Systems and methods for therapeutic cell manufacturing

Abstract

Described herein is a method for obtaining enucleated cells from nucleated cells. Further described herein is a method for cell processing, comprising providing a composition containing nucleated cells and enucleating at least a portion of the nucleated cells to generate an enucleated cell fraction. Further described herein is a method for cell processing, comprising expressing a heterologous gene product. Further provided is a pharmaceutical composition comprising the enucleated cells.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 254,920, filed October 12, 2021, and U.S. Provisional Patent Application No. 63 / 325,070, filed March 29, 2022, which are incorporated by reference in their entireties. Summary of the Invention

[0002] In some aspects, methods of cell processing are disclosed herein, comprising: a) providing a composition comprising nucleated cells; and b) enucleating a portion of the nucleated cells using continuous flow centrifugation to generate an enucleated cell fraction. In some embodiments, the portion of the nucleated cells comprises about 95% or more of nucleated cells. In some embodiments, the composition provided in a) has a volume comprising about 500 mL or more to about 10,000 mL. In some embodiments, the continuous flow centrifugation creates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition. In some embodiments, the density gradient comprises a polysaccharide density gradient. In some embodiments, the density gradient comprises a range of at least two, at least three, at least four, at least five, at least six, or at least seven density gradients. In some embodiments, the polysaccharide density gradient comprises about 25% polysaccharide, about 17% polysaccharide, about 16% polysaccharide, about 15% polysaccharide, or about 12.5% ​​polysaccharide. In some embodiments, the enucleated cell fraction generated by a single round of continuous flow centrifugation is approximately 6×10 7 More than 250 x 10 enucleated cells 7In some embodiments, the enucleating step b) further comprises creating a density gradient comprising centrifuging the polysaccharide at a maximum centrifugal force of about 30,000 RCF to about 200,000 RCF. In some embodiments, the continuous flow centrifugation creates a zonal centrifugation for separating the at least one enucleated cell from the nucleated cells. In some embodiments, the zonal centrifugation separates the at least one enucleated cell from the nucleated cells based on a size of the at least one enucleated cell. In some embodiments, the zonal centrifugation separates the at least one enucleated cell from the nucleated cells based on a mass of the at least one enucleated cell. In some embodiments, at least one density fraction is obtained from the density gradient, the at least one density fraction comprising a mixed population of nucleated cells and a subset of enucleated cells of the enucleated cell fraction. In some embodiments, the mixed population comprises at least 70% enucleated cells. In some embodiments, the use of continuous flow centrifugation increases the yield of enucleated cells from nucleated cells by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, or more, as compared to embodiments in which enucleated cells are obtained from nucleated cells by methods that do not use continuous flow centrifugation. In some embodiments, the nucleated cells comprise a heterologous polynucleotide. In some embodiments, the method comprises inducing cell death of nucleated cells that are not enucleated after b), wherein cell death is induced by expressing a heterologous gene product encoded by a heterologous polynucleotide in the nucleated cells.

[0003] In some aspects, methods of cell treatment are disclosed herein, comprising: a) providing a composition comprising enucleated cells derived from (i) a first subset of nucleated cells and (ii) a second subset of nucleated cells, the first subset of nucleated cells comprising a heterologous polynucleotide encoding a heterologous gene product; and b) expressing the heterologous gene product, thereby inducing cell death of at least one nucleated cell of the first subset of nucleated cells, and in some embodiments, the heterologous polynucleotide comprises a promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the heterologous gene product comprises herpes simplex virus-thymidine kinase (HSV-TK), cytosine deaminase (CD), varicella zoster TK (VZV-TK), nitroreductase, carboxypeptidase G2 (CPG2), cytochrome P450, or purine nucleoside phosphorylase. In some embodiments, the heterologous gene product comprises FKBP or a caspase. In some embodiments, the heterologous gene product comprises an antigen, and the antigen induces cell death of at least one nucleated cell of the first subset of nucleated cells by eliciting an immune response. In some embodiments, the immune response is an in vivo immune response. In some embodiments, the immune response is an in vitro immune response. In some embodiments, the heterologous polynucleotide is integrated into a chromosome of the nucleated cell. In some embodiments, the heterologous polynucleotide comprises a vector. In some embodiments, expression of the heterologous gene product increases the yield of obtaining enucleated cells from nucleated cells by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, or more, as compared to an embodiment in which the enucleated cells are obtained from the nucleated cells by a method that does not express the heterologous gene product. In some embodiments, the method further comprises cryopreserving the enucleated cell fraction to generate a cryopreserved enucleated cell fraction.In some embodiments, the method further comprises thawing the cryopreserved enucleated cell fraction, and after thawing, the enucleated cells of the cryopreserved enucleated cell fraction are as viable as other comparable enucleated cells that are not cryopreserved. In some embodiments, the nucleated cells comprise stem cells. In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs) from cell lines, adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells, or combinations thereof. In some embodiments, the nucleated cells comprise mesenchymal stromal cells. In some embodiments, the nucleated cells comprise immune cells. In some embodiments, the immune cells comprise lymphocytes or natural killer cells. In some embodiments, the enucleated cells lack a nucleus and comprise one or more intracellular organelles for synthesis or secretion of an exogenous polypeptide in the absence of a nucleus. In some embodiments, the exogenous polypeptide is encoded by a heterologous polynucleotide. In some embodiments, the exogenous polypeptide comprises a therapeutic agent. In some embodiments, the enucleated cells comprise at least one targeting moiety. In some embodiments, the enucleated cells comprise at least one fusion moiety. In some embodiments, the enucleated cells comprise at least one immune evasion moiety. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises 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 that comprises about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm or more. In some embodiments, the diameter comprises about 8 μm.

[0004] In some aspects, disclosed herein are compositions comprising: a) an enucleated cell obtained from a first subset of the plurality of nucleated cells; and b) a second subset of the plurality of nucleated cells, wherein the nucleated cells of the second subset of the plurality of nucleated cells comprise a heterologous polynucleotide encoding a heterologous gene product configured to induce cell death of the nucleated cells. In some embodiments, the heterologous polynucleotide comprises a promoter configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the promoter comprises an inducible promoter embodied such that, upon induction, the heterologous polynucleotide activates transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the constitutively active promoter is configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the heterologous gene product comprises Herpes Simplex Virus-Thymidine Kinase (HSV-TK), Cytosine Deaminase (CD), Varicella Zoster TK (VZV-TK), Nitroreductase, Carboxypeptidase G2 (CPG2), Cytochrome P450, or Purine Nucleoside Phosphorylase. In some embodiments, the heterologous gene product comprises FKBP or a caspase. In some embodiments, the heterologous gene product comprises an antigen, and the antigen induces cell death of at least one nucleated cell of the first subset of nucleated cells by eliciting an immune response. In some embodiments, the immune response is an in vivo immune response. In some embodiments, the immune response is an in vitro immune response. In some embodiments, the heterologous polynucleotide is integrated into a chromosome of the nucleated cell. In some embodiments, the heterologous polynucleotide comprises a vector. In some embodiments, the plurality of nucleated cells comprises stem cells. In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs) from cell lines, adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells, or combinations thereof. In some embodiments, the stem cells comprise mesenchymal stromal cells.In some embodiments, the plurality of nucleated cells comprises immune cells. In some embodiments, the immune cells comprise lymphocytes or natural killer cells. In some embodiments, the enucleated cells lack a nucleus and comprise one or more structural features of the plurality of nucleated cells. In some embodiments, the one or more structural features comprise one or more organelles, one or more tunneling nanotubes, or a combination thereof. In some embodiments, the enucleated cells lack a nucleus and comprise one or more organelles for synthesis or secretion of an exogenous polypeptide in the absence of a nucleus. In some embodiments, the one or more organelles comprise a Golgi apparatus, an endoplasmic reticulum, or a combination thereof. In some embodiments, the exogenous polypeptide comprises a therapeutic agent. In some embodiments, the enucleated cells comprise at least one targeting moiety. In some embodiments, the enucleated cells comprise at least one fusion moiety. In some embodiments, the enucleated cells comprise at least one immune evasion moiety. In some embodiments, the enucleated cells comprise at least one therapeutic moiety. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises about 10 μm to about 100 μm. In some embodiments, the diameter comprises about 8 μm. In some embodiments, the composition is in a dosage form suitable for intravenous administration. In some embodiments, the dosage form comprises a solid dosage form. In some embodiments, the composition comprises a tablet, pill, powder, capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulation, pulsatile release dosage form, multiparticulate dosage form, beads, pellets, or granules. In some embodiments, the enucleated cells are further cryopreserved to generate cryopreserved enucleated cells. In some embodiments, the cryopreserved enucleated cell fraction is thawed and, after thawing, the enucleated cells of the cryopreserved enucleated cell fraction are as viable as other comparable enucleated cells that are not cryopreserved, hi some embodiments, the enucleated cells exhibit viability following cryohibernation.In some embodiments, the enucleated cells exhibit a post-cryopause viability measured 24 hours after cryopreservation that is equal to or greater than the viability of a comparable enucleated cell that is not cryopreserved. In some embodiments, the enucleated cells exhibit viability following cryopreservation. In some embodiments, the enucleated cells exhibit a post-cryopause viability measured 24 hours after cryopreservation that is equal to or greater than the viability of a comparable enucleated cell that is not cryopreserved. In some embodiments, the composition is purified. In some embodiments, the composition is lyophilized. In some embodiments, the enucleated cell and the plurality of nucleated cells are at the same stage of cell differentiation. In some embodiments, the enucleated cell is not derived from the plurality of nucleated cells by cell differentiation. In some embodiments, the enucleated cell is not a terminally differentiated cell. In some embodiments, the enucleated cell is not a platelet. In some embodiments, the enucleated cell is not derived from a platelet lineage cell. In some embodiments, the enucleated cell is not an erythroid cell. In some embodiments, the enucleated cell is not derived from an erythroid lineage cell.

[0005] In some aspects, described herein are a plurality of enucleated cells comprising a plurality of the enucleated cells disclosed herein.

[0006] In some aspects, disclosed herein is a pharmaceutical composition comprising a) an enucleated cell disclosed herein and b) a pharma- ceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is in a unit dosage form. In some embodiments, the pharmaceutical composition is formulated for administration to a subject intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, intratumorally, intrapulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled aerosol form, by intraluminal-GI route, or combinations thereof. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition comprises at least one additional active agent. In some embodiments, the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or a combination thereof.

[0007] In some aspects, described herein is a kit that includes: a) a composition disclosed herein or a pharmaceutical composition disclosed herein; and b) a container.

[0008] In some aspects, methods of cell processing are disclosed herein, the methods comprising providing a composition comprising nucleated cells and enucleating a portion of the nucleated cells using continuous flow centrifugation to generate an enucleated cell fraction, the portion of the nucleated cells comprising about 70% or more of the nucleated cells. In some embodiments, the composition provided in the method has a volume comprising between about 10 milliliters (mL) or more and about 10,000 mL. In some embodiments, the composition has a volume comprising about 10 milliliters (mL), about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 80 mL, about 100 mL, about 200 mL, about 300 mL, about 500 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, about 9000 mL, or about 10,000 mL or more. In some embodiments, the continuous flow centrifugation creates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition. In some embodiments, the density gradient comprises a polysaccharide density gradient. In some embodiments, the polysaccharide density gradient comprises a Ficoll density gradient. In some embodiments, the Ficoll density gradient comprises at least two, at least three, at least four, at least five, at least six, or at least seven ranges of density gradients. In some embodiments, the Ficoll density gradient comprises about 25% Ficoll, about 17% Ficoll, about 16% Ficoll, about 15% Ficoll, or about 12.5% ​​Ficoll. In some embodiments, the portion of nucleated cells comprises about 75% or more of nucleated cells. In some embodiments, the portion of nucleated cells comprises about 80% or more of enucleated cells. In some embodiments, the portion of nucleated cells comprises about 90% or more of enucleated cells. In some embodiments, the enucleated cell fraction generated by a single run of continuous flow centrifugation comprises about (i) 6×10 7 of enucleated cells, (ii) 7 × 10 7enucleated cells, (iii) 8 × 10 7 enucleated cells, (iv) 9 × 10 7 of enucleated cells (v) 10 × 10 7 enucleated cells, (vi) 15 × 10 7 of enucleated cells, (vii) 20 × 10 7 of enucleated cells, (viii) 50 × 10 7 of enucleated cells, (ix) 100 × 10 7 of enucleated cells, (x) 150 × 10 7 of enucleated cells, (xi) 200 × 10 7 of enucleated cells, or (xii) 250 × 10 7or more enucleated cells. In some embodiments, the method includes generating a density gradient comprising centrifuging the polysaccharides at an acceleration ranging from 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 method includes generating a density gradient comprising centrifuging the polysaccharides at an acceleration ranging from about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, or about 50 minutes. In some embodiments, the method includes generating a density gradient comprising centrifuging the polysaccharides at an acceleration ranging from about 30 minutes. In some embodiments, the enucleating step of the method further comprises generating a density gradient comprising centrifuging the polysaccharides at a minimal deceleration. In some embodiments, the enucleating step of the method further comprises creating a density gradient comprising centrifuging the polysaccharides at a maximum centrifugal force of about 30,000 relative centrifugal force (RCF) to about 200,000 RCF. In some embodiments, the enucleating step of the method further comprises creating a density gradient comprising centrifuging the polysaccharide at a maximum centrifugal force of about 50,000 RCF to about 120,000 RCF. In some embodiments, the enucleating step of the method using continuous flow centrifugation to generate an enucleated cell fraction is performed using an ultracentrifuge. In some embodiments, the enucleating step of the method using continuous flow centrifugation to generate an enucleated cell fraction is performed using a fixed angle centrifuge or a swinging bucket centrifuge. In some embodiments, the nucleated cells comprise a heterologous polynucleotide. In some embodiments, the method comprises inducing cell death of the non-enucleated nucleated cells, the cell death being induced by expression of at least one heterologous gene encoded by the heterologous polynucleotide. In some embodiments, the continuous flow centrifugation creates zonal centrifugation to separate at least one enucleated cell from the nucleated cells. In some embodiments, the zonal centrifugation separates at least one enucleated cell from the nucleated cells based on the size of the at least one enucleated cell. In some embodiments, zonal centrifugation separates the at least one enucleated cell from the nucleated cells based on the mass of the at least one enucleated cell.In some embodiments, the zonal centrifugation separates at least one enucleated cell from nucleated cells based on the size and mass of the at least one enucleated cell. In some embodiments, at least one density fraction is obtained from the density gradient, and the at least one density fraction comprises a mixed population of nucleated cells and a subset of enucleated cells of the enucleated cell fraction. In some embodiments, the mixed population comprises at least 70% enucleated cells. In some embodiments, the mixed population comprises at least 99% enucleated cells.

[0009] In some embodiments, a method of cell treatment is disclosed herein, comprising providing a composition comprising enucleated cells derived from a first subset of nucleated cells and a second subset of said nucleated cells, said first subset of said nucleated cells comprising a heterologous polynucleotide encoding a heterologous gene product, and expressing said heterologous gene product, thereby inducing cell death of at least one nucleated cell of said first subset of said nucleated cells. In some embodiments, the heterologous polynucleotide comprises a promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is induced by contacting nucleated cells with a temperature less than 37° C. In some embodiments, the inducible promoter comprises dsrA or CIRP. In some embodiments, the inducible promoter is induced by contacting nucleated cells with a temperature greater than 37° C. In some embodiments, the inducible promoter comprises heat shock protein 70 (HSP70, e.g., NCBI gene ID 3308), heat shock protein 90 (HSP90, e.g., NCBI gene ID 3320), growth arrest and DNA damage inducible gene 153 (GADD153, e.g., NCBI gene ID 1649), multidrug resistance mutation 1 (MDR1, e.g., NCBI gene ID 5243), or cytomegalovirus (HSE-CMV, e.g., NCBI gene ID 3077513). In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a molecule. In some embodiments, the molecule comprises rtTA, TRE, TetR, Cumate, rapamycin, abscisic acid, IPTG, or metallothionein. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with light. In some embodiments, the inducible promoter comprises CIB1-CRY2 or GAL4-VVD. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a hormone. In some embodiments, the inducible promoter comprises estradiol-Gal4. In some embodiments, the promoter is a constitutively active promoter.In some embodiments, the constitutively active promoter is configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the heterologous gene product comprises Herpes Simplex Virus-Thymidine Kinase (HSV-TK), Cytosine Deaminase (CD), Varicella Zoster TK (VZV-TK), Nitroreductase, Carboxypeptidase G2 (CPG2), Cytochrome P450, or Purine Nucleoside Phosphorylase. In some embodiments, the heterologous gene product comprises FKBP or a caspase. In some embodiments, the heterologous gene product comprises an antigen. In some embodiments, the heterologous polynucleotide is integrated into a chromosome of the nucleated cell. In some embodiments, the heterologous polynucleotide comprises a vector. In some embodiments, the method further comprises cryopreserving the enucleated cell fraction to generate a cryopreserved enucleated cell fraction. In some embodiments, the method further comprises thawing the cryopreserved enucleated cell fraction, and after thawing, the enucleated cells of the cryopreserved enucleated cell fraction are as viable as other comparable enucleated cells that were not cryopreserved. In some embodiments, the nucleated cells comprise stem cells. In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs) from cell lines, adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells, or combinations thereof. In some embodiments, the nucleated cells comprise mesenchymal stromal cells. In some embodiments, the enucleated cells lack a nucleus and comprise one or more organelles for synthesis or secretion of an exogenous polypeptide in the absence of a nucleus. In some embodiments, the exogenous polypeptide comprises a therapeutic agent. In some embodiments, the enucleated cells comprise at least one targeting moiety. In some embodiments, the enucleated cells comprise at least one fusion moiety. In some embodiments, the enucleated cells comprise at least one immune evasion moiety. In some embodiments, the enucleated cells comprise at least one therapeutic moiety.In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises 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 provided in the method. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises about 70% or less of the average diameter of the nucleated cells provided in the method. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises about 5 micrometers (μm), about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm or more. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises about 10 μm to about 100 μm. In some embodiments, the diameter comprises about 8 μm.

[0010] In some aspects, compositions are described herein that include enucleated cells obtained from a first subset of the plurality of nucleated cells, and a second subset of the plurality of nucleated cells, where the nucleated cells of the second subset of the plurality of nucleated cells include a heterologous polynucleotide encoding a heterologous gene product configured to induce cell death of the nucleated cells. In some embodiments, the heterologous polynucleotide includes a promoter configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the promoter includes an inducible promoter configured, upon induction, to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the inducible promoter is induced by contacting the nucleated cells with a temperature less than 37° C. In some embodiments, the inducible promoter includes dsrA or CIRP. In some embodiments, the inducible promoter is induced by contacting the nucleated cells with a temperature greater than 37° C. In some embodiments, the inducible promoter comprises heat shock protein 70 (HSP70, e.g., NCBI gene ID 3308), heat shock protein 90 (HSP90, e.g., NCBI gene ID 3320), growth arrest and DNA damage inducible gene 153 (GADD153, e.g., NCBI gene ID 1649), multidrug resistance mutation 1 (MDR1, e.g., NCBI gene ID 5243), or cytomegalovirus (HSE-CMV, e.g., NCBI gene ID 3077513). In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a molecule. In some embodiments, the molecule comprises rtTA, TRE, TetR, Cumate, rapamycin, abscisic acid, IPTG, or metallothionein. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with light. In some embodiments, the inducible promoter comprises CIB1-CRY2 or GAL4-VVD. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a hormone, hi some embodiments, the inducible promoter comprises estradiol-Gal4.In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the constitutively active promoter is configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the heterologous gene product comprises Herpes Simplex Virus-Thymidine Kinase (HSV-TK), Cytosine Deaminase (CD), Varicella Zoster TK (VZV-TK), Nitroreductase, Carboxypeptidase G2 (CPG2), Cytochrome P450, or Purine Nucleoside Phosphorylase. In some embodiments, the heterologous gene product comprises FKBP or a caspase. In some embodiments, the heterologous gene product comprises an antigen. In some embodiments, the heterologous polynucleotide is integrated into a chromosome of a nucleated cell. In some embodiments, the heterologous polynucleotide comprises a vector.

