21 to 24 hours, about 24 to 36 hours, about 36 to 48 hours, and about 48 hours. Incubation is performed for approximately 60 hours, 60 to 72 hours, 72 to 84 hours, 84 to 96 hours, 96 to 108 hours, 108 to 120 hours, 120 to 132 hours, 132 to 144 hours, 144 to 156 hours, 156 to 168 hours, 7 to 8 days, 8 to 9 days, 9 to 10 days, 10 to 11 days, 11 to 12 days, 12 to 13 days, or 13 to 14 days.

[0216] In some embodiments, the fluid formation is incubated with the sample for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 15 hours, at least about 18 hours, at least about 21 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, at least about 120 hours, at least about 132 hours, at least about 144 hours, at least about 156 hours, at least about 168 hours, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days or longer.

[0217] In some embodiments, the fluid formation is left with the sample for up to approximately 14 days, up to approximately 13 days, up to approximately 12 days, up to approximately 11 days, up to approximately 10 days, up to approximately 9 days, up to approximately 8 days, up to approximately 168 hours, up to approximately 156 hours, up to approximately 144 hours, up to approximately 132 hours, up to approximately 120 hours, up to approximately 108 hours, up to approximately 96 hours, up to approximately 84 hours, up to approximately 72 hours, and so on. They are incubated for approximately 60 hours for large sizes, 48 ​​hours for maximum sizes, 36 hours for maximum sizes, 24 hours for maximum sizes, 21 hours for maximum sizes, 18 hours for maximum sizes, 15 hours for maximum sizes, 12 hours for maximum sizes, 11 hours for maximum sizes, 10 hours for maximum sizes, 9 hours for maximum sizes, 8 hours for maximum sizes, 7 hours for maximum sizes, 6 hours for maximum sizes, 5 hours for maximum sizes, 4 hours for maximum sizes, 3 hours for maximum sizes, 2 hours for maximum sizes, 1 hour for maximum sizes, or less.

[0218] In some embodiments, the fluid formation is incubated with the sample for approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0219] In some embodiments, the condition sufficient for delivering one or more therapeutic agents is administering a fluid compound to the patient.

[0220] In some embodiments, one or more therapeutic agents are delivered to the sample in vitro. In some embodiments, one or more therapeutic agents are delivered to the sample in vivo. In some embodiments, one or more therapeutic agents are delivered to the sample ex vivo.

[0221] In some embodiments, cryopreserved enucleated cells deliver one or more therapeutic agents to a sample. In some embodiments, the sample is a target cell. In some embodiments, the sample is a subject.

[0222] In some embodiments, cryopreserved enucleated cells deliver to a sample an amount of one or more therapeutic agents that is approximately equal to or greater than the amount of one or more therapeutic agents delivered to the same otherwise identical sample by non-cryopreserved enucleated cells. In some embodiments, cryopreserved enucleated cells deliver about 1 to about 50 times more of one or more therapeutic agents than equivalent enucleated cells. In some embodiments, cryopreserved enucleated cells deliver about 1 to 2 times, 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times more than the amount of one or more therapeutic agent delivered by otherwise identical enucleated cells. In some embodiments, cryopreserved enucleated cells deliver about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, or more amounts of one or more therapeutic agents compared to the amount of one or more therapeutic agents delivered by otherwise identical enucleated cells. In some embodiments, the amount of one or more therapeutic agents delivered by cryopreserved enucleated cells is equal to the amount of one or more therapeutic agents delivered by otherwise identical enucleated cells.

[0223] In some embodiments, the amount of one or more therapeutic agents delivered is measured in vitro. In some embodiments, the amount of one or more therapeutic agents delivered is measured in vivo. In some embodiments, the amount of one or more therapeutic agents delivered is measured ex vivo.

[0224] In some embodiments, one or more therapeutic agents are viruses. In some embodiments, the amount of one or more therapeutic agents delivered is a measure of viral titer in target cells. In some embodiments, viral titer is measured in plaque-forming units (PFUs). In some embodiments, PFUs are measured as PFUs per milliliter (mL). In some embodiments, PFUs are measured as PFUs per gram.

[0225] In some embodiments, the target cells are biological samples. Non-limiting examples of biological samples include isolated cells, cell supernatants, tissue biopsies, tumor biopsies, cell lines, cell cultures, and biological fluids (e.g., saliva, blood, plasma, serum, urine, feces, lymph, cerebrospinal fluid).