[0011] In some aspects, compositions are described herein that include enucleated cells obtained from a first subset of a plurality of nucleated cells, and about 0.1% or less by volume of the composition further includes a second subset of the plurality of nucleated cells. In some embodiments, the plurality of nucleated cells includes stem cells. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs) from a cell line, adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells, or combinations thereof. In some embodiments, the nucleated cells include mesenchymal stromal cells. In some embodiments, the enucleated cells lack a nucleus and include one or more structural features of a plurality of nucleated cells. In some embodiments, the one or more structural features include one or more organelles, one or more tunneling nanotubes, or combinations thereof. In some embodiments, the enucleated cells lack a nucleus and include one or more organelles for synthesis or secretion of an exogenous polypeptide in the absence of a nucleus. In some embodiments, the one or more organelles include a Golgi apparatus, an endoplasmic reticulum, or combinations thereof. In some embodiments, the exogenous polypeptide includes a therapeutic agent. In some embodiments, the enucleated cells comprise at least one targeting moiety. In some embodiments, the enucleated cells comprise at least one fusion moiety. In some embodiments, the enucleated cells comprise at least one immune evasion moiety. In some embodiments, the enucleated cells comprise at least one therapeutic moiety. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the average diameter of the nucleated cells provided in the composition. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises at least about 70% of the average diameter of the nucleated cells provided in the composition. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises about 10 μm to about 100 μm. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter comprising about 1 μm, about 5 μm, about 8 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm or more. In some embodiments, the diameter comprises about 8 μm.In some embodiments, the composition is in a dosage form suitable for intravenous administration. In some embodiments, the dosage form comprises a solid dosage form. In some embodiments, the composition comprises a tablet, a pill, a powder, a capsule, a solid dispersion, a solid solution, a biodegradable dosage form, a controlled release formulation, a pulsatile release dosage form, a multiparticulate dosage form, a bead, a pellet, or a granule. In some embodiments, the total number of enucleated cells in the composition comprises about 10 million enucleated cells, about 20 million enucleated cells, about 30 million enucleated cells, about 40 million enucleated cells, about 45 million enucleated cells, about 50 million enucleated cells, about 55 million enucleated cells, about 60 million enucleated cells, about 65 million enucleated cells, about 70 million enucleated cells, about 75 million enucleated cells, about 80 million enucleated cells, about 90 million enucleated cells, or about 100 million or more enucleated cells. In some embodiments, the enucleated cells are further cryopreserved to generate cryopreserved enucleated cells. In some embodiments, the cryopreserved enucleated cell fraction is thawed, and after thawing, the enucleated cells of the cryopreserved enucleated cell fraction are as viable as other comparable enucleated cells that were not cryopreserved. In some embodiments, the enucleated cells exhibit viability after cryopreservation. In some embodiments, the enucleated cells exhibit viability after cryopreservation measured after 24 hours of cryopreservation that is equal to or greater than the viability of comparable enucleated cells that were not cryopreserved. In some embodiments, the enucleated cells exhibit viability after cryopreservation measured after 24 hours of cryopreservation that is equal to or greater than the viability of comparable enucleated cells that were not cryopreserved. In some embodiments, the composition is purified. In some embodiments, the composition is lyophilized. In some embodiments, the enucleated cells and the plurality of nucleated cells are at the same stage of cell differentiation. In some embodiments, the enucleated cells are not obtained from the plurality of nucleated cells by cell differentiation. In some embodiments, the enucleated cells are not terminally differentiated cells. In some embodiments, the enucleated cells are not platelets. In some embodiments, the enucleated cells are not derived from a platelet lineage cell. In some embodiments, the enucleated cells are not red blood cells. In some embodiments, the enucleated cells are not derived from an erythroid lineage cell.

[0012] In some aspects, a plurality of enucleated cells are described herein, comprising a plurality of enucleated cells described herein.

[0013] In some aspects, described herein are pharmaceutical compositions comprising the enucleated cells described herein and a pharma- ceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is in unit dosage form. In some embodiments, the pharmaceutical composition is formulated for administration to a subject intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, intratumorally, intrapulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled aerosol form, by intraluminal-GI route, or combinations thereof. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition comprises at least one additional active agent. In some embodiments, the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or a combination thereof.

[0014] In some aspects, described herein is a kit comprising a composition described herein or a pharmaceutical composition described herein and a container.

[0015] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated 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. To the extent that the publications and patents or patent applications incorporated by reference conflict with disclosures contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0016] Several novel features of the methods and compositions disclosed herein are set forth in this disclosure. The features and advantages of the methods and compositions disclosed herein will be better understood by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosed compositions and methods are utilized, and the accompanying drawings.

[0017] [Figure 1] 1 illustrates a flow chart showing non-limiting steps of a process for an enucleated cell composition or pharmaceutical composition for delivery of a therapeutic agent according to embodiments of the present disclosure. [Diagram 2] A process for producing enucleated cells for delivery of therapeutic agents according to various embodiments described herein is described. [Diagram 3] 1 illustrates a timeline for generation of enucleated cells for delivery of single domain antibodies according to various embodiments compared to a typical biologics development timeline. [Figure 4A] 1 is a representative graph showing the relative fold change in viable or enucleated cells ("cytoplasts") over time. [Figure 4B] 1 is a representative graph showing viable cells and cytoplasts after recovery from cryopreservation (cryopreservation). [Figure 4C] Representative graph showing relative viability of cytoplasts 24 hours after enucleation (fresh cytoplasts) or 24 hours after recovery from cryopreservation after enucleation (cryopreservation), where fresh and cryopreserved cytoplasts are compared to the viability of cytoplasts 4 hours after enucleation. Mean ± SEM, n=10. [Figure 5A] 1 is a representative line graph showing the viability of MSCs and MSC-derived cytoplasts immediately after recovery from cryostasis at 4° C. for the indicated times. Viability was assessed by automated cell counting (Cell Countess) using trypan blue dye exclusion and expressed as a percentage of input cell number. [Figure 5B]Representative bar graph comparing migrated MSCs and MSC-derived cytoplasts in a Boyden chamber assay immediately after recovery from cryostasis at 4° C. for the indicated times. Cells and cytoplasts were allowed to migrate for 3 hours in the bottom chamber without serum (negative control) or with 10% Premium FBS (P-FBS) as a chemoattractant, and counts were normalized to a loading control. [Figure 6A] 13 is a representative flow cytometry graph showing the number of events counted versus the signal intensity of cell surface CXCR4 expression by fluorescent antibody on engineered cytoplasts and engineered parental MSCs as analyzed by FlowJo. [Figure 6B] Representative bar graph showing the percentage of migratory cells or cytoplasts that migrated to the underside of the Boyden chamber membrane compared to loading control. Mean ± SEM, n = 10. MSCs as well as MSC-derived cytoplasts with or without engineered CXCR4 receptors were allowed to migrate towards the indicated concentrations of SDF-1α for 2 hours in a Boyden chamber assay. [Figure 7A] 13 is a representative flow cytometry graph showing the number of events counted versus the signal intensity of cell surface PSGL1 expression with fluorescent antibody on engineered cytoplasmic and engineered parental MSCs as analyzed by FlowJo. [Figure 7B] 1 is a representative graph showing cell surface binding of P-selectin to engineered MSCs and MSC-derived cytoplasts as determined by flow cytometry. MSC control = parental MSCs. Engineered MSCs = MSCs engineered with PSGL1 / Fut7. Engineered cytoplasts = MSC-derived cytoplasts engineered with PSGL1 / Fut7. [Figure 8A] 13 is a representative flow cytometry graph showing the number of events counted versus the signal intensity of engineered cytoplasm and cell surface of mCD47 expression on engineered MSCs as analyzed by FlowJo. [Figure 8B]Representative bar graph showing the number of viable cytoplasm (DiD+) that were not phagocytosed by macrophages (F4 / 80- and CD11b-), indicating that cytoplasm escaped macrophage phagocytosis in the lung. Mean ± SEM, n=3. DiD dye-labeled control cytoplasm or engineered cytoplasm (mCD47 cytoplasm) was injected retro-orbitally into the vasculature of mice. After 24 hours, tissues were harvested and stained with two different pan-macrophage markers (F4 / 80 and CD11b). [Figure 8C] Representative bar graph showing viable cytoplasm (DiD+) that was not phagocytosed by macrophages (F4 / 80- and CD11b-), indicating that cytoplasm escaped macrophage phagocytosis in the liver. Mean ± SEM, n=3. DiD dye-labeled control cytoplasm or engineered cytoplasm (mCD47 cytoplasm) was injected retro-orbitally into the vasculature of mice. After 24 hours, tissues were harvested and stained with two different pan-macrophage markers (F4 / 80 and CD11b). [Figure 9A] Representative scatter plots showing the number of DiD-labeled MSCs or cytoplasts detected in the lung. To generate 3D cytoplasts, MSCs were cultured under standard adherent conditions (2D) or in suspension by the hanging drop method (3D). MSCs and cytoplasts were labeled with Vybrant® DiD dye and injected retro-orbitally into the vasculature of C57BL / 6 mice. Tissues were harvested 24 hours later and cell suspensions were analyzed by flow cytometry. Mean ± SEM, n=2. [Figure 9B] Representative scatter plots showing the number of DiD-labeled MSCs or cytoplasts detected in the liver. To generate 3D cytoplasts, MSCs were cultured under standard adherent conditions (2D) or in suspension by hanging drop method (3D). MSCs and cytoplasts were labeled with Vybrant® DiD dye and injected retro-orbitally into the vasculature of C57BL / 6 mice. Tissues were harvested 24 hours later and cell suspensions were analyzed by flow cytometry. Mean ± SEM, n=2. [Figure 9C]Representative scatter plots showing the number of Vybrant® DiD-labeled MSCs or cytoplasts detected in the spleen. To generate 3D cytoplasts, MSCs were cultured under standard adherent conditions (2D) or in suspension by hanging drop method (3D). MSCs and cytoplasts were labeled with DiD dye and injected retro-orbitally into the vasculature of C57BL / 6 mice. Tissues were harvested 24 hours later and cell suspensions were analyzed by flow cytometry. Mean ± SEM, n=2. [Figure 10] We describe cell surface staining of fluorescein isothiocyanate (FITC)-labeled annexin V on mesenchymal stromal stem cells (MSCs) or cytoplasts, analyzed by flow cytometry for the analysis of cell viability. [Figure 11A] 1 illustrates an exemplary gradient produced by the methods described herein. [Figure 11B] 11A illustrates an exemplary image of a viable enucleated cell obtained from a nucleated cell. FIG. 11B further illustrates an exemplary diameter reduction of the cell due to enucleation. The diameter of the cell decreases after enucleation (from 18.37 μm to 15.36 μm). [Figure 11C] Illustrates that the viability of nucleated cells did not change significantly before or after enucleation. Figure 11C further illustrates the enucleation efficiency and viability of cells following enucleation via the methods described herein. [Figure 11D] Fluorescence images of cells immediately after enucleation (top two images) and 24 hours after enucleation (bottom image) are illustrated. [Figure 12A] FIG. 1 illustrates the density gradient measured after continuous flow centrifugation, performed by ultracentrifugation runs (5 Ficoll layers in grey, 3 Ficoll layers in dark grey, and continuous flow indicated by callout lines). [Figure 12B] Representative images of the enucleation efficiency test are illustrated. Each field image was obtained using bright field (total cells) and Hoechst channel (total nucleated cells).

[0018] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments and the accompanying drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] overview Existing techniques for using centrifugal force to displace nuclei in adherent cells, i.e., enucleation, have drawbacks that make them inoperable on enucleated cell platforms for large-scale biomedical applications. Such drawbacks include, but are not limited to, the small swinging bucket volume, which varies from a few milliliters to hundreds of milliliters, and the overall length of time for pelleting, resulting in small-scale production with low efficiency. These drawbacks severely limit the number of cells that can be processed at one time, making it difficult to achieve optimal cell density in each centrifuge bucket for large-scale manufacturing. While large-scale techniques such as continuous-flow centrifugation have been used to separate cell types from one another, these techniques have not been used to displace nuclei in nucleated cells to generate enucleated cells.

[0020] To eliminate these drawbacks, the inventors of the present disclosure have developed a method for cell processing that involves enucleating cells using continuous-flow centrifugation. Continuous-flow centrifugation reduces processing time and allows large volumes of material to be centrifuged at high centrifugal forces without the tedium of repeatedly filling and decanting centrifuge tubes or frequently starting and stopping the rotor. The combination of high centrifugal forces and high throughput makes continuous-flow processing amenable to large-scale enucleation of cells for biomedical applications.

[0021] In addition, quality control of the enucleated cell platform for biomedical applications is a challenge that becomes more severe with large-scale manufacturing. Some of the many advantages of the enucleated cells disclosed herein result from the absence of a nucleus, such as unwanted gene transfer in vivo, limited life span in vivo, etc. However, with existing large-scale manufacturing techniques, some of the nucleated parent cells in the resulting therapeutic composition eliminate the advantages of the enucleated cell platform.

[0022] To improve quality control, the inventors of the present disclosure have engineered nucleated parent cells (from which the enucleated cells are derived) with a biomolecular "suicide switch" that functions to kill the nucleated parent cell when expression or activity of the suicide switch is induced. As disclosed herein, the utilization of a biomolecular suicide switch is a fail-safe for maximizing the enucleated cell fraction in the resulting composition. In some embodiments, a heterologous polynucleotide encoding a biomolecular suicide switch under the control of an inducible promoter is introduced into the nucleated parent cell using a suitable technique, such as transfection or transduction. After enucleation, if nucleated cells remain, the inducible promoter may be activated, expressing the biomolecular suicide switch and inducing cell death. Without being bound to any particular theory, as disclosed herein, the deployment of a biomolecular "suicide switch" as a measure of quality control is suitable for virtually any cell therapy or cell-based therapy delivery platform where nucleated cells are not required. Such cell therapies include, but are not limited to, tumor infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, natural killer (NK) cell therapy, etc. Such cell-mediated therapeutic delivery platforms include, but are not limited to, red blood cells, platelets, stem cells, white blood cells, and others, such as those disclosed in Yu H, et al. Cell-mediated targeting drugs delivery systems. Drug Deliv. 2020 Dec; 27(1): 1425-1437, which is incorporated by reference in its entirety. Such techniques for utilizing biomolecular suicide switches can result in increased enucleation efficiency compared to mechanical enucleation techniques that do not utilize programmed cell death. In some embodiments disclosed herein, enucleation efficiency is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, or 200% or more.

[0023] In addition to advances in manufacturing scalability and quality control, the enucleated cell platform described herein itself has certain advantages over existing cell-based therapeutic platforms that make it uniquely suitable for large-scale use as a therapeutic composition. The enucleated cells described herein can be found in U.S. Patent Application No. 10,927,349, which is incorporated herein by reference in its entirety. In addition, further utilities and advantages of the enucleated cells disclosed herein are discussed in International Application No. PCT / US2022 / 018007, filed February 25, 2022 and published as WO / 20221 / 83057A1, and U.S. Patent Application No. 17 / 885,867, filed August 11, 2022 and published as WO / 20211 / 63222A1, each of which is incorporated herein by reference in its entirety. For example, there are certain therapeutic applications of cell delivery platforms, such as in response to pathogen outbreaks, where existing manufacturing timelines may limit the scalability and speed required to address public health emergencies due to pathogen outbreaks. Existing therapeutic cell therapies, which require extensive engineering, take a minimum of about 12 months to develop. The enucleated cells disclosed herein can be extensively engineered before and after enucleation (e.g., with targeting moieties specific to target tissues, immune system evasion moieties to reduce phagocytosis in vivo, etc.), but can then be preserved without sacrificing viability once recovered by appropriate means disclosed herein (e.g., lyophilization, cryostasis, cryopreservation). When a new pathogen or a new strain of a known pathogen is identified, the biological activity of the enucleated cells (already engineered to express appropriate targeting moieties, immune system evasion moieties, immune activators, etc.) can be restored (e.g., rehydrated, thawed, etc.) and further engineered to express or deliver a therapeutic agent for the prevention or treatment of infection with that recently discovered pathogen or strain. These advantages can be seen in FIG. 3, which illustrates the process for producing enucleated cells of the present disclosure in approximately 2 months, compared to a suitable timeline of 12 months or more.

[0024] Existing platforms for red blood cells or platelets therapy are enucleated by erythropoiesis, in which blood cells undergo terminal differentiation and some of the organelles and ribosomes responsible for protein synthesis and secretion are removed. Thus, the resulting red blood cells or platelets lose cell-like functions after enucleation by erythropoiesis (e.g., protein expression, secretion, cell motility, chemokine sensing, homing ability, etc.) that may be important for therapeutic applications such as the production, delivery, and secretion of therapeutic agents in vivo. In contrast, the enucleated cells described herein retain one or more organelles endogenous to the parent cell after enucleation. In some embodiments, all of one or more organelles are retained. In some embodiments, less than all of one or more organelles are retained. In some embodiments, the Golgi apparatus and / or endoplasmic reticulum are retained, which are involved in protein synthesis and secretion. By retaining one or more intracellular organelles, the enucleated cell is capable of at least partially synthesizing or releasing a biomolecule disclosed herein (e.g., a single domain antibody or portion thereof, a targeting moiety, an immune evasion moiety, etc.) in the absence of a nucleus.

[0025] The enucleated cells disclosed herein can be derived from virtually any nucleated cell (referred to herein as a "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 cell is derived from an induced pluripotent stem cell (iPSC). In some embodiments, the parent cell is not an erythrocyte. In some embodiments, the parent cell is not an erythrocyte progenitor cell. In some embodiments, the parent cell is not a vascular endothelial cell. In some embodiments, the parent cell is not an endothelial precursor cell.

[0026] Methods are described herein for producing enucleated cells with increased quantity and purity, and the produced enucleated cells can be formulated into compositions or pharmaceutical compositions for treating a disease or condition in a subject in need of such treatment. FIG. 1 illustrates 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 expansion. In some embodiments, the nucleated cells (101) can further be immortalized or derived from a cell line. In some embodiments, the nucleated cells can be engineered (103) to include a heterologous polynucleotide (102), which encodes a suicide moiety (e.g., a suicide gene) to kill the nucleated cells when necessary. The nucleated cells can then be enucleated by continuous flow centrifugation (104). The use of continuous flow centrifugation to enucleate cells presents an improvement over currently available methods for enucleation, and enucleation conducted via continuous flow centrifugation increases the quantity (e.g., yield) or purity of enucleated cells obtained from nucleated cells. After obtaining a composition of enucleated cells (105) (which may have residual nucleated cells), the composition can be further purified for enucleated cells by selecting for a marker of enucleated cells (106) or by inducing cell death of the remaining residual nucleated cells (107) to obtain a portion of the enucleated cells (108). The portion of the enucleated cells can be cryostasized (109), cryopreserved (110), lyophilized (111), or combinations thereof, and formulated into a composition or pharmaceutical composition for delivery of a therapy to treat a disease or disorder in a subject.