[0226] In some embodiments, cryopreserved enucleated cells deliver to target cells an amount of virus approximately equal to or greater than that delivered to otherwise identical, non-cryopreserved enucleated cells to the same target cells. In some embodiments, cryopreserved enucleated cells deliver to target cells an amount of virus generally greater than that delivered to otherwise identical, non-cryopreserved enucleated cells to the same target cells. In some embodiments, cryopreserved enucleated cells deliver to target cells approximately 1 to 50 times more virus than that delivered to otherwise equivalent enucleated cells to the same target cells. In some embodiments, cryopreserved enucleated cells deliver to target cells approximately 1 to 2 times, 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times more virus than would be delivered to target cells by otherwise identical enucleated cells. In some embodiments, cryopreserved enucleated cells deliver to target cells about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, or more amounts of virus compared to the amount of virus delivered to otherwise identical enucleated cells to otherwise identical target cells. In some embodiments, the amount of virus delivered to target cells by cryopreserved enucleated cells is equal to the amount of virus delivered to otherwise identical enucleated cells to otherwise identical target cells.

[0227] In some embodiments, one or more therapeutic agents are cytokines or cytokine receptor-binding fragments thereof. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured by the amount of soluble cytokine or cytokine receptor-binding fragment present. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in micrograms (μg) per mL. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in nanograms (ng) per mL. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in picograms (pg) per mL. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in the supernatant of cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in the supernatant of otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in the supernatant of cryopreserved enucleated cells and in the supernatant of otherwise identical enucleated cells.

[0228] In some embodiments, cryopreserved nucleated cells deliver a greater amount of cytokines or cytokine receptor-binding fragments compared to otherwise identical enucleated cells. In some embodiments, cryopreserved enucleated cells deliver about 1 to about 50 times more cytokines or cytokine receptor-binding fragments than those delivered by otherwise equivalent enucleated cells. In some embodiments, cryopreserved enucleated cells deliver about 1 to 2 times, 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times more cytokines or cytokine receptor binding fragments than would be delivered by otherwise identical enucleated cells. In some embodiments, cryopreserved enucleated cells deliver about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, or more amounts of cytokines or cytokine receptor-binding fragments delivered by otherwise identical enucleated cells. In some embodiments, the amount of cytokines or cytokine receptor-binding fragments delivered by cryopreserved enucleated cells is equal to the amount of cytokines or cytokine receptor-binding fragments delivered by otherwise identical enucleated cells.

[0229] In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in target cells introduced into cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in target cells introduced into cryopreserved nucleated cells and otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment is measured as the amount of mRNA of the cytokine or cytokine receptor-binding fragment. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured by the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in target cells introduced into cryopreserved enucleated cells, and the amount of mRNA of the cytokine or cytokine receptor-binding fragment present in otherwise identical target cells introduced into otherwise identical enucleated cells.

[0230] In some embodiments, target cells introduced into cryopreserved enucleated cells have a higher amount of cytokine or cytokine receptor-binding fragment mRNA compared to otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells have approximately 1 to 50 times more amount of cytokine or cytokine receptor-binding fragment mRNA compared to otherwise identical target cells introduced into equivalent enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells have about 1 to 2 times, about 2 to 3 times, about 3 to 4 times, about 4 to 5 times, about 5 to 10 times, about 10 to 15 times, about 15 to 20 times, about 20 to 25 times, about 25 to 30 times, about 30 to 35 times, about 35 to 40 times, about 40 to 45 times, or about 45 to 50 times compared to the amount of mRNA of cytokines or cytokine receptor binding fragments present in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments, target cells introduced into cryopreserved enucleated cells contain approximately 1 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 25 times, approximately 30 times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times, or more amounts of mRNA of cytokines or cytokine receptor-binding fragments compared to target cells introduced into otherwise identical enucleated cells. In some embodiments, the amount of mRNA of cytokines or cytokine receptor-binding fragments present in target cells introduced into cryopreserved enucleated cells is equal to the amount of mRNA of cytokines or cytokine receptor-binding fragments present in otherwise identical target cells introduced into otherwise identical enucleated cells.