[0027] In some embodiments, the methods of enucleation disclosed herein result in a composition comprising enucleated cells (also referred to herein as the "enucleated cell fraction" of the composition). In some embodiments, the composition comprises about one (1) percent (%) or less by volume of remaining nucleated cells that were not enucleated (also referred to herein as the "nucleated cell fraction" of the composition). In some embodiments, the nucleated cell fraction comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or 0.9% or less by volume of the composition. In some embodiments, the nucleated cell fraction comprises about 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% by volume of the composition. In some embodiments, the nucleated cell fraction comprises 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% by volume 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% by volume of the composition. In some embodiments, the nucleated cell fraction comprises about 0.6% to about 0.7%, about 0.6% to about 0.8%, about 0.6% to about 0.9%, or about 0.6% to about 1.0% by volume of the composition. 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% by volume of the composition.In some embodiments, the nucleated cell fraction comprises about 0.8% to about 0.9%, or 0.8% to about 1.0% by volume of the composition. In some embodiments, the nucleated cell fraction comprises about 0.9% to about 1.0% by volume of the composition. In some embodiments, the nucleated cell fraction is eliminated by induced cell death following enucleation. In some embodiments, induced cell death is utilized using a biomolecular suicide switch that is expressed in response to exposure to an external stimulus, e.g., a small molecule drug (e.g., a rimiducid), a prodrug (e.g., ganciclovir), etc.

[0028] Also described herein are pharmaceutical compositions and formulations comprising the compositions described herein and pharma- ceutical acceptable carriers, excipients, diluents, or nebulizers.The pharmaceutical compositions are provided in pharmaceutical formulations.In some embodiments, the pharmaceutical formulations are formulated for administration to subjects as combination therapy (e.g., prodrugs, adjuvants, additional therapeutic agents, or other therapies) or as monotherapy.In some embodiments, the pharmaceutical formulations are formulated for administration to the site of action, such as systemic or intratumoral administration.

[0029] Disclosed herein is a kit comprising a composition disclosed herein and a packaging material configured to deliver the composition to an individual. The kit disclosed herein may include an enucleated cell fraction and a composition comprising less than 0.1% of a nucleated cell fraction. In some embodiments, the kit further comprises instructions for further manipulation of the enucleated cells in the enucleated cell fraction, e.g., producing or secreting a therapeutic agent disclosed herein. In some embodiments, the kit further comprises a stimulant used to trigger the expression or activity of a biomolecular suicide switch in the nucleated cell fraction of the composition. In either case, the instructions may further comprise instructions for how to formulate the resulting composition into a pharmaceutical formulation for administration to a subject as disclosed herein.

[0030] composition Disclosed herein are compositions and formulations thereof comprising enucleated cells that can be extensively engineered to express an active agent or a portion thereof in the absence of a nucleus. Such enucleated cells are living cell-like entities that can synthesize, release (e.g., secrete), or deliver an active agent to a target cell or tissue in the absence of a nucleus. The compositions disclosed herein can be stored in an arrested biological stage by means such as cryostasis, cryopreservation, or lyophilization for any period of time without affecting the viability of the enucleated cells even if biological activity is restored. Additionally, the compositions disclosed herein contain about 0.1% or less nucleated cells (e.g., parent cells that were not enucleated during the enucleation process), optimizing the compositions disclosed herein for therapeutic use. The enucleated cells (also referred to herein as "cytoplasts") may further comprise naturally occurring cell surface molecules that were retained from the parent cells. In some embodiments, the enucleated cells further comprise exogenous molecules such as targeting moieties, transmembrane moieties, additional therapeutic agents (e.g., other than the active agent), such as those disclosed herein.

[0031] enucleated cells The enucleated cells of the present disclosure are obtained or derived from a corresponding nucleated cell (referred to herein as a "parent cell"). The parent cell may be derived from a variety of different cell types, including eukaryotic cells. For example, the enucleated cells may be derived from adult stem cells, mesenchymal stromal stem cells (MSCs), natural killer (NK) cells, macrophages, myoblasts, neutrophils, endothelial cells, endothelial progenitor cells, and / or fibroblasts. In some embodiments, the enucleated cells are derived from mesenchymal stromal cells. In some embodiments, the enucleated cells are derived from induced pluripotent stem cells (iPSCs). In some embodiments, the parent cell is derived from a cell that has been immortalized using a suitable method. In some embodiments, the enucleated cells comprise or retain one or more structural features of the parent cell, including intracellular organelles, one or more tunneling nanotubes, or a combination thereof. In some embodiments, the enucleated cells comprise one or more intracellular organelles for synthesis or secretion of an exogenous polypeptide (e.g., a therapeutic agent) in the absence of a nucleus. In some embodiments, the one or more intracellular organelles comprises the Golgi apparatus, the endoplasmic reticulum, or a combination thereof. In some embodiments, the enucleated cells comprise or express any one of the therapeutic agents described herein.

[0032] In some embodiments, the cells may be 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, protozoan cells, cells of plants (e.g., cells of crop plants, fruits, vegetables, cereals, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, coniferous trees), cells of gymnosperms, ferns, club mosses, mosses, liverworts, mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh), seaweed (e.g., kelp), fungal cells (e.g., cells from yeast, mushrooms), animal cells, invertebrate (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.) cells, vertebrate (e.g., fish, amphibian, reptile, bird, mammal) cells, mammalian (e.g., pig, cow, goat, sheep, rodent, rat, mouse, non-human primate, human, etc.) cells, and others. The cell is a cell that is not derived from a natural organism (e.g., a synthetically produced cell, sometimes also referred to as an artificial cell). In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem or progenitor cell. In some cases, the cell is a mesenchymal stem or progenitor cell. In some cases, the cell is a hematopoietic stem or progenitor cell. In some embodiments, the cell is a muscle cell, a skin cell, a blood cell, or an immune cell. Other non-limiting examples of cells include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, and cytokine-induced killer (CIK) cells; myeloid cells such as granulocytes (basophilic granulocytes, eosinophils, neutrophilic granulocytes / hypersegmented neutrophils), monocytes / macrophages, red blood cells (reticulocytes), mast cells, platelets / megakaryocytes, and dendritic cells; thyroid (thyroid epithelial cells,Cells of the endocrine system, including parathyroid (chief parathyroid cells, eosinophilic cells), parathyroid (chromaffin cells), and pineal (pineal cells) cells; cells of the nervous system, including glial cells (astrocytes, microglia), large neurosecretory cells, stellate cells, Bechtel cells, and pituitary gland (gonadotropes, corticotropes, thyrotropes, growth hormones, and prolactin-producing cells); cells of the respiratory system, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, and dust cells cells of the circulatory system including myocardiocytes and pericytes; cells of the digestive system including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells including silver-reducing cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts); cartilage cells including chondrocytes, chondrocytes cells), skin cells including fibrocytes, keratinocytes, melanocytes (nevus cells), muscle cells including myocytes, podocytes, juxtaglomerular cells, glomerular mesangial cells / extraglomerular mesangial cells, kidney proximal tubule brush border cells, urinary system cells including macula densa cells, reproductive system cells including sperm, Sertoli cells, Leydig cells, and eggs, as well as adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, epidermal hair stem cells, epidermal root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, Outer hair root sheath cells, hair matrix basal cells (stem cells), moist stratified barrier epithelial cells, surface epithelial cells of stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, bile duct epithelial basal cells (stem cells) of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urinary epithelial cells (inner bladder and ureter), exocrine gland secretory epithelial cells, salivary gland mucous cells (polysaccharide-rich secretion), salivary gland serous cells (glycoprotein enzyme-rich secretion), von Ebner's gland cells of the tongue (taste bud lavage), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), earwax gland cells of the ear (earwax secretion), eccrine gland dark cells (glycoprotein secretion), eccrine gland clear cells (small molecule secretion),Apocrine gland cells (scent secretion, sex hormone sensitivity), Moll cell glands in the eyelids (certain sweat glands), sebaceous gland cells (secretion of lipid-rich sebum), nose (washes olfactory epithelium) Bowman's gland cells in the epithelium, Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secrete seminal fluid components including fructose for swimming sperm), prostate cells (secrete seminal fluid components), bulbourethral gland cells (secrete mucus), Bartholin's gland cells (secrete vaginal fluid), Littré gland cells (secrete mucus), endometrial cells of the uterus (secrete carbohydrates), isolated goblet cells of the respiratory system and digestive tract (secrete mucus), mucous cells lining the stomach (secrete mucus), gastric gland zymogen cells (secrete pepsinogens), gastric gland acid secreting cells (secrete hydrochloric acid), pancreatic acinar cells (secrete bicarbonate and digestive enzymes), Paneth cells of the small intestine (secrete lysozyme), type II pneumocytes of the lungs (secrete surfactant), Clara cells of the lungs, hormone secreting cells, anterior pituitary cells, growth Hormone-producing cells, lactotrophs, thyrotrophs, gonadotrophs, corticotrophs, cells of the intermediate lobe of the pituitary gland, large cell neurosecretory cells, cells of the intestine and respiratory system, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, acidophilic cells, adrenal cells, chromaffin cells, Leydig cells of the testes, theca interna cells of the follicle, luteal cells of ruptured follicles, granulosa lutein cells, theca luteal cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands, and genitourinary tract), kidney, type I pneumocytes (in the inner wall of the lung), pancreatic duct cells (cardiac atrial cells), nonstriated duct cells (sweat glands, salivary glands, mammary glands, etc.) duct cells, duct cells (e.g. seminal vesicles, prostate), epithelial cell walls closing internal body cavities, ciliated cells with propulsive functions, extracellular matrix secreting cells, other cells including contractile cells, skeletal muscle cells, stem cells, cardiac muscle cells, blood and immune cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the 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, stem cells and committed progenitors committed to the blood and immune systems (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic neuronal cells, sensory organs and peripheral nerve supporting cells, central nervous system neurons and glial cells (lens cells), pigment cells, melanocytes, retinal pigmented epithelial cells, germ cells, oogonia / oocytes, sperm cells, spermatocytes, spermatogonia (stem cells for spermatocytes), sperm, nurse cells, follicle cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0033] In some embodiments, the cell is a eukaryotic cell. Non-limiting examples of eukaryotic cells include mammalian (e.g., rodent, non-human primate, or human), non-mammalian (e.g., fish, bird, reptile, or amphibian), invertebrate, insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as a mammalian, avian, plant, or insect cell. In some embodiments, the nucleated cell is a primary cell. In some embodiments, the nucleated cell is an immune cell (e.g., lymphocyte (e.g., T cell, B cell), macrophage, natural killer cell, neutrophil, mast cell, basophil, dendritic cell, monocyte, myeloid-derived suppressor cell, eosinophil). In some embodiments, the nucleated cell is a phagocyte or a polymorphonuclear leukocyte. In some embodiments, the nucleated cells are stem cells (e.g., adult stem cells (e.g., hematopoietic stem cells, mammary 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, induced pluripotent stem cells (iPS)). In some embodiments, the nucleated cells are progenitor cells. In some embodiments, the nucleated cells are from a cell line. In some embodiments, the nucleated cells are suspension cells. In some embodiments, the nucleated cells are adherent cells. In some embodiments, the nucleated cells are cells immortalized by expression of an oncogene. In some embodiments, the nucleated cells are immortalized by expression of human telomerase reverse transcriptase (hTERT) or any oncogene. In some embodiments, the nucleated cells are cells derived from a patient or individual (e.g., autologous patient-derived cells or allogeneic patient-derived cells).In some embodiments, before the nucleated cells are enucleated, the 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 viral (AAV) vector, a vesicular viral vector (e.g., a vesicular stomatitis virus (VSV) vector) or a hybrid viral vector), a plasmid) using any of the enucleation techniques described herein and known in the art.

[0034] In some embodiments, the cytoplasts are derived from cells that are autologous to the subject. In some embodiments, the cytoplasts are derived from cells that are allogeneic to the subject.

[0035] In some embodiments, the cytoplast is derived from an immune cell, hi some embodiments, the cytoplast is derived from a natural killer (NK) cell, a neutrophil, a macrophage, a lymphocyte, a fibroblast, an adult stem cell (e.g., a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchymal stem cell, a mesenchymal stromal cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a skin stem cell, or a testicular cell), a mast cell, a basophil, an eosinophil, an endothelial cell, an endothelial cell progenitor cell, or a pluripotent stem cell.

[0036] In some embodiments, the parent cells may be enucleated and engineered for therapeutic use. In some embodiments, the 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 body by high-speed centrifugation in a gradient of polysaccharides to create enucleated cells. In some embodiments, the polysaccharide is Ficoll for the creation of a Ficoll gradient to create enucleated cells. Since enucleated cells and intact nucleated cells settle into different layers in the Ficoll gradient, the enucleated cells may be isolated and prepared for therapeutic purposes or fused to other cells (nucleated or enucleated). The enucleation process may be clinically scalable to process tens of millions of cells by utilizing the methods described herein. In some embodiments, the enucleated cells may be used as disease-homing vehicles to deliver clinically relevant cargo / payloads to treat various diseases.

[0037] In some embodiments, the enucleated cell comprises at least one therapeutic agent. In some embodiments, the enucleated cell disclosed herein expresses the therapeutic agent with one or more intracellular organelles in the absence of a nucleus. 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 the enucleated cell surface. In some embodiments, the therapeutic agent is secreted by the enucleated cell into the extracellular space (e.g., microenvironment) of the target tissue. In some embodiments, the therapeutic agent is the cargo of the enucleated cell (e.g., packaged by the enucleated cell).

[0038] In some embodiments, the enucleated cells are obtained from a first subset of a plurality of nucleated cells. In some embodiments, the enucleated cells are in a composition, further comprising a second subset of a plurality of nucleated cells. In some embodiments, the second subset of nucleated cells comprises less than about 0.1% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 0.5% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 1% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 5% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 10% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 15% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 20% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 25% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 30% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 40% by volume of the composition. In some embodiments, the second subset of nucleated cells comprises less than about 50% by volume of the composition.

[0039] In one aspect, a nucleated cell (e.g., a parent cell prior to enucleation to obtain an enucleated cell as described herein) comprises a heterologous polynucleotide encoding a heterologous gene product configured to induce cell death of the nucleated cell. In some embodiments, the heterologous polynucleotide comprises a promoter. In some embodiments, the promoter is configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is configured, upon induction, to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product.

[0040] In some embodiments, the enucleated cells described herein can be cryopreserved, cryopreserved, freeze-dried, or a combination thereof. In some embodiments, cryopreserved enucleated cells, upon thawing, are as viable as other equivalent enucleated cells that are not cryopreserved. In some embodiments, freeze-dried enucleated cells are as viable as other equivalent enucleated cells that are not freeze-dried. In some embodiments, cryopreserved enucleated cells are as viable as other equivalent enucleated cells that are not cryopreserved.

[0041] In some embodiments, the enucleated cells or compositions comprising the enucleated cells may be cryopreserved (e.g., storing the enucleated cells or compositions comprising the enucleated cells at freezing temperatures) or cryohibernated (e.g., storing the enucleated cells or compositions comprising the enucleated cells at temperatures between ambient and freezing temperatures). The period of cryopreservation or cryohibernation may be for a period of about 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 more. In some embodiments, the enucleated cells exhibit viability after the same period of cryopreservation or cryohibernation that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been cryopreserved or cryohibernated). In some embodiments, the enucleated cells exhibit a post-cryopause viability measured 24 hours after cryopreservation that is equal to or greater than the viability of a comparable enucleated cell that is not cryopreserved. In some embodiments, the enucleated cells exhibit a post-cryopause viability measured 24 hours after cryopreservation that is equal to or greater than the viability of a comparable enucleated cell that is not cryopreserved. Viability in this context can be measured by trypan blue dye exclusion as described herein. In some embodiments, trypan blue dye exclusion is performed by (a) centrifuging an aliquot of the denucleated cells in suspension to create a cell pellet, (b) resuspending the cell pellet in serum-free medium to generate a serum-free cell suspension, (c) mixing one part trypan blue dye with one part serum-free cell suspension, and (d) counting the denucleated cells within 3-5 minutes of (c), where at least some of the denucleated cells are not stained with trypan blue dye, indicating viability. In some embodiments, viability is measured using Annexin-V cell surface staining. In some embodiments, viability is measured by expression of an exogenous polypeptide. For example, the viability of the enucleated cells may be determined by expression of an exogenous antibody or single domain antibody expressed by the enucleated cells.In some embodiments, viability is measured by expression of any one of the cell surface markers described herein, such as CD105, CD90, CD45, CXCR4, PSGL-1, or CCR2. In some embodiments, viability is measured by the cellular activity of the enucleated cells. In some embodiments, viability is measured by the homing ability of the enucleated cells, as determined by chemosensing or chemokine homing activity as described herein.

[0042] In some embodiments, the enucleated cells or compositions comprising the enucleated cells may be lyophilized. In some embodiments, the enucleated cells exhibit viability following reconstitution from lyophilization that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that is not lyophilized).

[0043] In some embodiments, the enucleated cells or compositions comprising the enucleated cells may be dehydrated. In some embodiments, the enucleated cells exhibit viability after rehydration from lyophilization that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been dehydrated).

[0044] In some embodiments, the enucleated cells or compositions comprising the enucleated cells are stable for a period of about 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 more at 4° C. In some embodiments, the compositions are stable for a period of about 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 more at room temperature. In some embodiments, the compositions are stable for a period of about 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 more at 37° C. In some embodiments, the enucleated cells or compositions comprising the enucleated cells may remain viable after being administered to a subject in need of disease or condition treatment to treat a disease or condition described herein, in some embodiments, the enucleated cells or compositions comprising the enucleated cells may remain viable after being administered to a subject for a period of about 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, weeks, 1 month, 2 months, 3 months, or more.

[0045] In some embodiments, the enucleated cells may be obtained from parent cells that are autologous to the subject in need of treatment with the enucleated cells described herein. In some embodiments, the enucleated cells may be obtained from parent cells that are allogeneic to the subject in need of treatment with the enucleated cells described herein.

[0046] Enucleated cells may be smaller than their nucleated counterparts (e.g., nucleated parent cells) and therefore may be better able to migrate through small openings in the vasculature and tissue parenchyma. In addition, removal of the large density nucleus alleviates a major physical barrier, allowing the cells to migrate freely through small openings in blood vessels and tissue parenchyma. Thus, enucleated cells have improved in vivo biodistribution and movement to target tissues. In some embodiments, the enucleated cells comprise a diameter of at least 1 μm. In some embodiments, the enucleated cells are greater than 1 μm in diameter. In some embodiments, the enucleated cells are 1-100 μm in diameter (e.g., 1-90 μm, 1-80 μm, 1-70 μm, 1-60 μm, 1-50 μm, 1-40 μm, 1-30 μm, 1-20 μm, 1-10 μm, 1-5 μm, 5-90 μm, 5-80 μm, 5-70 μm, 5-60 μm, 5-50 μm, 5-40 μm, 5-30 μm, 5 ~20μm, 5~10μm, 10~90μm, 10~80μm, 10~70μm, 10~60μm, 10~50μm, 10~40μm, 10~30μm, 10~20μm, 10~15μm, 15~90μm, 15~80μm, 15~70μm, 15~60μm, 15~50μm, 15~40μm, 15~30μm, 15~20μm. In some embodiments, the enucleated cells are 10-30 μm in diameter. In some embodiments, the enucleated cells are between 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 about 8 μm. Some enucleated cells may advantageously be small enough to allow better homing or delivery to the target site. For example, the enucleated cells described herein may pass through passages in narrow lung tissue or lung structures such as alveolar ducts or microcapillaries that most cells, such as parent cells, cannot pass through.

[0047] In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises 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 of the enucleated cell fraction have a diameter that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises about 90% or less of the average diameter of the nucleated cells.

[0048] In some embodiments, enucleated cells have significant therapeutic value because they remain viable, do not differentiate into other cell types, do not secrete bioactive molecules, can physically migrate / home for about 5 days or less, may be extensively enucleated ex vivo, perform specific therapeutic functions, or may be fused to the same or other cell types to transfer desired productions, either natural or enucleated. Thus, enucleated cells have broad utility as cellular vehicles for delivery of therapeutically important biomolecules and disease targeting cargoes, including genes, viruses, bacteria, mRNA, shRNA, siRNA, polypeptides (including antibodies and antigen-binding fragments), plasmids, gene editing mechanisms, or nanoparticles. The present disclosure enables the creation of safe (e.g., undesirable DNA is not transferred to the subject) and controllable (e.g., cell death occurs in approximately 3-4 days) cell-based carriers that can be genetically enucleated to deliver specific disease-treating and health-promoting cargoes to humans. In some embodiments, the enucleated cells remain viable and retain the ability to migrate or home for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or more after administration to a subject in need thereof.

[0049] In some embodiments, the enucleated cells are engineered to express at least one of 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 a single-stranded DNA, a double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or a combination thereof. In some embodiments, the exogenous RNA molecule is a messenger RNA (mRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an RNA virus, or a combination thereof. In some embodiments, the exogenous protein is a cytokine, a growth factor, a hormone, an antibody or an antigen-binding fragment thereof, an enzyme, or a combination thereof. In some embodiments, the antibody is a single domain antibody or an antigen-binding fragment thereof. In some embodiments, the parent cell (e.g., a nucleated cell) is genetically enucleated prior to enucleation (e.g., pre-enucleation). In some embodiments, the parent cell is genetically enucleated following enucleation (eg, post-enucleation).

[0050] Transmembrane part In some aspects, described herein is an enucleated cell or a composition comprising an enucleated cell, comprising at least one transmembrane moiety. In some embodiments, the enucleated cell is 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 mutant or fragment thereof. In some embodiments, the transmembrane moiety is endogenous to the parent cell that is enucleated to obtain the enucleated cell. In some embodiments, the transmembrane moiety may be a transmembrane moiety exogenous to the parent cell or the enucleated cell. In some embodiments, the transmembrane moiety is selected from transmembrane proteins comprising a single transmembrane alpha helix (rotatory membrane proteins). The transmembrane moiety comprises a polymorphic transmembrane alpha helix protein. In some embodiments, the transmembrane moiety comprises a polymorphic transmembrane P-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 CD4, CD14, glycophorin alpha (GPA), or any combination of integrins.

[0051] In some embodiments, the transmembrane portion is added to the exogenous polypeptide by modification.For example, the transmembrane portion can be added to the N-terminus or C-terminus of the exogenous polypeptide to insert the exogenous polypeptide into the cell membrane of the enucleated cell described herein.Non-limiting examples of the modification added to the exogenous polypeptide to add the transmembrane portion can include adding glycosylphosphatidylinositol, famecil, palmitate, myristic acid, or a combination thereof to the exogenous polypeptide.

[0052] In some embodiments, the transmembrane portion is genetically modified to fuse or complex with at least one exogenous therapeutic agent as described herein. In some embodiments, the transmembrane portion is genetically modified to fuse with at least one exogenous therapeutic agent as described herein. In some embodiments, the enucleated cell comprises an immune evasion portion. In some aspects, the immune evasion comprises a "don't eat me" signaling peptide, such as CD47 (e.g., NCBI gene ID 961), programmed cell death 1 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-1, e.g., NCBI gene ID 3135), fragments thereof, or combinations thereof.

[0053] targeting part In some aspects, enucleated cells comprising a targeting moiety are described herein. The targeting moiety described herein is designed to direct the enucleated cell to a target cell or target environment (e.g., tissue) in a subject after delivery (e.g., systemic delivery) of the enucleated cell to the subject. 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, the enucleated cell comprising the targeting moiety localizes 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold more in the target cell or target environment compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells containing a targeting moiety localize with a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% increase in the target cell or target environment compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the target cell or target environment is in vivo. In some embodiments, the target cell or target environment is ex vivo.

[0054] In some embodiments, the targeting moiety comprises an exogenous antibody or an exogenous antigen-binding fragment for targeting a biomarker described herein. In some embodiments, the targeting moiety comprises an exogenous antibody or an exogenous antigen-binding fragment for targeting a chemokine receptor or chemokine ligand, or a portion thereof, involved in chemokine signaling. In some embodiments, the exogenous antibody is an exogenous single domain antibody or a fragment thereof.

[0055] In some embodiments, the targeting moiety targets a biomarker expressed by or associated with a target cell or microenvironment. In some embodiments, the biomarker may be released by a target cell. The biomarker may indicate the presence of a disease or condition. In some embodiments, the biomarker is expressed by an immune cell that responds to a target cell or microenvironment associated with a disease or condition. In some embodiments, the biomarker may be an epitope or antigen. In some embodiments, a biomarker that includes an epitope may be bound by an antibody that is distinct from the antibody or antigen-binding fragment thereof that confers a therapeutic property (e.g., a therapeutic agent).

[0056] In some embodiments, the targeting moiety targets a biomarker 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 / testis antigen 83 (CXorf61, e.g., NCBI gene ID 203413), desmoglein 3 (DSG3, e.g., NCBI gene ID 1830), FAT variant cadherin 2 (FAT2, e.g., NCBI gene ID 2196), G protein-coupled receptor (GPR87, e.g., NCBI gene ID 53836), KISS1, and the like. receptor (KISS1R, e.g., NCBI Gene ID 84634), LY6 / PLAUR domain containing 3 (LYPD3, e.g., NCBI Gene ID 27076), solute carrier family 7 member 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), secreted phosphoprotein 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 fragment (CYFRA21-1, e.g., NCBI Gene ID 3880), serpin family B member 3 (SCC, e.g., NCBI Gene ID 6317), receptor for advanced glycosylation end product (A GER, e.g., NCBI Gene ID 177), adipogenesis regulator (ClOorf116, e.g., NCBI Gene ID 10974), adducin 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), synnemin (SYNM, e.g., NCBI Gene ID 23336), spectrin alpha, erythroid 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 (CAI, e.g., NCBI Gene ID 759), tenascin XB (TNXB, e.g., NCBI Gene ID 7148), multimer 2 (MMRN2, e.g., NCBI Gene ID 79812), 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 (Clorf198, e.g., NCBI gene ID 84886), chloride intracellular channel 2 (CLIC2, e.g., NCBI gene ID 1193), transcriptional regulator of the SdpC synthesis operon (ArsR family) (SDPR, e.g., NCBI gene ID 1111), 8436), EH domain containing 2 (EHD2, e.g., NCBI Gene ID 30846), apolipoprotein A2 (APOA2, e.g., NCBI Gene ID 336), NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7, e.g., NCBI Gene ID 4713), protein kinase C delta binding protein (PRKCDBP, e.g., NCBI Gene ID 112464), laminin subunit alpha 3 (LAMA3, e.g., NCBI Gene ID), EvC ciliary complex subunit 2 (LBN, e.g., NCBI Gene ID 101616), ribosomal protein 1 (RIRP1, e.g., NCBI Gene ID 101625), ribosomal protein 2 (RIRP2, e.g., NCBI Gene ID 101636), ribosomal protein 1 (RIRP1, e.g., NCBI Gene ID 101646), ribosomal protein 2 (RIRP1, e.g., NCBI Gene ID 101655), ribosomal protein 1 (RIRP1, e.g., NCBI Gene ID 101666), ribosomal protein 2 (RIRP1, e.g., NCBI Gene ID 101671), ribosomal protein 1 (RIRP1, e.g., NCBI Gene ID 101682), ribosomal protein 2 (RIRP1, e.g., NCBI Gene ID 101691), ribosomal protein 1 (RIRP1, e.g., NCBI Gene ID 101692), ribosomal protein 2 (RIRP1, e.g., NCBI Gene ID 101693). Gene ID 132884), serpin family A member 3 (ACT, e.g., NCBI Gene ID 12), insulin-like growth factor binding protein 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 Cluster A (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), fibrinogen alpha chain (FGA, e.g., NCBI Gene ID 2243), gelsoh phospholipid (GSN, e.g., NCBI Gene ID 2934), haptoglobin (HP, e.g., NCBI Gene ID 3240), ficolin 3 (FCN3, e.g., NCBI Gene ID 8547), carnosin 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), chromogranin B (CHGB, e.g., NCBI Gene ID 1374), chromoglobin B (CHG, e.g., NCBI Gene ID 1375), chromoglobin B (CHG, e.g., NCBI Gene ID 1376), chromoglobin B (CHG, e.g., NCBI Gene ID 1377), chromoglobin C (CHG, e.g., NCBI Gene ID 1379), chromoglobin D (CHG, e.g., NCBI Gene ID 1379), chromoglobin D (CHG, e.g., NCBI Gene ID 1379), chromoglobin E (CHG, e.g., NCBI Gene ID 1379), chromoglobin F (CHG, e.g., NCBI Gene ID 1379), chromoglobin G (CHG, e.g., NCBI Gene ID 1379), chromoglobin H (CHG, e.g., NCBI Gene ID 1379), chromoglobin I ... I gene ID 1114), involucrin (IVL, e.g., NCBI gene ID 3713), anterior gradient 2 (AGR2, e.g., NCBI gene ID 10551), nuclear autoantigenic 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 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 associated protein (STRAP, e.g., NCBI gene ID 1011), and the like. 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 IB (CKMT1B, e.g., NCBI Gene ID 1159), aldo-ketoreductase family 1 member B10 (AKR1B10, e.g., NCBI Gene ID 57016), carboxypeptidase D (CPD, e.g., NCBI Gene ID 13 62), proteasome activator subunit 3 (PSME3, e.g., NCBI gene ID 10197), villin 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), plakophilin 1 (PKP1, e.g., NCBI gene ID 5317), ribosomal protein L10 (RPL10, e.g., NCBI gene ID 6134), aldo-keto reductase family 1 member 5 (ALPHA ...10197), villin 1 (VIL1, e.g., NCBI gene ID 10197), villin 1 (VIL1, e.g., NCBI gene ID 10197), villin 1 (VIL1, 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 6187), aldo-keto reductase family 1 member C3 (AKR1C3, e.g., NCBI gene ID 8644), acyl-CoA binding domain containing 3 (ACBD3, e.g., NCBI gene ID 64746),Visinin-like 1 (VSNL1, e.g., NCBI Gene ID 7447), adenosylhomocysteinase (AHCY, e.g., NCBI Gene ID 191), IMMP10, activating 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-ATPase 2 (PSMD2, e.g., NCBI Gene ID 5708), guanylate-binding protein and NCBI gene ID 1011162, NCBI gene ID 1011163, NCBI gene ID 101117, NCBI gene ID 101118, NCBI gene ID 101119, NCBI gene ID 101120, NCBI gene ID 101130, NCBI gene ID 101140, NCBI gene ID 101150, NCBI gene ID 101161, NCBI gene ID 101172, NCBI gene ID 101183, NCBI gene ID 101194, NCBI gene ID 101195, NCBI gene ID 101196, NCBI gene ID 101197, NCBI gene ID 10119 ... 9542), carnosine dipeptidase 1 (CNDP1, e.g., NCBI Gene ID 84735), ubiquitin cross-reactive protein (UCRP, e.g., NCBI Gene ID 9636), crammer (CER, e.g., NCBI Gene ID 8110), plasminogen activator (UPA, e.g., NCBI Gene ID 5328), matrix metallopeptidase 14 (MT1-MMP, e.g., NCBI Gene ID 4323), stratifin (SFN, e.g., NCBI Gene ID 2810), transferrin (TF, e.g., NCBI Gene ID 2810), NCBI Gene ID 7018), albumin (ALB, e.g., NCBI Gene ID 213), S100 calcium binding protein A9 (S100A9, e.g., NCBI Gene ID 6280), stathmin 1 (STMN, e.g., NCBI Gene ID 3925), 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).

[0057] In some embodiments, the targeting moiety targets a biomarker expressed or released by metastatic cancer cells. For example, cancer cells may originate from one tissue and then metastasize to a different location. In some embodiments, metastatic cancer cells express non-limiting examples of cancer biomarkers described herein. In some embodiments, the metastatic cancer cells express an antigen that is a member of the 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 5133), 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 5329), or a combination thereof. Gene ID 7015), PRAME nuclear receptor transcription factor (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 (MAGE-A4, e.g., NCBI Gene ID 4103), survivin, six transmembrane epithelial antigen of the prostate 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).

[0058] In some embodiments, the targeting moiety targets a biomarker expressed or released by a vascular endothelial cell. In some embodiments, the vascular endothelial cell is a blood vessel cell. In some embodiments, the vascular endothelial cell is a lymphatic vessel cell. In some embodiments, the biomarker is expressed or released by a blood vessel cell. In some embodiments, the biomarker is expressed or released by a lymphatic vessel cell. Non-limiting examples of endothelial cell biomarkers include angiotensin I converting enzyme (ACE / CD143, e.g., NCBI Gene ID 1636), CD93 molecule (ClqR1 / 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).

[0059] CD300 molecule-like family member g (CD300g / 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 1033 2), discoidin, CUB and LCCL domain containing 2 (DCBLD2 / ESDN, e.g., NCBI Gene ID 131566), endothelial cell surface expressed chemotaxis and apoptosis regulator (ECSCR, e.g., NCBI Gene ID 641700), basidin (Ok blood group) (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 vascular cell adhesion molecule (ESAM, e.g., NCBI Gene ID 90952), fatty acid binding protein 5 (FABP5ZE-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-13Rα1, e.g., NCBI Gene ID 3597), integrin alpha 4 / CD49d (e.g., NCBI Gene ID 3676), integrin alpha 4 beta 1 (e.g., NCBI Gene ID 3688), integrin alpha 4 beta 7 / LPAM-1 (e.g., NCBI Gene ID 3676), integrin beta 2 / CD18 (e.g., NCBI Gene ID 3689), KLF transcription factor 4 (KLF4, e.g., NCBI Gene ID 9314), lymphatic endothelial hyaluronan 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 18 90), podocalyxin (e.g., 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 9294), For example, NCBI gene ID 1903), sphingosine-1-phosphate receptor 4 (S1P4 / EDG-6, for example, NCBI gene ID 8698), sphingosine-1-phosphate receptor 5 (S1P5 / EDG-8, for example, NCBI gene ID 53637), E-selectin / CD62E (for example, NCBI gene ID 6401), P-selectin / CD62P (for example, NCBI gene ID 6403), slow asmolasses (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 57125), 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), basigin (Ok blood group) (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: NCBI Gene ID 2321), kinase insert 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), angiogenic factor with G-patch and FHA domain 1 (VG5Q, e.g., NCBI Gene ID 55109), or von Willebrand factor domain 2 (vWF-A2, e.g., NCBI Gene ID 7450).

[0060] In some embodiments, the targeting moiety comprises a chemokine receptor or chemokine ligand or portion thereof involved in chemokine signaling, such as SDF-1α / CXCR4, CCL2 / CCR2, or an adhesion molecule, such as PSGL-1. As shown herein, enucleated cells may be enucleated to express functional CXCR4, CCR2, and glycosylated PSGL-1, which may greatly facilitate specific targeting of the enucleated cells. In some embodiments, targeting moieties such as CXCR4, CCR2, or PSGL-1 may be expressed on the surface of the enucleated cells. Non-limiting examples of cell surface proteins that may be expressed on the cell surface of the enucleated cells as targeting moieties include chemokines such as CXCR4, CCR2, CCR1, CCR5, CXCR7, CXCR2, and CXCR1. In some embodiments, the enucleated cells may be enucleated to secrete the targeting moiety or be tethered to the extracellular matrix, such as SDF1-α or CCL2. Non-limiting examples of targeting moieties that may be secreted by enucleated cells include SDF1-α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11, and CXCL12. In some embodiments, the enucleated cells comprise cell-matrix receptors and cell-cell adhesion molecules include integrins, cadherins, glycoproteins, and heparin sulfate proteoglycans.

[0061] In some embodiments, the enucleated cells may further comprise surface markers (e.g., by engineering or from the cells from which they are derived) that aid in evading the subject's immune system. In some embodiments, for example, the enucleated cells may comprise CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof. Without being bound to any particular theory, it is believed that CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof help prevent the enucleated cells from being phagocytosed by macrophages. Non-limiting examples of cell-matrix receptors and cell-cell adhesion molecules include integrins, cadherins, glycoproteins, or heparin sulfate proteoglycans. In some embodiments, the cell-matrix receptors or cell-cell adhesion molecules include PD-L1, HLA-E, or HLA-G. Non-limiting examples of therapeutic molecules include tumor antigens and immune-modulating peptides, polyamines, and ATP. In some embodiments, the therapeutic molecules may be recognized by immune cells and induce an immune response. For example, the therapeutic molecule can be 4-IBB or any one of the cytokines described herein for inducing an immune response.

[0062] Therapeutic Agents In some embodiments, the enucleated cells of the present disclosure include at least one therapeutic agent. In some embodiments, the enucleated cells of the present disclosure include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more therapeutic agents. In some embodiments, the therapeutic agent includes an active agent. In some embodiments, the therapeutic agent is exogenous to the enucleated cells or their parent cells. The active agent includes at least one of a DNA molecule, an RNA molecule, a protein (e.g., an enzyme, an antibody, an antigen, a toxin, a cytokine, a protein hormone, a growth factor, a cell surface receptor, or a vaccine), a peptide (e.g., a peptide hormone or an antigen), a small molecule (e.g., a steroid, a polyketide, an alkaloid, a toxin, an antibiotic, an antiviral drug, colchicine, taxol, mitomycin, or emtansine), a gene editing factor, a nanoparticle, or another active agent (e.g., a bacterium, a bacterial spore, a bacteriophage, a bacterial component, a virus (e.g., an oncolytic virus), an exosome, a lipid, or an ion). In some embodiments, the enucleated cells are engineered to produce (e.g., express, and in some cases, release or secrete) a therapeutic agent. In some embodiments, the parent may be engineered to produce a therapeutic agent prior to enucleation to generate the enucleated cells. Non-limiting examples of oncolytic viruses include talimogene laherparepvec, Onyx-015, GL-ONC1, CV706, Voyager-V1, and HSV-1716. Several wild-type viruses, such as vaccinia virus, vesicular stomatitis virus, poliovirus, reovirus, Seneca virus, ECHO-7, and Semliki Forest virus, also exhibit oncolytic behavior.

[0063] The therapeutic agent may be or may include a targeting moiety described herein. Non-limiting examples of targeting moieties that may be produced by or contained in an enucleated cell include chemokine receptors, adhesion molecules, and antigens. In some embodiments, the therapeutic agent may be or may include a transmembrane moiety described herein.

[0064] In some embodiments, the therapeutic agent is recombinantly expressed by the enucleated cell or a parent cell thereof. In some embodiments, the parent cell from which the enucleated cell is derived or obtained is engineered to produce or express the therapeutic agent. In some embodiments, the expression of the therapeutic agent is stable (e.g., permanent). In some embodiments, the expression of the therapeutic agent by the parent cell is transient (e.g., non-permanent). In some embodiments, the parent cell is enucleated prior to engineering the enucleated cell to recombinantly express the therapeutic agent.

[0065] In some embodiments, the therapeutic agent is not naturally expressed (e.g., in the absence of manipulation) in the cell from which the enucleated cell is derived or obtained (e.g., the therapeutic agent is exogenous to the parent cell). In some embodiments, the therapeutic agent is not naturally expressed in the subject (e.g., the therapeutic agent is exogenous to the subject). In some embodiments, the therapeutic agent is not naturally expressed in the subject at the intended site of treatment (e.g., a tumor or a particular tissue such as the brain, intestine, lung, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is exogenous to the intended site of treatment). In some embodiments, the level of the therapeutic agent is not naturally present in the enucleated cell of the parent cell.