[0231] In some embodiments, the amount of cytokine or cytokine receptor-bound fragment delivered is measured through the release of another cytokine or cytokine receptor-bound fragment by target cells introduced into cryopreserved enucleated cells. In some embodiments, the other cytokine or cytokine receptor-bound fragment is a soluble cytokine or cytokine receptor-bound fragment. In some embodiments, the other cytokine or cytokine receptor-bound fragment is any cytokine or cytokine receptor-bound fragment of this disclosure. In some embodiments, the other cytokine or cytokine receptor-bound fragment is IFN-γ. In some embodiments, the other cytokine or cytokine receptor-bound fragment is TNF-α. In some embodiments, the other cytokine or cytokine receptor-bound fragment is IL-6.

[0232] In some embodiments, the amount of cytokine or cytokine receptor-binding fragment delivered is measured in otherwise identical target cells introduced into otherwise identical enucleated cells. In some embodiments,...

Claims

1. A method for delivering a therapeutic agent to target cells of a subject, the method comprising introducing a plurality of enucleated cells containing the therapeutic agent into the subject or a sample of the subject in vivo or ex vivo, under conditions sufficient to deliver the therapeutic agent to the target cells of the subject, wherein the plurality of enucleated cells are obtained from a cryopreserved composition or a cryopreserved composition, and the therapeutic agent is delivered to the target cells in an amount approximately equal to or greater than the amount of the therapeutic agent delivered to otherwise equivalent target cells of the subject by otherwise equivalent enucleated cells that are neither cryopreserved nor cryopreserved.

2. The method according to claim 1, further comprising preparing a fluid composition containing the plurality of enucleated cells from the cryopreserved composition.

3. The method according to claim 1 or 2, wherein the cryopreserved composition is cryopreserved in liquid nitrogen.

4. The method according to any one of claims 1 to 3, wherein the frozen composition is frozen for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 1 month, at least about 1 month, or at least about 1 year.

5. The method according to any one of claims 1 to 4, wherein the frozen composition is stored at a maximum temperature of about -80°C before being frozen.

6. The method according to claim 5, wherein the frozen composition is stored at a temperature of about -80°C or lower for at least about 24 hours.

7. The method according to claim 1, further comprising preparing a fluid composition containing the plurality of enucleated cells from the cryogenically dormant composition.

8. The method according to claim 1 or claim 7, wherein the composition subjected to cryogenic dormancy is stored at a temperature of approximately 4°C or lower.

9. The method according to claim 1, claim 7, or claim 8, wherein the composition subjected to cryopreservation is cryopreserved for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 1 month, at least about 1 month, or at least about 1 year.

10. The method according to any one of claims 1 to 9, wherein the plurality of enucleated cells from the cryopreserved composition are suspended in a xenofree medium.

11. The method according to any one of claims 1 to 10, wherein the plurality of enucleated cells from the cryopreserved composition are suspended in a freezing medium.

12. The method according to claim 11, wherein the freezing medium contains at least 2%, at least 5%, or at least 10% DMSO.

13. The method according to claim 11 or claim 12, wherein the frozen culture medium comprises CryoStor® culture medium.

14. The method according to claim 13, wherein the CryoStor® culture medium is CryoStor® CS5 or CryoStor® CS10.

15. The method according to claim 11, wherein the freezing medium comprises DMSO, sucrose, sodium hydroxide, potassium hydroxide, or a combination thereof.

16. The method according to claim 15, wherein the freezing medium contains approximately 2% to approximately 15% DMSO.

17. The method according to claim 15, wherein the freezing medium contains approximately 0.5% to approximately 2% sucrose.

18. The method according to claim 17, wherein the freezing medium contains approximately 1% sucrose.

19. The method according to claim 15, wherein the freezing medium contains about 0.5% to about 1% sodium hydroxide.

20. The method according to claim 19, wherein the freezing medium contains approximately 0.6% sodium hydroxide.

21. The method according to claim 15, wherein the freezing medium contains approximately 0.05% to approximately 0.5% potassium hydroxide.

22. The method according to claim 21, wherein the freezing medium contains approximately 0.1% potassium hydroxide.

23. The method according to claim 2, wherein the preparation of the fluid composition includes thawing the frozen composition.

24. The method according to claim 23, wherein the thawing of the frozen composition is carried out at room temperature or 37°C.

25. The method according to claim 23 or claim 24, further comprising reconstituting the plurality of enucleated cells from the cryopreserved composition after thawing.

26. The method according to claim 25, wherein the reconstitution of the plurality of enucleated cells from the cryopreserved composition is performed using phosphate buffer solution (PBS).

27. The method of claim 25, wherein the reconstitution of the plurality of enucleated cells from the cryopreserved composition is performed using a sodium lactate solution.