[0066] In some embodiments, the therapeutic agent is naturally expressed (e.g., in the absence of manipulation) in the cells from which the enucleated cells are derived or obtained (e.g., the therapeutic agent is endogenous to the enucleated cells). In some embodiments, the therapeutic agent is naturally expressed in the subject (e.g., the therapeutic agent is endogenous to the subject). In some embodiments, the therapeutic agent is naturally expressed in the subject at the intended site of treatment (e.g., a tumor or a particular tissue such as the brain, intestine, lung, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is endogenous to the intended site of treatment).

[0067] In some embodiments, the therapeutic agent is derived from a synthetic cell and loaded into the enucleated cell. For example, the therapeutic agent may be endocytosed into the cell. Alternatively, the therapeutic agent may be synthesized by the cell and subsequently delivered to the target cell.

[0068] In some embodiments, the therapeutic agent comprises a modified, truncated, or non-mutated version and / or copy of a DNA molecule, an RNA molecule, a protein, a peptide, a small molecule active agent, and / or a gene editing factor compared to the cell from which the enucleated cell is derived or obtained. For example, the therapeutic agent can correct mutated p53 or EGFR in the target cell as part of the treatment of lung cancer.

[0069] In some embodiments, the therapeutic agent comprises at least two (e.g., at least 2, 3, 4, 5, or more) different therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule active agents, or therapeutic gene editors, in any combination. For example, in some embodiments, the therapeutic agent comprises a therapeutic DNA molecule and a small molecule active agent. For example, in some embodiments, the therapeutic agent comprises two different small molecule active agents. For example, in some embodiments, the therapeutic agent comprises a chemokine receptor (e.g., for targeting) and a small molecule active agent.

[0070] In some embodiments, the therapeutic agent comprises an RNA molecule, including messenger RNA (mRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA, long non-coding RNA (IncRNA), or an RNA virus. In some embodiments, the therapeutic agent comprises a DNA molecule that is single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, or a DNA virus. In some embodiments, the therapeutic agent comprises a protein or a portion thereof. In some embodiments, the protein is a cytokine, a growth factor, a hormone, an antibody or an antigen-binding fragment thereof, a small peptide-based drug, or an enzyme. In some embodiments, the 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 permanent.

[0071] In some embodiments, the therapeutic agent comprises an exogenous agent. In some embodiments, the exogenous agent is an exogenous polypeptide. In some embodiments, the exogenous polypeptide is encoded by an exogenous polynucleotide delivered to the parent cell or the 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 of 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 that expresses a cancer biomarker of any cancer described herein. In some embodiments, the target cell is a vascular endothelial cell that expresses a vascular endothelial biomarker described herein. In some embodiments, the vascular endothelial cell is a vascular cell. In some embodiments, the vascular endothelial cell is a lymphatic cell.

[0072] In some embodiments, the exogenous polypeptide comprises any one of the cytokines described herein. In some embodiments, the exogenous polypeptide comprises a soluble cytokine. For example, the exogenous polypeptide can comprise an extracellular domain or fragment of a cytokine. In some embodiments, the exogenous polypeptide comprises a solubility determined by a turbidimetric solubility assay or a thermodynamic solubility assay by dissolving the exogenous polypeptide in a solvent such as an organic solvent including dimethylsulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc., or an inorganic solvent including water or phosphate buffered saline (PBS). In some embodiments, the exogenous polypeptide comprises a solubility that is 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.

[0073] In some embodiments, the exogenous polypeptide comprises a tumor necrosis factor (TNF) superfamily member or a catalytically active fragment thereof. Non-limiting examples of TNF superfamily members include lymphotoxin alpha (TNFβ), tumor necrosis factor (TNFα), lymphotoxin beta (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 (4-1BBL), TNF-related apoptosis-inducing ligand (CD253 or APO-2L), nuclear factor κB ligand, and the like. receptor activator of cytokines (CD254, OPGL, TRANCE, or ODF), TNF-related weak inducer of apoptosis (APO-3L or DR3L), proliferation-inducing ligand (CD256, TALL-2, or TRDL1), B cell-activating factor (CD257, BLyS, TALL-1, or TNFSF20), LIGHT (CD258 or HVEML), vascular endothelial growth inhibitor (TL1 or TL-1A), TNF superfamily member 18 (GITRL, AITRL, or TL-6), or ectodysplasin A (ED1-A1 or ED1-A2).

[0074] 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 called CD276), A2AR, CD27, LAG3, TIM-3, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD73, NKG2A, PVRIG, PVRL2, CEACAM1, CEACAM5, CEACAM6, FAK, CCR-2, CCL-2, LIF, CD47, SIRPα, M-CSF, IL-3, IL-1RAP, IL-8, SEMA4D, angiopoietin-2, CLEVER-1, Axl, phosphatidylserine, or a fragment thereof.

[0075] In some embodiments, the enucleated cells comprise an additional therapeutic agent, such as those disclosed herein. In some embodiments, a composition comprising the enucleated cells is formulated for administration to a subject with an additional therapeutic agent disclosed herein. In some embodiments, the additional therapeutic agent is administered to the subject sequentially, simultaneously, substantially sequentially, or substantially simultaneously.

[0076] Biomolecular suicide switches Described herein is an enucleated cell comprising one or more biomolecules that induce cell death, such as the biomolecular suicide switch disclosed herein. In some embodiments, the biomolecule is encoded by a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide comprises a promoter configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the biomolecular suicide switch. In some embodiments, expression of the biomolecular suicide switch is sufficient to cause cell death. In some embodiments, the heterologous polynucleotide is integrated into a chromosome of a nucleated cell. In some embodiments, the heterologous polynucleotide comprises a vector. In some embodiments, the heterologous polynucleotide is not integrated into a chromosome of a nucleated cell. In such cases, the heterologous polynucleotide can be induced for expression of a heterologous gene product in the absence of a nucleus. For example, a heterologous polynucleotide that is not integrated into a chromosome of a cell can be induced in a nucleated or enucleated cell.

[0077] In some embodiments, the promoter driving the expression of the biomolecular suicide switch is compatible with mammalian gene expression in the presence of its induction stimulus, and can provide rapid and strong gene expression. In some embodiments, the heterologous gene product is a suicide gene or any gene product that induces cell death. Non-limiting examples of suicide genes include, but are not limited to, caspases, DNA crosslinkers, synthetic NOTCH receptor-inducing death, toxins, and inducers for inducing apoptosis, autophagy, entosis, necrosis, necroptosis, ferroptosis, or combinations thereof.

[0078] In some embodiments, the promoter is a hypothermic promoter. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature less than 40° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature less than 39° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature less than 38° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature less than 37° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature less than 36° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature less than 35° C. In some embodiments, examples of inducible promoters include, but are not limited to, dsrA or CIRP.

[0079] In some embodiments, the promoter is a hyperthermic promoter. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 35° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 36° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 37° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 38° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 39° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 40° C. In some embodiments, examples of inducible promoters include, but are not limited to, HSP70, HSP90, GADD153, MDR1, or HSE-CMV.

[0080] In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a molecule, in some embodiments, examples of molecules include, but are not limited to, rtTA, TRE, TetR, Cumate, rapamycin, abscisic acid, IPTG, or metallothionein.

[0081] In some embodiments, the inducible promoter is induced by contacting nucleated cells with light. In some embodiments, examples of inducible promoters include, but are not limited to, CIB1-CRY2 or GAL4-VVD.

[0082] In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a hormone. In some embodiments, an example of an inducible promoter includes, but is not limited to, estradiol-Gal4.

[0083] In some embodiments, the promoter is a constitutively active promoter. The promoter is continuously active, but suicide is induced under certain circumstances. In some embodiments, the constitutively active promoter is configured to activate transcription of a heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, examples of heterologous gene products include, but are not limited to, herpes simplex virus-thymidine kinase (HSV-TK), cytosine deaminase (CD), varicella zoster TK (VZV-TK), nitroreductase, carboxypeptidase G2 (CPG2), cytochrome P450, or purine nucleoside phosphorylase. In some embodiments, examples of heterologous gene products include, but are not limited to, FKBP or caspase. In some embodiments, examples of heterologous gene products include, but are not limited to, antigens. In some embodiments, the heterologous polynucleotide is integrated into the chromosome of a nucleated cell. In some embodiments, examples of heterologous polynucleotides include, but are not limited to, vectors.

[0084] Pharmaceutical Compositions In some aspects, disclosed herein is a pharmaceutical composition comprising the composition disclosed herein and a pharma- ceutical acceptable carrier, excipient, diluent, or nebulizer. In some embodiments, the composition disclosed herein comprises one or more additional active agents or therapeutic agents.

[0085] In some embodiments, the composition comprises two or more active agents or two or more therapeutic agents as disclosed herein. In some embodiments, the two or more active agents are included in a single dosage unit, for example, when the enucleated cells comprise two or more therapeutic agents. In embodiments, the two or more active agents are included in separate dosage units, such as when the enucleated cells are administered separately from the additional therapeutic agent or adjuvant. In some embodiments, the pharmaceutical composition described herein comprises at least one additional active agent other than the enucleated cells described herein. In some embodiments, the at least one additional active agent is a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an antihormonal agent, an antiangiogenic agent, a cardioprotectant, and / or a checkpoint inhibitor.Non-limiting examples of checkpoint inhibitors include IMP321 / Eftilagimod alpha (Immutep), lilatolimab BMS-986016, ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), ipilimumab (Yervoy), LAG525, MK-4280, irinotecan, oxaliplatin, REGN3767, TSR-033, BI754111, S ym022, FS118 (bispecific anti-LAG3 / PD-L1 antagonistic mAb), MGD013 (bispecific anti-LAG3 / PD-1 antagonistic mAb), TSR-022, Niraparib, Bevacizumab, MBG453, Decitabine, Spartalizumab, Sym023, INCAGN2390, LY3321367, Ramucirumab, Abemaciclib, Merestinib, BMS-986258, SHR-1702, Camrelizumab, MK-7684, Etigilimab / OMP-313 M32, tiragolumab / MTIG7192A / RG-6058, BMS-986207, AB-154, ASP-8374, JNJ-61610588, CA-170d, enoblitutumab / MGA271, MGD009, I-8H9 / omburtamab, trastuzumab, MGD013 (anti-PD-1, anti-LAG-3 dual checkpoint inhibitor), BGB-A1217, CM-24 (MK-6 018), BMS986178, MEDI6469, PF-04518600, GSK3174998, MOXR0916, utomirimab (PF-05082566), urelumab (BMS-663513) ES101, BMS-986156, TRX-518, AMG228, JTX-2011, GSK3359609, BMS-986226, MEDI-570, or valilumab (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, allowing activation of T cells and APCs), monalizumab (to inhibit NKG2A), COM701 (to inhibit PVRIG / PVRL2, allowing activation of T cells), CM24 (to inhibit CEACAM1, allowing activation of T cells and NK cells), NEO-201 (to inhibit CEACAM5 and CEACAM6, allowing T cell activation while interfering with tumor cell proliferation), defactinib (to inhibit FAK), and erythropoietinib (to inhibit NKG2A). and disrupt tumor growth), PF-04136309 (to inhibit CCR-2 and CCL-2 and enable T cell recruitment and activation), MSC-1 (to inhibit LIF while disrupting cancer growth and enable T cell and APC activation), Hu5F9-G4 (5F9), ALX148, TTI-662, and RRx-001 (to inhibit CD47 or SIRPα and enable T cell and APC activation), Lacnotuzumab (MCS-110), LY3022 855, SNDX-6352, 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 while interfering with tumor growth and reduce the immunosuppressive tumor microenvironment), pepinemab (VX15 / 2503) (to interfering with tumor growth while interfering with S These include trebananib (to inhibit EMA4D and reduce the immunosuppressive tumor microenvironment), trebananib (to inhibit angiopoietin-2 and enable APC activation while interfering with cancer growth), FP-1305 (to inhibit CLEVER-1 and enable APC activation), enapotamab vedotin (EnaV) (to inhibit Axl and enable APC activation while interfering with cancer growth), or bavituximab (to inhibit phosphatidylserine and enable T cell and APC activation while interfering with cancer growth).

[0086] The composition may contain at least the exogenous therapeutic agent as an active ingredient in free acid or free base form, or in the form of a pharmaceutically acceptable salt.In addition, the methods and compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds that also have the same type of activity.In some embodiments, the therapeutic agent is present in a nonsolvated form or in a solvated form that includes a pharmaceutically acceptable solvent, such as water, ethanol, etc.Solvated forms of the therapeutic agent are also considered to be disclosed herein.

[0087] In certain embodiments, the compositions provided herein include one or more preservatives that inhibit microbial activity.Suitable preservatives include mercury-containing substances such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0088] In some embodiments, the compositions described herein may benefit from antioxidants, metal chelators, thiol-containing compounds, and other general stabilizers. Examples of such stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0089] Formulation for administration The compositions described herein are 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, fast dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations. In one aspect, the therapeutic agents discussed herein, e.g., therapeutic agents, are formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders that are rehydrated into sterile injectable or dispersion solutions. Examples of suitable water-soluble and non-water-soluble carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. In some cases, it is desirable to include isotonic agents such as sugars, sodium chloride, etc. Prolonged absorption of the injectable dosage form can be brought about by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0090] For intravenous injection or drip or infusion, the compositions described herein are formulated in aqueous solution, preferably in physiologically compatible buffer such as Hanks' solution, Ringer's solution or buffered saline.For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.

[0091] Parenteral injection may include bolus injection or continuous infusion. Compositions for injection may be presented in unit dosage form, for example in 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 formulatory agents such as suspending, stabilizing, and / or dispersing agents. In one embodiment, the active ingredient is in powder form for constitution with a suitable vehicle, for example pyrogen-free distilled water, before use.

[0092] For administration by inhalation, the therapeutic agents are formulated for use as an aerosol, mist, or powder. The pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. By way of example only, capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agents described herein and a suitable powder base such as lactose or starch. Formulations containing the compositions can be prepared using methods known in the art. The nasal dosage form is prepared as a solution in saline, utilizing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents, which are known to be effective in treating nasal ulcers. Preferably, these compositions and formulations are prepared using suitable non-toxic pharmaceutically acceptable ingredients. The selection of suitable carriers depends on the exact nature of the nasal dosage form desired, e.g., solution, suspension, ointment, or gel. Nasal dosage forms generally contain a large amount of water in addition to the active ingredient. Small amounts of other ingredients are optionally present, e.g., pH adjusting agents, emulsifiers, or dispersing agents, preservatives, surfactants, gelling agents, or buffers, and other stabilizers and solubilizers. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[0093] Orally used pharmaceutical preparations are mixed by mixing one or more solid excipients with one or more compositions described herein, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries to obtain tablets or dragee cores, if necessary.Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, or others such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.If necessary, disintegrants such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or its salts, for example, sodium alginate, are added. In some embodiments, dyestuffs or pigments are added to the tablets or dragee coatings for identification or to characterize different combinations of active therapeutic agent doses.

[0094] In some embodiments, the composition of the exogenous therapeutic agent is in the form of a capsule, including push-fit capsules made of gelatin and sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules contain 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 a soft capsule, the active therapeutic agent is dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In some embodiments, stabilizers are added. Capsules can be prepared, for example, by placing a bulk mix of the therapeutic agent formulation inside the capsule. In some embodiments, the formulation (non-aqueous suspensions and solutions) 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 into a sprinkle capsule, where the capsule is either swallowed whole or opened and its contents sprinkled onto food prior to a meal.

[0095] The composition for oral administration is in a dosage suitable for such administration. In one aspect, the solid oral dosage form is prepared by mixing the composition with one or more of antioxidants, flavorings, and carrier substances, such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some embodiments, the solid dosage form disclosed herein is in the form of a tablet (including suspension tablets, fast-dissolving tablets, bite-disintegration tablets, rapid-disintegration tablets, effervescent tablets, or caplets), pill, powder, capsule, solid dispersion, solid solution, biodegradable dosage form, controlled release formulation, pulsatile release dosage form, multiparticulate dosage form, beads, pellets, 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 tablet includes one or more flavorings. In other embodiments, the tablet includes a film surrounding the final compressed tablet. In some embodiments, the film coating can provide delayed release of the therapeutic agent from the formulation. In other embodiments, the film coating aids in patient compliance. The film coating may vary from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms, such as 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 easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, the individual unit doses include a film coating.

[0096] In another aspect, the dosage form comprises a microencapsulated formulation. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Non-limiting examples of materials include pH adjusters, erosion promoters, antifoaming agents, antioxidants, flavoring agents, and carrier materials, such as binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0097] The dosage form of the liquid formulation for oral administration is optionally an aqueous suspension selected from the group including, but not limited to, pharma- ceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form optionally contains additives such as (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.

[0098] In some embodiments, the compositions described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are made by vigorous mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only light mixing is required to distribute the droplets throughout the solution. In addition, water or an aqueous phase is optionally added immediately prior to administration, thereby ensuring the stability of unstable or hydrophobic active ingredients. Thus, SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improved bioavailability of hydrophobic active ingredients.

[0099] The compositions (e.g., pharmaceutical compositions) described herein may be formulated for administration to a subject by a route of administration, including, but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal. The compositions described herein may include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0100] In some embodiments, buccal formulations are administered using various formulations known in the art.In addition, the buccal dosage forms described herein may further comprise a biodegradable (hydrolyzable) polymer carrier that also serves to attach the dosage form to buccal mucosa.For buccal or sublingual administration, the composition may take the form of a tablet, lozenge, or gel that is appropriately formulated.

[0101] For intravenous injection, the composition is optionally formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological buffered saline.For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients.

[0102] Parenteral injections optionally include bolus injections or continuous infusions. Preparations for injection are optionally provided in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives. In some embodiments, the compositions described herein are in a form suitable for parenteral injection as a sterile suspension, aqueous solution, or emulsion in an oily or aqueous vehicle, and contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Compositions for parenteral administration include aqueous solutions of the agents that modulate the activity of carotid body in aqueous form. In addition, suspensions of the agents that modulate the activity of carotid body are optionally prepared as needed (e.g., oily injection suspensions).

[0103] Traditional formulation techniques include, for example, one or a combination of: (1) dry blending, (2) direct compression, (3) milling, (4) dry or nonaqueous 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., Worcester coating), tangential coating, top spraying, tabletting, extrusion, and the like.

[0104] In some embodiments, compositions are provided that include particles of a therapeutic agent and at least one dispersing or suspending agent for oral administration to a subject. The formulation may be a powder and / or granules for suspension, which upon mixing with water results in a substantially uniform suspension.

[0105] In addition, the compositions optionally contain one or more pH adjusting or buffering agents, 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 trishydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffering agents are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0106] In addition, the composition optionally contains one or more salts in an amount required to bring the osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, and suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0107] Other compositions optionally include one or more preservatives that inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0108] In one embodiment, the aqueous suspension and dispersion described herein remain homogeneous for at least 4 hours.In one embodiment, the aqueous suspension is resuspended into a homogeneous suspension by physical stirring lasting less than 1 minute.In yet another embodiment, stirring is not required to maintain a homogeneous aqueous dispersion.

[0109] Aerosol formulations for nasal administration are generally aqueous solutions designed to be administered to the nasal passages in drops or sprays.Nasal solutions can be similar to nasal mucus in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values ​​outside this range can also be used.Antibacterial agents or preservatives can also be included in the formulation.