28. The method of claim 25, wherein the reconstitution of the plurality of enucleated cells from the cryopreserved composition is performed using physiological saline.

29. The method according to any one of claims 1 to 28, wherein the therapeutic agent comprises a virus, an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, an exogenous peptide, or any combination thereof.

30. The method according to claim 29, wherein the therapeutic agent comprises the virus.

31. The method according to claim 30, wherein the virus is an adeno-associated virus (AAV), an adenovirus, a reovirus, a coxsackievirus, a retrovirus, a poxvirus, a baculovirus, or a herpesvirus.

32. The method according to claim 30, wherein the virus includes an oncolytic virus.

33. The method according to claim 32, wherein the oncolytic virus is adenovirus, human immunodeficiency virus, marabavirus, measles virus, Newcastle disease virus, poliovirus, Seneca Valley virus, parvovirus, Semryqui Forest virus, vesicular stomatitis virus, Sindbisvirus, or any combination thereof.

34. The method according to any one of claims 30 to 33, wherein the amount of the virus delivered to the target is measured by the viral titer in the target cells.

35. The method according to claim 34, wherein the viral titer measured in the target cells is greater than the viral titer measured in target cells otherwise equivalent.

36. The method according to claim 34, wherein the viral titer measured in the target cells is substantially equal to the viral titer measured in target cells otherwise equivalent.

37. The method according to claim 29, wherein the exogenous protein comprises a cytokine or a cytokine receptor-binding fragment thereof.

38. The method according to claim 37, wherein the amount of the cytokine or the cytokine receptor-binding fragment delivered to the target is measured by the secretion of the cytokine or the cytokine receptor-binding fragment from the plurality of enucleated cells.

39. The method according to claim 38, wherein the secretion of the cytokine or the cytokine receptor-binding fragment measured is substantially equal to or greater than the secretion of the cytokine or the cytokine receptor-binding fragment by otherwise equivalent, non-cryopreserved enucleated cells.

40. The method according to claim 38, wherein the secretion of the cytokine or the cytokine receptor-binding fragment measured is substantially equal to or greater than the secretion of the cytokine or the cytokine receptor-binding fragment by cryopreserved nucleated cells that are otherwise equivalent.

41. The method according to claim 29, wherein the exogenous protein comprises an immune checkpoint inhibitor.

42. The method according to claim 41, wherein the immune checkpoint inhibitor comprises an inhibitor specific to PD-L1, PD-1, or a combination thereof.

43. The method according to claim 29, wherein the exogenous protein includes an antigen.

44. The method according to claim 29, wherein the exogenous protein includes an immunomodulatory protein.

45. The method according to claim 29, wherein the therapeutic agent comprises the exogenous RNA molecule.

46. The method according to claim 45, wherein the exogenous RNA molecule encodes a cytokine or a cytokine receptor-binding fragment thereof, a chemokine, or any combination thereof.

47. The method according to claim 46, wherein the exogenous RNA molecule encodes the cytokine or the cytokine receptor-binding fragment thereof.

48. The method according to claim 47, wherein the cytokine or the cytokine receptor binding fragment comprises interleukin-12 (IL-12), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-7 (IL-7), interleukin-21 (IL-21), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-15 (IL-15), or any combination thereof.

49. The method according to claim 46, wherein the exogenous RNA molecule encodes the chemokine.

50. The method according to claim 49, wherein the chemokine comprises stromal cell-derived factor 1α (SDF1α), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 3 (CCL3), C-C motif chemokine ligand 5 (CCL5), C-C motif chemokine ligand 8 (CCL8), C-C motif chemokine ligand 1 (CCL1), CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), C-C motif chemokine ligand 11 (CCL11), CXC motif chemokine ligand 12 (CXCL12), or any combination thereof.

51. The method according to claim 45, wherein the exogenous RNA molecule encodes an immune checkpoint inhibitor, an antigen, or an immunomodulatory protein.

52. The method according to claim 51, wherein the immune checkpoint inhibitor comprises an inhibitor specific to PD-L1, PD-1, or a combination thereof.

53. The method according to any one of claims 1 to 52, further comprising treating a disease or condition in the subject.

54. The method according to claim 53, wherein the disease is cancer.

55. The method according to claim 54, wherein the cancer includes a solid tumor.

56. The method according to claim 54, wherein the cancer is lung cancer, cancer metastasis in lung tissue, liver cancer, or cancer metastasis in liver tissue.