[0110] Inhalants and aerosol formulations for inhalation drugs can be designed so that the drug or drug combination can be delivered to the respiratory tree of a subject when administered via the nasal or oral respiratory route. Inhalation liquids can be administered, for example, by a nebulizer. Inhalants or insufflations, including finely powdered or liquid drugs, can be delivered to the respiratory system as medicinal aerosols of a solution or suspension of the drug or drug combination in a propellant, for example, to aid in disbursement. Propellants can be liquefied gases, including fluorocarbons, such as halocarbons, e.g., fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

[0111] The aerosol formulation may also contain other ingredients, such as ethanol, isopropanol, propylene glycol, as well as other ingredients such as surfactants or oils and detergents, which may serve to stabilize the formulation and / or lubricate valve parts.

[0112] Aerosol formulations can be packaged under pressure and can be formulated as aerosols using solutions, suspensions, emulsions, powders, and semi-solid formulations.For example, solution aerosol formulations include solutions of agents such as transporters, carriers, or ion channel inhibitors in (substantially) pure propellants or as a mixture of propellants and solvents. Solvents can be used to dissolve agents and / or retard evaporation of propellants. Solvents can include, for example, water, ethanol, and glycol. Any combination of suitable solvents can be used, optionally in combination with preservatives, antioxidants, and / or other aerosol components.

[0113] Aerosol formulations can be dispersions or suspensions.Suspension aerosol formulations include a suspension of a drug or drug combination, such as a transporter, carrier, or ion channel inhibitor, and a dispersing agent.Dispersing agents can include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin, and corn oil.Suspension aerosol formulations can also include lubricants, preservatives, antioxidants, and / or other aerosol components.

[0114] Aerosol formulations can also be formulated as emulsions.Aerosol formulations of emulsions can also include, in addition to alcohol, such as ethanol, surfactant, water and propellant, a drug or combination of drugs, such as a transporter, carrier or ion channel.The surfactants used can be non-ionic, anionic or cationic.An example of an aerosol formulation of emulsions includes, for example, ethanol, surfactant, water and propellant.Another example of an aerosol formulation of emulsions includes, for example, vegetable oil, glycerin monostearate and propane.

[0115] method In some embodiments, methods of producing or using the compositions disclosed herein are disclosed herein. In some embodiments, the methods include high-throughput techniques for enucleating cells to produce compositions comprising enucleated cells for biomedical applications with minimal residual nucleated parent cells. In some embodiments, the methods include inducing expression of a suicide gene under conditions suitable to kill residual nucleated parent cells in the composition. The methods disclosed herein also provide methods of using enucleated cells as fusion partners (e.g., fusion to another cell in vivo or ex vivo), or therapeutic delivery vehicles, or combinations thereof.

[0116] In some aspects, disclosed herein are methods of generating enucleated cells as described herein, the methods comprising enucleating nucleated parent cells. In some embodiments, the parent cells can be treated with an exogenous molecule to soften the cytoskeleton of the parent cells. For example, the parent cells can 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 generate enucleated cells. In some embodiments, the centrifugation comprises the use of a density gradient, where at least the enucleated cells and the intact nucleated cells sediment in different layers in the density gradient, thus isolating the enucleated cells. In some embodiments, the centrifugation comprises continuous-flow centrifugation. Example 8 describes an exemplary continuous-flow centrifugation experiment to obtain enucleated cells from nucleated cells. In some embodiments, the 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, the use of continuous-flow centrifugation increases the volume that can be centrifuged compared to swinging bucket centrifugation (to generate a comparable density gradient). In some embodiments, the centrifugation comprises zonal centrifugation, and the enucleated cells are separated from the nucleated cells based on size difference, mass difference, or a combination thereof. Example 7 describes generating enucleated cells by zonal centrifugation. In some embodiments, the method comprises inducing cell death of the nucleated cells after centrifugation or enucleation. For example, the nucleated cells can be engineered to have a heterologous polynucleotide encoding a heterologous gene product described herein, and expression of the heterologous gene product induces cell death of at least one nucleated cell.

[0117] In some embodiments, the methods disclosed herein result in a composition comprising tens of millions of enucleated cells (the "enucleated cell fraction"). In some embodiments, the composition also comprises remaining nucleated cells (the "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 in a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, excipient, or diluent.

[0118] In some aspects, the method of producing an enucleated cell does not consist of or include differentiation of a parent cell. For example, the enucleated cell is not obtained by differentiating a nucleated red blood cell progenitor cell into an enucleated red blood cell. In some embodiments, the enucleated cell is not a terminally differentiated cell. In some embodiments, the enucleated cell is not a platelet. In some embodiments, the enucleated cell is not obtained from a platelet lineage cell. In some embodiments, the enucleated cell is not an red blood cell. In some embodiments, the enucleated cell is not obtained from an erythroid lineage cell.

[0119] In some embodiments, a parent cell containing a nucleus is engineered to express at least one of the therapeutic agents, transmembrane moieties, immune evasion moieties, or targeting moieties described herein, and then the nucleus of the parent cell is removed. In some embodiments, a parent cell containing a nucleus is enucleated, and the enucleated cell is engineered to express the therapeutic agents, transmembrane moieties, immune evasion moieties, or targeting moieties described herein. In some embodiments, a parent cell is engineered to express one or more of the above biomolecules (e.g., immune evasion moieties and / or targeting moieties), and the resulting enucleated cell (e.g., already expressing an immune evasion moiety and / or targeting moiety) is further engineered to express a second of the above biomolecules (e.g., a therapeutic agent). In this way, the enucleated cells of the present disclosure can be extensively engineered prior to enucleation, stored for long periods of time as needed (e.g., via lyophilization, cryostasis, cryopreservation), and rapidly engineered to express a therapeutic agent closer to the time of need.

[0120] In some embodiments, the composition has a volume of about 10 milliliters (mL) or more to about 10,000 mL. In some embodiments, the composition has a volume of about 10 mL or more 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, about 100 mL ~4,000mL, 100mL~5,000mL, 100mL~6,000mL, 100mL~7,000mL, 100mL~8,000mL, 100mL~9,000mL, 100mL~10,000mL, 1,000mL~2, 000mL, 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 1000mL to approximately 7,000mL, approximately 1000mL to approximately 8,000mL, approximately 1000mL ~9,000mL, 1000mL~10,000mL, 2000mL~3,000mL, 2000mL~4,000mL, 2000mL~5,000mL, 2000mL~6,000mL, 2000mL~7,000mL, 2000mL mL~about 8,000mL, about 2000mL~about 9,000mL, about 2000mL~about 10,000mL, about 3000mL~about 4,000mL, about 3000mL~about 5,000mL, about 3000mL~about 6,000mL, about 3000mL~about 7,000mL, about 300 0mL to approx. 8,000mL, approx. 3000mL to approx. 9,000mL, approx. 3000mL to approx. 10,000mL, approx. 4000mL to approx. 5,000mL, approx. 4000mL to approx. 6,000mL, approx. 4000mL to approx. 7,000mL, approx. 4000mL to approx. 8,000mL, approx. 4, 000mL to about 9,000mL, about 4,000mL to about 10,000mL, about 5,000mL to about 6,000mL, about 5,000mL to about 7,000mL, about 5,000mL to about 8,000mL, about 5,000mL to about 9,000mL, about 5,000mL to about 10,The volume may be from about 6,000 mL, about 6,000 mL to about 7,000 mL, about 6,000 mL to about 8,000 mL, about 6,000 mL to about 9,000 mL, about 6,000 mL to about 10,000 mL, about 7,000 mL to about 8,000 mL, about 7,000 mL to about 9,000 mL, about 7,000 mL to about 10,000 mL, about 8,000 mL to about 9,000 mL, about 8,000 mL to about 10,000 mL, or about 9,000 mL to about 10,000 mL. In some embodiments, the composition has a volume including about 10 mL or more, about 100 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, about 9000 mL, or about 10000 mL. In some embodiments, the composition has a volume including at least about 10 mL or more, about 100 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, or about 9000 mL. In some embodiments, the composition has a volume of at least about 100 mL, including at most about 1000 mL, at most about 2000 mL, at most about 3000 mL, at most about 4000 mL, at most about 5000 mL, at most about 6000 mL, at most about 7000 mL, at most about 8000 mL, at most about 9000 mL, or at most about 10000 mL.

[0121] In some embodiments, the composition has a volume between about 10 mL and about 10,000 mL. In some embodiments, the composition has a volume between about 10 mL and about 100 mL, about 10 mL and about 1,000 mL, about 10 mL and about 2,000 mL, about 10 mL and about 3,000 mL, about 10 mL and about 4,000 mL, about 10 mL and about 5,000 mL, about 10 mL and about 6,000 mL, about 10 mL and about 7,000 mL, about 10 mL and about 8,000 mL, about 10 mL and about 9,000 mL, about 10 mL and about 10,000 mL, about 100 mL and about 1,000 mL, about 100 mL and about 2,000 mL, about 100 mL and about 3,000 mL, about 100 mL and about 4, 000mL, approximately 100mL to approximately 5,000mL, approximately 100mL to approximately 6,000mL, approximately 100mL to approximately 7,000mL, approximately 100mL to approximately 8,000mL, approximately 100mL to approximately 9,000mL, approximately 100mL to approximately 10,000mL, approximately 1,000mL to approximately 2,000m L, 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 1000mL to approximately 7,000mL, approximately 1000mL to approximately 8,000mL, approximately 1000mL to approximately 9,0 00mL, about 1000mL to about 10,000mL, about 2000mL to about 3,000mL, about 2000mL to about 4,000mL, about 2000mL to about 5,000mL, about 2000mL to about 6,000mL, about 2000mL to about 7,000mL, about 2000mL to about 8, 000mL, approximately 2000mL to approximately 9,000mL, approximately 2000mL to approximately 10,000mL, approximately 3000mL to approximately 4,000mL, approximately 3000mL to approximately 5,000mL, approximately 3000mL to approximately 6,000mL, approximately 3000mL to approximately 7,000mL, approximately 3000mL to approximately 8, 000mL, about 3000mL to about 9,000mL, about 3000mL to about 10,000mL, about 4000mL to about 5,000mL, about 4000mL to about 6,000mL, about 4000mL to about 7,000mL, about 4000mL to about 8,000mL, about 4,000mL to about 9 ,000mL, approximately 4,000mL to approximately 10,000mL, approximately 5,000mL to approximately 6,000mL, approximately 5,000mL to approximately 7,000mL, approximately 5,000mL to approximately 8,000mL, approximately 5,000mL to approximately 9,000mL, approximately 5,000mL to approximately 10,000mL, approximately 6,The volume may range from about 7,000mL to about 7,000mL, about 6,000mL to about 8,000mL, about 6,000mL to about 9,000mL, about 6,000mL to about 10,000mL, about 7,000mL to about 8,000mL, about 7,000mL to about 9,000mL, about 7,000mL to about 10,000mL, about 8,000mL to about 9,000mL, about 8,000mL to about 10,000mL, or about 9,000mL to about 10,000mL. In some embodiments, the composition has a volume including between about 10 mL, about 100 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, about 9000 mL, or about 10000 mL. In some embodiments, the composition has a volume including between at least about 10 mL, about 100 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, or about 9000 mL. In some embodiments, the composition has a volume of up to about 100 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, about 9000 mL, or about 10000 mL.

[0122] In some embodiments, described herein are methods for cell processing by enucleating a portion of nucleated cells (parent cells) using continuous flow centrifugation to generate an enucleated cell fraction, where the continuous flow centrifugation is fixed angle centrifugation. In some embodiments, the continuous flow centrifugation is swinging bucket centrifugation.

[0123] In some embodiments, the resulting composition comprises an enucleated cell fraction, which may be 100% of the composition. In other embodiments, there may be a nucleated cell fraction of the composition that is comprised of nucleated parent cells that were not enucleated. In some embodiments, the enucleated cell fraction is about 30% or more of the composition. In some embodiments, the enucleated cell fraction is about 35% or more of the composition. In some embodiments, the enucleated cell fraction is about 40% or more of the composition. In some embodiments, the enucleated cell fraction is about 45% or more of the composition. In some embodiments, the enucleated cell fraction is about 50% or more of the composition. In some embodiments, the enucleated cell fraction is about 55% or more of the composition. In some embodiments, the enucleated cell fraction is about 60% or more of the composition. In some embodiments, the enucleated cell fraction is about 65% or more of the composition. In some embodiments, the enucleated cell fraction is about 70% or more of the composition. In some embodiments, the enucleated cell fraction is about 75% or more of the composition. In some embodiments, the enucleated cell fraction is about 80% or more of the composition. In some embodiments, the enucleated cell fraction is about 85% or more of the composition. In some embodiments, the enucleated cell fraction is about 90% or more of the composition. In some embodiments, the enucleated cell fraction is about 95% or more of the composition. In some embodiments, the enucleated cell fraction is about 96% or more of the composition. In some embodiments, the enucleated cell fraction is about 97% or more of the composition. In some embodiments, the enucleated cell fraction is about 98% or more of the composition. In some embodiments, the enucleated cell fraction is about 99% or more of the composition.

[0124] In some embodiments, cell separation, cell isolation, or cell sorting is a process for isolating one or more specific cell populations from a heterogeneous mixture of cells. In some embodiments, the cell enucleation method disclosed herein is performed on an isolated population of homogeneous cells. In some embodiments, the cell enucleation method disclosed herein is performed on a heterogeneous mixture of cells. In some embodiments, the method disclosed herein comprises isolating a homogeneous population of cells from a heterogeneous mixture of cells using a suitable cell separation technique, including but 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.

[0125] In centrifugation, denser particles can migrate to the outer edge of the mixture, while less dense objects further congregate as the sample is spun. The biological sample can be centrifuged until the cell types are isolated into layers. During centrifugation, each cell type can settle to its isothermal point, which is the place in the media gradient where the density of the cells and the media are equal. Examples of density gradient media include Lymphoprep™, Lympholyte™, Ficoll-Paque™, Percoll™, OptiPrep™, Accuspin™, Aystem-Histopaque™ media cell separation, Histopaque™ media, Histopaque™ iodized gradient media, inorganic salts, non-ionic iodized density gradient media, polyhydric alcohols, polysaccharides, and the like. For example, Lymphoprep™, Lympholyte™, and Ficoll-Paque™ consist 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 separate cells, organelles, viruses, and other intracellular particles. OptiPrep™ is a medium consisting of iodixanol in water and is used to isolate viruses, organelles, macromolecules, and cells.

[0126] In some embodiments, disclosed herein are methods for cell processing by enucleating a portion of nucleated cells to generate an enucleated cell fraction using continuous flow centrifugation. In some embodiments, disclosed herein are methods for cell processing by enucleating a portion of nucleated cells to generate an enucleated cell fraction using zonal centrifugation. In some embodiments, the continuous flow centrifugation is fixed angle centrifugation. In some embodiments, the continuous flow centrifugation is swinging bucket centrifugation. In some embodiments, the continuous flow centrifugation creates a density gradient. In some embodiments, the density gradient separates the enucleated cell fraction from the nucleated cells in the composition. In some embodiments, the density gradient comprises a polysaccharide density gradient. In some embodiments, the polysaccharide density gradient comprises a Ficoll density gradient. In some embodiments, the method further comprises generating a Ficoll gradient by polymerizing sucrose molecules with epichlorohydrin to obtain an osmotically inactive polysaccharide.

[0127] In some embodiments, the gradient comprises a density gradient of 2 ranges to 20 ranges. In some embodiments, the gradient comprises a density gradient of 2 ranges to 3 ranges, 2 ranges to 4 ranges, 2 ranges to 5 ranges, 2 ranges to 6 ranges, 2 ranges to 8 ranges, 2 ranges to 10 ranges, 2 ranges to 12 ranges, 2 ranges to 14 ranges, 2 ranges to 16 ranges, 2 ranges to 18 ranges, 2 ranges to 20 ranges, 3 ranges to 4 ranges, 3 ranges to 5 ranges, 3 ranges to 6 ranges, 3 ranges to 8 ranges, 3 ranges to 10 ranges, range, 3 ranges~12 ranges, 3 ranges~14 ranges, 3 ranges~16 ranges, 3 ranges~18 ranges, 3 ranges~20 ranges, 4 ranges~5 ranges, 4 ranges~6 ranges, 4 ranges~8 ranges, 4 ranges~10 ranges, 4 ranges~12 ranges, 4 ranges~14 ranges, 4 ranges~16 ranges, 4 ranges~18 ranges, 4 ranges~20 ranges, 5 ranges~6 ranges, 5 ranges~8 ranges, 5 ranges~10 ranges range, 5 range~12 range, 5 range~14 range, 5 range~16 range, 5 range~18 range, 5 range~20 range, 6 range~8 range, 6 range~10 range, 6 range~12 range, 6 range~14 range, 6 range~16 range, 6 range~18 range, 6 range~20 range, 8 range~10 range, 8 range~12 range, 8 range~14 range, 8 range~16 range, 8 range~18 range , 8 ranges to 20 ranges, 10 ranges to 12 ranges, 10 ranges to 14 ranges, 10 ranges to 16 ranges, 10 ranges to 18 ranges, 10 ranges to 20 ranges, 12 ranges to 14 ranges, 12 ranges to 16 ranges, 12 ranges to 18 ranges, 12 ranges to 20 ranges, 14 ranges to 16 ranges, 14 ranges to 18 ranges, 14 ranges to 20 ranges, 16 ranges to 18 ranges, 16 ranges to 20 ranges, or 18 ranges to 20 ranges. In some embodiments, the gradient comprises between 2 ranges, 3 ranges, 4 ranges, 5 ranges, 6 ranges, 8 ranges, 10 ranges, 12 ranges, 14 ranges, 16 ranges, 18 ranges, or 20 ranges.In some embodiments, the gradient comprises a density gradient between at least 2 ranges, 3 ranges, 4 ranges, 5 ranges, 6 ranges, 8 ranges, 10 ranges, 12 ranges, 14 ranges, 16 ranges, or 18 ranges. In some embodiments, the gradient comprises a density gradient between up to 3 ranges, 4 ranges, 5 ranges, 6 ranges, 8 ranges, 10 ranges, 12 ranges, 14 ranges, 16 ranges, 18 ranges, or 20 ranges. In some embodiments, the gradient comprises a density gradient of 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. In some embodiments, the gradient comprises a density gradient of at least 7 ranges. In some embodiments, the gradient comprises a density gradient of at least 5 ranges. In some embodiments, the gradient comprises a density gradient of at least 3 ranges. In some embodiments, the gradient comprises a seven range density gradient. In some embodiments, the gradient comprises a five range density gradient. In some embodiments, the gradient comprises a three range density gradient.

[0128] In some embodiments, the gradient comprises about 7.5% density gradient medium to about 30% density gradient medium. 7.5% density gradient medium to about 10% density gradient medium, about 7.5% density gradient medium to about 12.5% ​​density gradient medium, about 7.5% density gradient medium to about 15% density gradient medium, about 7.5% density gradient medium to about 16% density gradient medium, about 7.5% density gradient medium to about 17% density gradient medium, about 7.5% density gradient medium to about 18% density gradient medium, about 7.5% density gradient medium to about 19% density gradient medium, about 7.5% density gradient medium to about 20% density gradient medium, about 7.5% density gradient medium to about 25% density gradient medium, about 7. 5% density gradient medium to about 27.5% density gradient medium, about 7.5% density gradient medium to about 30% density gradient medium, about 10% density gradient medium to about 12.5% ​​density gradient medium, about 10% density gradient medium to about 15% density gradient medium, about 10% density gradient medium to about 16% density gradient medium, about 10% density gradient medium to about 17% density gradient medium, about 10% density gradient medium to about 18% density gradient medium, about 10% density gradient medium to about 19% density gradient medium, about 10% density gradient medium to about 20% density gradient medium, about 10% density gradient medium ~25% density gradient medium, ~27.5% density gradient medium, ~30% density gradient medium, ~12.5% ​​density gradient medium ~15% density gradient medium, ~12.5% ​​density gradient medium ~16% density gradient medium, ~12.5% ​​density gradient medium ~17% density gradient medium, ~12.5% ​​density gradient medium ~18% density gradient medium, ~12.5% ​​density gradient medium ~19% density gradient medium, ~12.5% ​​density gradient medium ~20% density gradient medium, ~12.5% ​​density gradient medium Distribution medium to about 25% density gradient medium, about 12.5% ​​density gradient medium to about 27.5% density gradient medium, about 12.5% ​​density gradient medium to about 30% density gradient medium, about 15% density gradient medium to about 16% density gradient medium, about 15% density gradient medium to about 17% density gradient medium, about 15% density gradient medium to about 18% density gradient medium, about 15% density gradient medium to about 19% density gradient medium, about 15% density gradient medium to about 20% density gradient medium, about 15% density gradient medium to about 25% density gradient medium, about 15% density gradient medium to about 27.5% density gradient medium, about 15% to about 30% density gradient medium, about 16% to about 17% density gradient medium, about 16% to about 18% density gradient medium, about 16% to about 19% density gradient medium, about 16% to about 20% density gradient medium, about 16% to about 25% density gradient medium, about 16% to about 27.5% density gradient medium, About 16% density gradient medium to about 30% density gradient medium, about 17% density gradient medium to about 18% density gradient medium, about 17% density gradient medium to about 19% density gradient medium, about 17% density gradient medium to about 20% density gradient medium, about 17% density gradient medium to about 25% density gradient medium, about 17% density gradient medium to about 27.5% density gradient medium, about 17% density gradient medium to about 30% density gradient medium, about 18% density gradient medium ~19% density gradient medium, ~18% density gradient medium ~20% density gradient medium, ~18% density gradient medium ~25% density gradient medium, ~18% density gradient medium ~27.5% density gradient medium, ~18% density gradient medium ~30% density gradient medium, ~19% density gradient medium ~20% density gradient medium, ~19% density gradient medium ~25% density gradient medium, ~19% density gradient medium ~27.5% density gradient medium Distribution medium, about 19% density gradient medium to about 30% density gradient medium, about 20% density gradient medium to about 25% density gradient medium, about 20% density gradient medium to about 27.5% density gradient medium, about 20% density gradient medium to about 30% density gradient medium, about 25% density gradient medium to about 27.5% density gradient medium, about 25% density gradient medium to about 30% density gradient medium, or about 27.5% density gradient medium to about 30% density gradient medium.

[0129] In some embodiments, the gradient is a Ficoll gradient. In some embodiments, the Ficoll gradient comprises a density Ficoll gradient between 2 ranges and 20 ranges. In some embodiments, the Ficoll gradient comprises a density Ficoll gradient between 2 ranges and 3 ranges, between 2 ranges and 4 ranges, between 2 ranges and 5 ranges, between 2 ranges and 6 ranges, between 2 ranges and 8 ranges, between 2 ranges and 10 ranges, between 2 ranges and 12 ranges, between 2 ranges and 14 ranges, between 2 ranges and 16 ranges, between 2 ranges and 18 ranges, between 2 ranges and 20 ranges, between 3 ranges and 4 ranges, between 3 ranges and 5 ranges, between 3 ranges and 6 ranges, between 3 ranges and 8 ranges, between 3 ranges and 10 ranges, between 3 ranges and 12 ranges, between 3 ranges and 14 ranges, between 3 ranges and 16 ranges, between 2 ranges and 18 ranges, between 2 ranges and 20 ranges, between 3 ranges and 4 ranges, between 3 ranges and 5 ranges, between 3 ranges and 6 ranges, between 3 ranges and 8 ranges, between 3 ranges and 10 ranges, 10 range, 3 ranges~12 ranges, 3 ranges~14 ranges, 3 ranges~16 ranges, 3 ranges~18 ranges, 3 ranges~20 ranges, 4 ranges~5 ranges, 4 ranges~6 ranges, 4 ranges~8 ranges, 4 ranges~10 ranges, 4 ranges~12 ranges, 4 ranges~14 ranges, 4 ranges~16 ranges, 4 ranges~18 ranges, 4 ranges~20 ranges, 5 ranges~6 ranges, 5 ranges~8 ranges, 5 ranges~10 range, 5 range~12 range, 5 range~14 range, 5 range~16 range, 5 range~18 range, 5 range~20 range, 6 range~8 range, 6 range~10 range, 6 range~12 range, 6 range~14 range, 6 range~16 range, 6 range~18 range, 6 range~20 range, 8 range~10 range, 8 range~12 range, 8 range~14 range, 8 range~16 range, 8 range~18 range, Include density Ficoll gradients ranging from 8 ranges to 20 ranges, 10 ranges to 12 ranges, 10 ranges to 14 ranges, 10 ranges to 16 ranges, 10 ranges to 18 ranges, 10 ranges to 20 ranges, 12 ranges to 14 ranges, 12 ranges to 16 ranges, 12 ranges to 18 ranges, 12 ranges to 20 ranges, 14 ranges to 16 ranges, 14 ranges to 18 ranges, 14 ranges to 20 ranges, 16 ranges to 18 ranges, 16 ranges to 20 ranges, or 18 ranges to 20 ranges.In some embodiments, the Ficoll gradient comprises between 2 ranges, 3 ranges, 4 ranges, 5 ranges, 6 ranges, 8 ranges, 10 ranges, 12 ranges, 14 ranges, 16 ranges, 18 ranges, or 20 ranges. In some embodiments, the Ficoll gradient comprises a density Ficoll gradient of at least 2 ranges, 3 ranges, 4 ranges, 5 ranges, 6 ranges, 8 ranges, 10 ranges, 12 ranges, 14 ranges, 16 ranges, or 18 ranges. In some embodiments, the Ficoll gradient comprises a density Ficoll gradient of at most 3 ranges, 4 ranges, 5 ranges, 6 ranges, 8 ranges, 10 ranges, 12 ranges, 14 ranges, 16 ranges, 18 ranges, or 20 ranges. In some embodiments, the Ficoll gradient comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty ranges of density Ficoll gradients. In some embodiments, the Ficoll gradient comprises at least seven ranges of density Ficoll gradients. In some embodiments, the Ficoll gradient comprises at least five ranges of density Ficoll gradients. In some embodiments, the Ficoll gradient comprises at least three ranges of density Ficoll gradients. In some embodiments, the Ficoll gradient comprises seven ranges of density Ficoll gradients. In some embodiments, the Ficoll gradient comprises five ranges of density Ficoll gradients. In some embodiments, the Ficoll gradient comprises three ranges of density Ficoll gradients.

[0130] In some embodiments, the Ficoll density gradient is from about 7.5% Ficoll to about 10% Ficoll, from about 7.5% Ficoll to about 12.5% ​​Ficoll, from about 7.5% Ficoll to about 15% Ficoll, from about 7.5% Ficoll to about 16% Ficoll, from about 7.5% Ficoll to about 17% Ficoll, from about 7.5% Ficoll to about 18% Ficoll, from about 7.5% Ficoll to about 19% Ficoll, from about 7.5% Ficoll to about 20% Ficoll, from about 7.5% Ficoll to about 25% Ficoll, from about 7.5% Ficoll to about 27.5% Ficoll Ficoll, about 7.5% Ficoll to about 30% Ficoll, about 10% Ficoll to about 12.5% ​​Ficoll, about 10% Ficoll to about 15% Ficoll, about 10% Ficoll to about 16% Ficoll, about 10% Ficoll to about 17% Ficoll, about 10% Ficoll to about 18% Ficoll, about 10% Ficoll to about 19% Ficoll, about 10% Ficoll to about 20% Ficoll, about 10% Ficoll to about 25% Ficoll, about 10% Ficoll to about 27.5% Ficoll, about 10% Ficoll to about 30% Ficoll, about 12.5% ​​Ficoll Ficoll to about 15% Ficoll, about 12.5% ​​Ficoll to about 16% Ficoll, about 12.5% ​​Ficoll to about 17% Ficoll, about 12.5% ​​Ficoll to about 18% Ficoll, about 12.5% ​​Ficoll to about 19% Ficoll, about 12.5% ​​Ficoll to about 20% Ficoll, about 12.5% ​​Ficoll to about 25% Ficoll, about 12.5% ​​Ficoll to about 27.5% Ficoll, about 12.5% ​​Ficoll to about 30% Ficoll, about 15% Ficoll to about 16% Ficoll, about 15% Ficoll to about 17% Ficoll, about 15% Ficoll to about 18% Ficoll, about 15% Ficoll to about 19% Ficoll, about 15% Ficoll to about 20% Ficoll, about 15% Ficoll to about 25% Ficoll, about 15% Ficoll to about 27.5% Ficoll, about 15% Ficoll to about 30% Ficoll, about 16% Ficoll to about 17% Ficoll, about 16% Ficoll to about 18% Ficoll, about 16% Ficoll to about 19% Ficoll, about 16% Ficoll to about 20% Ficoll, about 16% Ficoll to about 25% Ficoll, about 16% Ficoll to about 27.5% Ficoll, about 16% Ficoll to about 30% Ficoll, about 17% Ficoll to about 18% Ficoll, about 17% Ficoll to about 19% Ficoll, about 17% Ficoll to about 20% Ficoll, about 17% Ficoll to about 25% Ficoll, about 17% Ficoll to about 27.5% Ficoll, about 17% Ficoll to about 30% Ficoll, about 18% Ficoll to about 19% Ficoll. The ficoll may be about 18% to about 20% ficoll, about 18% to about 25% ficoll, about 18% to about 27.5% ficoll, about 18% to about 30% ficoll, about 19% to about 20% ficoll, about 19% to about 25% ficoll, about 19% to about 27.5% ficoll, about 19% to about 30% ficoll, about 20% to about 25% ficoll, about 20% to about 27.5% ficoll, about 20% to about 30% ficoll, about 25% to about 27.5% ficoll, about 25% to about 30% ficoll, or about 27.5% to about 30% ficoll.

[0131] In some embodiments, the Ficoll density gradient comprises about 7.5% Ficoll, about 10% Ficoll, about 12.5% ​​Ficoll, about 15% Ficoll, about 16% Ficoll, about 17% Ficoll, about 18% Ficoll, about 19% Ficoll, about 20% Ficoll, about 25% Ficoll, about 27.5% Ficoll, or about 30% Ficoll. In some embodiments, the Ficoll density gradient comprises at least about 7.5% Ficoll, about 10% Ficoll, about 12.5% ​​Ficoll, about 15% Ficoll, about 16% Ficoll, about 17% Ficoll, about 18% Ficoll, about 19% Ficoll, about 20% Ficoll, about 25% Ficoll, or about 27.5% Ficoll. In some embodiments, the ficoll density gradient comprises up to about 10% ficoll, about 12.5% ​​ficoll, about 15% ficoll, about 16% ficoll, about 17% ficoll, about 18% ficoll, about 19% ficoll, about 20% ficoll, about 25% ficoll, about 27.5% ficoll, or about 30% ficoll. In some embodiments, the ficoll density gradient comprises about 25% ficoll, about 17% ficoll, about 16% ficoll, about 15% ficoll, or about 12.5% ​​ficoll. In some embodiments, the ficoll density gradient comprises about 25% ficoll. In some embodiments, the ficoll density gradient comprises about 17% ficoll. In some embodiments, the ficoll density gradient comprises about 16% ficoll. In some embodiments, the ficoll density gradient comprises about 15% ficoll. In some embodiments, the ficoll density gradient comprises about 12.5% ​​ficoll.

[0132] In some embodiments, methods for cell processing disclosed herein include enucleating a portion of the nucleated cells using continuous flow centrifugation to generate an enucleated cell fraction, wherein the portion of the nucleated cells that are enucleated is greater than or equal to about 10% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 20% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 25% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 30% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 35% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 40% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 45% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 50% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 55% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 60% of the nucleated cells. In some embodiments, the portion of the nucleated cells is greater than or equal to about 65% of the nucleated cells. In some embodiments, the portion of nucleated cells is about 70% or more of the nucleated cells. In some embodiments, the portion of nucleated cells is about 75% or more of the nucleated cells. In some embodiments, the portion of nucleated cells is about 80% or more of the nucleated cells. In some embodiments, the portion of nucleated cells is about 85% or more of the nucleated cells. In some embodiments, the portion of nucleated cells is about 90% or more of the nucleated cells. In some embodiments, the portion of nucleated cells is about 95% or more of the nucleated cells.

[0133] In some embodiments, the enucleated cell fraction produced by the methods disclosed herein is about 1×10 5 Enucleated cells, 1 × 10 6 Enucleated cells, approximately 1 x 10 7 of enucleated cells, 3 × 10 5 of enucleated cells, 5 × 10 5 of 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, approximately 20 × 10 7Enucleated cells, 50 × 10 7 of enucleated cells, approximately 70 × 10 7 Enucleated cells, 90 × 10 7 of enucleated cells, 100 × 10 7 Enucleated cells, 150 × 10 7 of enucleated cells, 200 × 10 7 of enucleated cells, 250 × 10 7 of enucleated cells, 300 × 10 7 of enucleated cells, or 500 × 10 7 It contains at least one enucleated cell.

[0134] In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises about 90% or less of the average diameter of the nucleated cells.

[0135] In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter including about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm or more. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter ranging from about 1 μm to about 10 μm. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter of about 1 μm to about 2 μm, about 1 μm to about 3 μm, about 1 μm to about 4 μm, about 1 μm to about 5 μm, about 1 μm to about 6 μm, about 1 μm to about 7 μm, about 1 μm to about 8 μm, about 1 μm to about 9 μm, about 1 μm to about 10 μm, about 2 μm to about 3 μm, about 2 μm to about 4 μm, about 2 μm to about 5 μm, about 2 μm to about 6 μm, about 2 μm to about 7 μm, about 2 μm to about 8 μm, about 2 μm to about 9 μm, about 2 μm to about 10 μm, about 3 μm to about 4 μm, about 3 μm to about 5 μm, about 3 μm to about 6 μm, about 3 μm to about 7 μm, about 3 μm to about 8 μm, about The diameter is in the range of 3 μm to about 9 μm, about 3 μm to about 10 μm, about 4 μm to about 5 μm, about 4 μm to about 6 μm, about 4 μm to about 7 μm, about 4 μm to about 8 μm, about 4 μm to about 9 μm, about 4 μm to about 10 μm, about 5 μm to about 6 μm, about 5 μm to about 7 μm, about 5 μm to about 8 μm, about 5 μm to about 9 μm, about 5 μm to about 10 μm, about 6 μm to about 7 μm, about 6 μm to about 8 μm, about 6 μm to about 9 μm, about 6 μm to about 10 μm, about 7 μm to about 8 μm, about 7 μm to about 9 μm, about 7 μm to about 10 μm, about 8 μm to about 9 μm, about 8 μm to about 10 μm, or about 9 μm to about 10 μm. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter in the range of about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter in the range of at least about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, or about 9 μm. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter in the range of at most about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter of about 8 μm.

[0136] In some embodiments, the method for cell processing further comprises creating a density gradient by centrifuging the density gradient medium at an acceleration rate over 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 method for cell processing further comprises creating a density gradient by centrifuging the polysaccharide at an acceleration rate over 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 method for cell processing further comprises creating a density gradient by centrifuging the polysaccharide at an acceleration 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 method for cell processing further comprises creating a density gradient by centrifuging the polysaccharide at an acceleration for at least about 30 minutes.

[0137] In some embodiments, the method of enucleating a portion of nucleated cells to produce an enucleated cell fraction using continuous flow centrifugation for cell processing further comprises creating a density gradient by centrifuging the polysaccharide at a minimum deceleration. In some embodiments, the density gradient comprises centrifuging the polysaccharide at a maximum centrifugal force of about 20,000 relative centrifugal force (RCF) to about 250,000 RCF. In some embodiments, the density gradient is from about 20,000 RCF to about 30,000 RCF, from about 20,000 RCF to about 40,000 RCF, from about 20,000 RCF to about 50,000 RCF, from about 20,000 RCF to about 60,000 RCF, from about 20,000 RCF to about 70,000 RCF, from about 20,000 RCF to about 80,000 RCF, from about 20,000 RCF to about 100,000 RCF, from about 20,000 RCF to about 120,000 RCF, from about 20,000 RCF to about 30,000 RCF, from about 20,000 RCF to about 40,000 RCF, from about 20,000 RCF to about 50,000 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~about 100,000RCF, about 60,000RCF~about 120,000RCF, about 60,000RCF~about 150,000RCF, about 60,000RCF~about 200,0 00RCF, 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 120,0 00RCF, about 70,000RCF~about 150,000RCF, about 70,000RCF~about 200,000RCF, about 70,000RCF~about 250,000RCF, about 80,000RCF~about 100,0 00RCF, about 80,000RCF~about 120,000RCF, about 80,000RCF~about 150,000RCF, about 80,000RCF~about 200,000RCF, about 80,000RCF~about 250, 000RCF, about 100,000RCF to about 120,000RCF, about 100,000RCF to about 150,000RCF, about 100,000RCF to about 200,000RCF, about 100,000RCF to about The method includes centrifuging the polysaccharide at a maximum centrifugal force of about 250,000 RCF, about 120,000 RCF to about 150,000 RCF, about 120,000 RCF to about 200,000 RCF, about 120,000 RCF to about 250,000 RCF, about 150,000 RCF to about 200,000 RCF, about 150,000 RCF to about 250,000 RCF, or about 200,000 RCF to about 250,000 RCF. In some embodiments, the density gradient comprises centrifuging the polysaccharide at a maximum centrifugal force of between 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.In some embodiments, the density gradient comprises centrifuging the polysaccharide at a maximum centrifugal force of between about 10,000 RCF, about 80,000 RCF, about 100,000 RCF, about 120,000 RCF, about 150,000 RCF, or about 200,000 RCF. In some embodiments, the density gradient comprises centrifuging the polysaccharide at a maximum centrifugal force of up to 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.

[0138] Cellular Modifications of the Present Disclosure As shown in FIG. 2, the nucleated ("parent") cells may be engineered to express one or more exogenous factors prior to enucleation, or after enucleation, or combinations thereof. In some embodiments, the one or more exogenous biomolecules comprise a targeting moiety, a transmembrane moiety, a biomolecular suicide switch, or a therapeutic agent, or combinations thereof. In some embodiments, the targeting moiety comprises an adhesion molecule, a chemokine or retention receptor, or both. In some embodiments, the targeting moiety is engineered to target a target tissue, cell, or environment (e.g., lymphoid tissue of a subject) as disclosed herein. Additionally or alternatively, the resulting enucleated cells are engineered to express and optionally secrete a therapeutic agent. In some embodiments, the therapeutic agent comprises an antibody or antigen-binding fragment thereof (e.g., a single domain antibody). In some embodiments, the enucleated cells can be administered to a subject in need thereof to treat a disease or condition in the subject.

[0139] Various methods can be used to introduce biomolecules (e.g., therapeutic agents, transmembrane moieties, immune evasion moieties, and / or targeting moieties described herein) into parental or enucleated cells. Non-limiting examples of methods that can be used to introduce biomolecules into parental or enucleated cells include: liposome-mediated transfer, adenovirus, adeno-associated virus, herpes virus, retrovirus-based vectors, lentivirus vectors, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, infection, hydrodynamic delivery, magnetic transfection, nanoparticle transfection, or combinations thereof. In some embodiments, any of the therapeutic agents, viruses, antibodies, or nanoparticles of the compositions and methods provided herein can be introduced into enucleated cells.