57. The method according to claim 56, wherein the liver cancer is hepatocellular carcinoma or cholangiocarcinoma.

58. The method according to claim 54, wherein the cancer is lung cancer.

59. The method according to claim 58, wherein the lung cancer is small cell lung cancer, non-small cell lung cancer, or bronchial carcinoid.

60. The method according to claim 58, wherein the lung cancer is small cell lung cancer.

61. The method according to claim 58, wherein the lung cancer is a bronchial carcinoid.

62. The method according to claim 58, wherein the lung cancer is non-small cell lung cancer.

63. The method according to claim 62, wherein the non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

64. The method according to any one of claims 1 to 63, further comprising administering the plurality of enucleated cells intravenously to the subject.

65. The method according to any one of claims 1 to 64, wherein the target cells of the subject include cancer cells.

66. The method according to any one of claims 1 to 64, wherein the target cells of the subject include solid tumor cells.

67. The method according to any one of claims 1 to 64, wherein the target cells of the subject include lung cells.

68. The method according to any one of claims 1 to 64, wherein the target cells of the subject include hepatocytes.

69. A composition comprising a plurality of enucleated cells formulated from a cryopreserved composition or a cryopreserved composition, wherein the cryopreserved composition or the cryopreserved composition comprises the plurality of enucleated cells that are cryopreserved or cryopreserved, and at least a subset of the plurality of enucleated cells comprises (i) a therapeutic agent and (ii) an intracellular organelle sufficient to release the therapeutic agent in vivo or ex vivo in an amount approximately equal to or greater than the amount released by otherwise identical, non-cryopreserved and non-cryopreserved enucleated cells.

70. The composition according to claim 69, wherein the plurality of enucleated cells include a diameter that is about 70% or less of the average diameter of the nucleated parent cell.

71. The composition according to claim 69 or claim 70, wherein the plurality of enucleated cells include a diameter ranging from about 1 micrometer (μm) to about 100 μm.

72. The composition according to claim 71, wherein the plurality of enucleated cells include a diameter of approximately 5 μm to approximately 25 μm.

73. The composition according to claim 72, wherein the plurality of enucleated cells include a diameter of approximately 8 μm.

74. The composition according to any one of claims 69 to 73, wherein the therapeutic agent comprises a virus, an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein or exogenous peptide, or any combination thereof.

75. The therapeutic agent comprises the virus, as described in claim 74.

76. The composition according to claim 75, wherein the virus is an adeno-associated virus (AAV), an adenovirus, a reovirus, a coxsackievirus, a retrovirus, a poxvirus, a baculovirus, or a herpesvirus.

77. The composition according to claim 75, wherein the virus includes an oncolytic virus.

78. The composition according to claim 77, wherein the oncolytic virus is adenovirus, human immunodeficiency virus, marabavirus, measles virus, Newcastle disease virus, poliovirus, Seneca Valley virus, parvovirus, Semryqui Forest virus, vesicular stomatitis virus, Sindbisvirus, or any combination thereof.

79. The composition according to claim 75, wherein the amount of the released virus is measured by the viral titer in target cells.

80. The composition according to claim 79, wherein the viral titer measured in the target cells is greater than the viral titer measured in otherwise equivalent target cells.

81. The composition according to claim 79, wherein the viral titer measured in the target cells is substantially equal to the viral titer measured in otherwise equivalent target cells.

82. The composition according to claim 74, wherein the therapeutic agent comprises a cytokine or a cytokine receptor-binding fragment thereof.

83. The composition according to claim 82, wherein the amount of cytokine or cytokine receptor-binding fragment released in vivo or ex vivo is a measure of the secretion of cytokine or cytokine receptor-binding fragment from the plurality of enucleated cells.

84. The composition according to claim 83, wherein the amount of the cytokine or the cytokine receptor-binding fragment measured is approximately equal to or greater than the amount of cytokine or cytokine receptor-binding fragment secreted by enucleated cells that are otherwise equivalent, have not been cryopreserved, and have not undergone cryopreservation or cryopreservation.

85. The composition according to claim 83, wherein the amount of the cytokine or the cytokine receptor-binding fragment measured is approximately equal to or greater than the secretion of the cytokine or the cytokine receptor-binding fragment by cryopreserved or cryopreserved nucleated cells otherwise equivalent.

86. The composition according to claim 74, wherein the exogenous protein comprises an immune checkpoint inhibitor.

87. The composition according to claim 74, wherein the exogenous protein comprises an antigen.

88. The composition according to claim 74, wherein the exogenous protein comprises an immunomodulatory protein.

89. The composition according to claim 86, wherein the immune checkpoint inhibitor comprises an inhibitor specific to PD-L1, PD-1, or a combination thereof.

90. The composition according to claim 74, wherein the therapeutic agent comprises an exogenous RNA molecule.

91. The composition according to claim 90, wherein the exogenous RNA molecule encodes a cytokine or a cytokine receptor-binding fragment thereof, a chemokine, or any combination thereof.

92. The composition according to claim 90, wherein the exogenous RNA molecule encodes a cytokine or a cytokine receptor-binding fragment thereof.

93. The composition according to claim 92, wherein the cytokine or the cytokine receptor binding fragment comprises IL-12, IFN-α, IFN-β, IFN-γ, IL-7, IL-21, TNF-α, GM-CSF, IL-15, or any combination thereof.

94. The composition according to claim 90, wherein the exogenous RNA molecule encodes a chemokine.

95. The composition according to claim 94, wherein the chemokine comprises SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11, CXCL12, or a combination thereof.

96. The composition according to claim 90, wherein the exogenous RNA molecule encodes an immune checkpoint inhibitor, an antigen, or an immunomodulatory protein.

97. The composition according to claim 96, wherein the immune checkpoint inhibitor comprises an inhibitor specific to PD-L1, PD-1, or a combination thereof.

98. The composition according to any one of claims 69 to 97, wherein each of the plurality of enucleated cells lacks a nucleus and includes one or more structural features of a nucleated cell.

99. The composition according to claim 98, wherein the one or more structural features include one or more tunnel nanotubes.

100. The composition according to claim 69, wherein the intracellular organelle comprises the Golgi apparatus, the endoplasmic reticulum, or any combination thereof.

101. A pharmaceutical composition, (a) A composition according to any one of claims 69 to 100, (b) The pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent or carrier.

102. The pharmaceutical composition according to claim 101, wherein the pharmaceutical composition is in the form of a unit dose.

103. The pharmaceutical composition according to claim 101 or claim 102, which is formulated for administration to a target by intrathecal cavity, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or any combination thereof.

104. The pharmaceutical composition according to claim 103, wherein the pharmaceutical composition is formulated for intravenous administration.

105. A pharmaceutical composition according to any one of claims 101 to 104, further comprising at least one additional activator.

106. The pharmaceutical composition according to claim 105, wherein the at least one additional activator comprises a cytokine, growth factor, hormone, enzyme, small molecule, compound, or any combination thereof.

107. It's a kit, (a) The composition according to claims 69 to 100; or (b) The pharmaceutical composition according to any one of claims 101 to 106; and (c) The kit comprising a container for storing the composition or the pharmaceutical composition.

108. The kit according to claim 107, further comprising a resuspension buffer.

109. The kit according to claim 108, wherein the resuspension buffer comprises PBS.

110. The kit according to claim 108, wherein the resuspension buffer contains physiological saline.

111. The kit according to claim 108, wherein the resuspension buffer comprises a sodium lactate solution.

112. The kit according to any one of claims 107 to 111, further comprising instructions for a method of delivering the composition or the pharmaceutical composition to target cells, the method comprising introducing the composition or the pharmaceutical composition to the target cells in vivo or ex vivo under conditions sufficient to deliver the therapeutic agent to the target cells.

113. The kit according to claim 112, further comprising the method of treating the disease or condition of the subject by administering the therapeutic agent to the target cells of the subject.

114. The kit according to claim 113, wherein the disease or condition includes cancer.

115. The kit according to claim 113, wherein the cancer includes a solid tumor.

116. The kit according to claim 114, wherein the cancer is lung cancer, cancer metastasis in lung tissue, liver cancer, or cancer metastasis in liver tissue.

117. The kit according to any one of claims 107 to 116, wherein introducing the composition or the pharmaceutical composition into the target cells comprises administering the composition or the pharmaceutical composition to the target by intrathecal, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray, intracavitary gastrointestinal route, or any combination thereof.

118. The kit according to any one of claims 107 to 117, further comprising at least one additional activator, wherein the at least one additional activator comprises a cytokine, growth factor, hormone, enzyme, small molecule, compound, or any combination thereof.

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