[0140] In some embodiments, the enucleated cells are preserved by cryopreservation, cryopreservation, or freeze-drying. Cryopreservation involves freezing the enucleated cells, while cryopreservation involves storing the enucleated cells at temperatures below room temperature without freezing the enucleated cells. In some embodiments, the enucleated cells are freeze-dried. In some embodiments, the freeze-dried enucleated cells can be reconstituted, and the reconstituted enucleated cells exhibit viability equivalent to that of non-freeze-dried enucleated cells. In some embodiments, freeze-drying involves freezing the components, the cells, and drying using a vacuum under very low pressure (e.g., less than 3000 mTorr). The dried components can result in sublimation and dehydrate the cells while maintaining cell viability and biological function. In some embodiments, the freezing step involves balancing the duration and temperature of freezing to maintain cell viability and stability, proper crystal formation, and rate of reconstitution. The triple point of a substance is the temperature and pressure at which the sublimation curve, melting curve, and vaporization curve meet. The achievement of the triple point, which varies for different substances, ensures that sublimation, rather than melting, occurs in the subsequent drying step. To promote faster and more efficient freeze-drying, larger ice crystals are preferred because they form a network structure within the product that promotes faster removal of water vapor during sublimation. To produce larger crystals, the product should be frozen slowly, or the temperature can be raised and lowered in a process called annealing. Fresh or frozen living tissues or cells do not have a single homogenous melting point (eutectic point), and as a result, the freezing stage of the material (cells or tissues) is cooled below its triple point, which represents the temperature and pressure at which the solid, liquid and gas phases of the material can coexist. Living cells have a critical point on the phase diagram where both the liquid and gas phases of the object or substance have the same density and are therefore indistinguishable. The product critical point temperature must be maintained to prevent meltback or cake collapse that occurs during primary and secondary drying, which reflects incomplete sublimation. For materials where preservation of structure is required, such as living cells, large ice crystals can be harmful and can destroy cell walls, which can lead to poorer texture and loss of nutritional content.In this case, freezing should be done quickly to quickly bring the material below its critical point and thus avoid the formation of large ice crystals. The freezing temperature for cells or tissues can vary, but generally ranges from -50 °C (-58 °F) to -80 °C (-112 °F).

[0141] During the drying stage, the ambient pressure is lowered to the range of a few millibars, and then heat is supplied to the material for the ice to sublimate, by conduction or radiation. The amount of heat required can be calculated using the latent heat of sublimation of the sublimating molecules. In this initial drying stage, about 95% of the water in the material or substance sublimes. This stage is often time-consuming and can even last for days depending on the substance and technique used, but the application of excess heat can quickly change the structure of the material. In this stage, the pressure is controlled through the application of a partial vacuum. The vacuum accelerates the sublimation, making it useful as an intentional drying process. A low-temperature condenser chamber and / or condenser plates are used as a surface for the water vapor to re-liquefy and solidify. It is important to note that at pressures in this range, heat cannot be supplied by convection effects due to the low air density. Since the ice induced by freezing should be removed during the primary drying stage, the drying stage also aims to remove the remaining unfrozen water molecules. This part of the freeze-drying process is governed by the adsorption isotherm of the material. In this stage, the temperature is raised higher than in the primary drying stage, and can even exceed 0 °C (32 °F), to destroy any physicochemical interactions formed between the water molecules and the frozen material. Usually, the pressure is also reduced in this stage to facilitate desorption. However, some products benefit from an increase in pressure. After the freeze-drying process is completed, the vacuum is usually broken with an inert gas such as nitrogen before sealing the material. At the end of the operation, the residual water content in the product should be extremely low, ranging from less than 1% to 4% of the original concentration.

[0142] In some embodiments, freeze-drying of enucleated cells involves the use of a lyoprotectant to preserve cell viability and biological function. Lyoprotectants include the addition of reagents, salts, or additives that protect cells during the drying process. Common lyoprotectants include trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Additionally, methods to increase the transport of cryoprotectants inside cells in suspension can be utilized as a way to improve cell viability and function after freeze-drying. These methods include electroporation, the addition of reagents that enhance intracellular transport, genetic modification of cells to upregulate the expression of pores on the cell membrane, and mechanical microfluidic devices that partially disrupt the integrity of the cell membrane and potentially facilitate intracellular transport of the lyoprotectant.

[0143] In some embodiments, the nucleated cells described herein can be modified to express a targeting moiety (e.g., an antibody or antigen-binding fragment thereof), a therapeutic agent, a transmembrane moiety, a heterologous gene product, or a combination thereof. In some embodiments, the nucleated cells can be modified to express at least one heterologous polynucleotide, where the at least one heterologous polynucleotide encodes a targeting moiety, a therapeutic agent, a transmembrane moiety, a heterologous gene product, or a combination thereof.

[0144] In one aspect, disclosed herein is a method of modifying a cell by introducing at least one heterologous polynucleotide into the cell. In some embodiments, the heterologous polynucleotide encodes a promoter, a heterologous gene product, or a combination thereof. In some embodiments, the method includes providing a composition comprising a first subset of nucleated cells and an enucleated cell derived from a second subset of nucleated cells. In some embodiments, the first subset of nucleated cells comprises a heterologous polynucleotide encoding a heterologous gene product. In some embodiments, the method includes expressing the heterologous gene product, thereby inducing cell death of at least one nucleated cell of the first subset of nucleated cells. The heterologous polynucleotide can be introduced into any type of cell that can be enucleated, for example, but not limited to, hTERT-immobilized mesenchymal stem cells, by transfection, such as plasmids, transposons, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-Cas technology, viral transduction, and the like.

[0145] In some embodiments, the heterologous polynucleotide comprises a promoter. In some embodiments, the promoter comprises an inducible promoter. In some embodiments, the promoter should be compatible with mammalian gene expression and provide rapid and strong gene expression only in the presence of its inducing stimulus. The relevant suicide genes may include, but are not limited to, caspases, "eat me" signals, DNA crosslinkers, death-inducing synthetic NOTCH receptors, toxins, and apoptosis / autophagy / entosis / necrosis / necroptosis / ferroptosis inducers. Each promoter has its own activation protocol in terms of temperature, incubation time, light wavelength, inducer concentration, etc.

[0146] In some embodiments, the inducible promoter is hypothermia. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature of less than about 40° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature of less than about 39° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature of less than about 38° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature of less than about 37° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature of less than about 36° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature of less than about 35° C. In some embodiments, examples of inducible promoters include, but are not limited to, dsrA or CIRP.

[0147] In some embodiments, the inducible promoter is hyperthermia. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 35° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 36° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 37° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 38° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 39° C. In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 40° C. In some embodiments, examples of inducible promoters include, but are not limited to, heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), growth arrest and DNA damage inducible gene 153 (GADD153), multidrug resistance mutation 1 (MDR1), or cytomegalovirus (HSE-CMV).

[0148] In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a molecule, examples of which include, but are not limited to, rtTA, TRE, TetR, Cumate, rapamycin, abscisic acid, IPTG, or metallothionein.

[0149] In some embodiments, the inducible promoter is induced by contacting nucleated cells with light. In some embodiments, examples of inducible promoters include, but are not limited to, CIB1-CRY2 or GAL4-VVD.

[0150] In some embodiments, the inducible promoter is induced by contacting a nucleated cell with a hormone. In some embodiments, an example of an inducible promoter includes, but is not limited to, estradiol-Gal4.

[0151] In some embodiments, the promoter comprises a constitutively active promoter. The promoter is continuously active, but suicide is induced under certain circumstances. In some embodiments, the constitutively active promoter is configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product. In some embodiments, examples of heterologous gene products include, but are not limited to, herpes simplex virus-thymidine kinase (HSV-TK), cytosine deaminase (CD), varicella zoster TK (VZV-TK), nitroreductase, carboxypeptidase G2 (CPG2), cytochrome P450, or purine nucleoside phosphorylase. In some embodiments, examples of heterologous gene products include, but are not limited to, FKBP or caspase. In some embodiments, examples of heterologous gene products include, but are not limited to, antigens. In some embodiments, the heterologous polynucleotide is integrated into the chromosome of a nucleated cell. In some embodiments, examples of heterologous polynucleotides include, but are not limited to, vectors.

[0152] In some embodiments, the enucleated cells described herein may be cryopreserved, cryohiberlated, freeze-dried, or a combination thereof. In some embodiments, cryopreserved enucleated cells, upon thawing, are as viable as other equivalent enucleated cells that are not cryopreserved. In some embodiments, freeze-dried enucleated cells are as viable as other equivalent enucleated cells that are not freeze-dried. In some embodiments, cryopreserved enucleated cells are as viable as other equivalent enucleated cells that are not cryopreserved.

[0153] Treatment In some embodiments, disclosed herein are methods of using the enucleated cells, compositions, or pharmaceutical compositions described herein to treat a disease or condition. In some embodiments, the methods include treating a disease or condition in a subject by administering to the subject a composition described herein (e.g., a pharmaceutical composition containing enucleated cells engineered to express a therapeutic agent). In some embodiments, the enucleated cells disclosed herein may be loaded, transfected, or transduced with a therapeutic agent disclosed herein or any existing therapeutic agent and formulated into a pharmaceutical formulation, which may be delivered to a subject according to various embodiments herein. The pharmaceutical formulations disclosed herein increase the biodistribution and / or homing of the therapeutic agent to target cells or tissues in vivo compared to administration of a therapeutic agent that is not encapsulated or expressed by the enucleated cells disclosed herein.

[0154] The present disclosure also provides methods for using enucleated cells (natural or enucleated) as fusion partners with other cells (therapeutic or natural) to enhance and / or transfer biomolecules, such as therapeutic agents, described herein. In some embodiments, biomolecules include DNA / genes, RNA (mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, and plasmids, bacteria, viruses, small molecule drugs, ions, cytokines, growth factors, and hormones. In some embodiments, the enucleated cells are engineered to express a fusogenic moiety. The fusogenic moiety can be any biomolecule (e.g., sugar, lipid, or protein) that promotes membrane fusion. In some embodiments, the fusion moiety is a fusogenic protein. The fusion protein allows the enucleated cell expressing the fusion protein to fuse with a target cell. In some embodiments, the fusion protein promotes fusion between the enucleated cell expressing the fusion protein and the target cell, allowing the contents of the enucleated cell to enter the target cell. In some embodiments, the fusion protein is a variant such as a viral class I-III or HAP2 / GCS1 or SNARE. In some embodiments, the fusion protein is homoleptic, such as EFF-1 / AFF-1. Other non-limiting examples of fusion proteins are Izumo1 or Syncytin. In some embodiments, the fusion protein is a viral protein. In some embodiments, the virus-derived fusion protein is VSV-g, hERV-W-ENV (syncytin), or MV-Ed-F+MV-Ed-H (hemagglutinin). Unlike nucleated cells, fusion of enucleated cells to the same or another cell type of similar or different origin generates unique cell hybrids that lack problematic nuclear transfer while maintaining desirable therapeutic attributes, including but not limited to cell surface proteins, signaling molecules, secreted proteins, and epigenetic changes.

[0155] subject The methods disclosed herein, in some embodiments, include administering or delivering a composition (e.g., a pharmaceutical composition) to a subject. In some embodiments, the subject has a disease, disorder, or condition (e.g., cancer, idiopathic pulmonary fibrosis). In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is an adolescent, adult, or elderly subject. In some embodiments, the human subject is at least 18 years old. In some embodiments, the human subject is between 18 and about 55 years old. In some embodiments, the human subject is greater than 55 years old. In some embodiments, the subject is between 18 and about 65 years old. In some embodiments, the subject is greater than 65 years old. In some embodiments, the subject is a female. In some embodiments, the subject is a male. In some embodiments, the subject is immunocompromised or at high risk of being immunocompromised.

[0156] Disease or illness Provided herein is a method of treating a disease or condition in a subject by administering a composition described herein to the subject. In some embodiments, administration is by any suitable mode of administration, including systemic administration (e.g., intravenous, inhalation, etc.). In some embodiments, the subject is a human. In some embodiments, the disease or condition comprises an infectious disease (e.g., human immunodeficiency virus (HIV) infection, Chagas disease, tuberculosis), a neurological disease (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease), an autoimmune disease (e.g., diabetes, Crohn's disease, multiple sclerosis, sickle cell anemia), a cardiovascular disease (e.g., acute myocardial infarction, heart failure, refractory angina), an ophthalmological disease, a skeletal disease, a metabolic disease (e.g., phenylketonuria, glycogen storage deficiency type 1A, Gaucher disease), an inflammatory disease (e.g., cancer, inflammatory bowel disease), or a disease caused by an external pathogen or toxin in the subject. In some embodiments, the disease or condition comprises idiopathic pulmonary fibrosis. In some embodiments, the subject is in need of, or has been determined to be in need of, such an enucleation cell treatment.

[0157] In some embodiments, the cancer may be lung cancer, including non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), or any other lung cancer type. For example, lung cancer may include adenocarcinoma, squamous cell carcinoma, large cell (undifferentiated) carcinoma, large cell neuroendocrine carcinoma, adenosquamous cell carcinoma, sarcomatoid carcinoma, lung cancer tumor, or adenoid cystic carcinoma. Other non-limiting examples of lung cancer include lymphoma, sarcoma, benign lung tumor, or hamartoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer has metastasized to the lung from a different tissue or source. For example, metastatic cancers that may be found in the lung may include breast cancer, colon cancer, prostate cancer, sarcoma, bladder cancer, neuroblastoma, and Wilms' tumor.

[0158] In some embodiments, the enucleated cells described herein comprise a targeting moiety described herein for binding to an epitope expressed by a cancer cell or associated with the tumor microenvironment. In some embodiments, the targeting moiety comprises an antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof comprises a single domain antibody. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope expressed by a cancer cell or associated with the tumor microenvironment. In some embodiments, the binding of the targeting moiety (e.g., an antibody or antigen-binding fragment thereof) to the epitope provides a therapeutic effect for treating the subject's cancer. In some embodiments, the binding of the targeting moiety (e.g., an antibody or antigen-binding fragment thereof) to the epitope recruits immune cells to activate an immune response against the cancer.

[0159] In some embodiments, described herein are enucleated cells and methods of using these enucleated cells to treat a disease or condition associated with abnormal vasculature in a subject. Abnormal vasculature can be associated with a disease or condition such as inflammation and cancer (e.g., any one of the cancers described herein). In some embodiments, the enucleated cells described herein, upon contact with abnormal vasculature, increase normalization of the abnormal vasculature, increasing adhesion between endothelial cells to prevent leakage of intravascular factors from the vasculature. In some embodiments, normalization of abnormal vasculature includes a reduction in damage, such as cell death of endothelial cells of the vasculature. In some embodiments, normalization of abnormal vasculature includes angiogenesis of immature or leaky blood vessels. In some embodiments, normalization exerted by the enucleated cells can include normalization of blood vessels, lymphatic vessels, or a combination thereof.

[0160] In some embodiments, the disease or illness may be caused by a pathogen. In some embodiments, the enucleated cells described herein include an antibody or antigen-binding fragment thereof or single domain antibody that binds to an epitope expressed by a pathogen or associated with a microenvironment associated with the pathogen. In some cases, the binding of the antibody or antigen-binding fragment thereof or single domain antibody to the epitope confers a therapeutic property against the pathogen. In some embodiments, the binding of the antibody or antigen-binding fragment thereof or single domain antibody to the epitope recruits immune cells to activate an immune response, conferring a therapeutic property against the pathogen. For example, the disease or illness may be caused by a virus, a bacterium, a fungus, a parasite, or a molecule resulting from detoxification. In some embodiments, the pathogen may be disseminated or transmitted from individual to individual, resulting in high mortality rates, has the potential for major public health impacts, may cause social panic and social unrest, and requires special actions for public health preparedness. Examples of these pathogens may include anthrax (Bacillus anthracis), botulism (Clostridium botulinum toxin), plague (Yersinia pestis), smallpox (Valiola major), tularemia (Fragaria tularensis), or viral hemorrhagic fevers, including filoviruses (Ebola, Marburg) and arenaviruses (Lassa, Machupo).

[0161] In some embodiments, pathogens may be disseminated and may result in moderate morbidity and low mortality, thus requiring specific enhancement of diagnostic capabilities and enhanced disease surveillance. Examples of these pathogens include brucellosis (Brucella species), epsilon toxin of Clostridium perfringens, food safety threats (e.g., Salmonella species, Escherichia coli O157:H7, or Shigella), glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), Ricinus commmunis (castor bean), ricin toxin, Staphylococcal enterotoxin B, typhoid fever (Rickettsia prowazeki), viral encephalitis (alphaviruses such as Eastern equine encephalitis, Venezuelan equine encephalitis, and Western equine encephalitis), or water safety threats (e.g., Vibrio cholerae and Cryptosporidium parvum).

[0162] In some embodiments, the pathogen may include emerging pathogens that have a high potential for mortality and morbidity, but whose spread is not fully understood. Non-limiting examples of these pathogens may include Nipah virus and Hantavirus.

[0163] The enucleated cells described herein, or compositions containing such enucleated cells (referred to in this section as "compositions"), can be administered to a subject at a suitable dose, mode of administration, and frequency, depending on the intended effect.

[0164] In some embodiments, the composition is administered at least once during a period of time (e.g., every other day, twice a week, once a week, weekly, three times a month, twice a month, once a month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, once a year). In some embodiments, the composition is administered more than once during a period of time (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times).

[0165] In some embodiments, the composition is administered in a therapeutically effective amount by various forms and routes, including, for example, oral or topical administration. In some embodiments, the composition can be administered parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, intrathecally, intraocularly, intrasternally, ocularly, endothelially, topically, intranasally, intrapulmonary, rectally, intraarterially, intrathecally, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcutaneous, subarachnoid, or intraspinal administration, for example, by injection or infusion. In some embodiments, the composition can be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa administration). In some embodiments, the composition is delivered via multiple administration routes.

[0166] In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the composition is administered by slow continuous infusion over an extended period of time, such as more than 24 hours. In some embodiments, the composition is administered as an intravenous injection or short-term infusion.

[0167] The composition can be administered in a localized manner, for example, by injecting the agent directly into an organ, optionally in a depot or sustained release formulation or implant. The composition can be provided in the form of a rapid release formulation, a sustained release formulation, or an intermediate release formulation. The rapid release form can provide immediate release. The sustained release formulation can provide controlled release or sustained delayed release. In some embodiments, a pump can be used for the delivery of the composition. In some embodiments, a pen delivery device can be used, for example, for subcutaneous delivery of the composition of the present disclosure. The compositions provided herein can be administered in conjunction with other therapies, for example, antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents.

[0168] The compositions (e.g., enucleated cells or pharmaceutical compositions comprising enucleated cells described herein) can be administered before, during, or after the onset of a disease or condition, and the timing of administering a composition containing a therapeutic agent can vary. In some cases, the compositions can be used as prophylactics and can be administered continuously to subjects (e.g., immunized or treated subjects) with a susceptibility to coronavirus or a predisposition to a coronavirus-related disease or condition. Prophylactic administration can reduce the likelihood of infection, disease, or illness occurring, or can reduce the severity of infection, disease, or illness.

[0169] The composition may be administered to a subject prior to the onset of symptoms. The composition may be administered to a subject (e.g., an immunized or treated subject) following (e.g., as soon as possible) a test result, e.g., a test result providing a diagnosis, a test showing the presence of coronavirus in a subject (e.g., an immunized or treated subject), or a test showing disease progression, e.g., a drop in blood oxygen levels. The composition may be administered after (e.g., as soon as practicable) a disease or disease onset is detected or suspected. The composition may be administered after (e.g., as soon as practicable) a potential exposure to coronavirus, e.g., after the subject (e.g., an immunized or treated subject) comes into contact with an infected subject or knows that they have come into contact with an infected subject who may be infectious.

[0170] The actual dosage level of the agents of the present disclosure (e.g., antibodies or antigen-binding fragments thereof, or therapeutic agents) can be varied without toxicity to the subject (e.g., immunized or treated subjects) to obtain an amount of agent that achieves the desired therapeutic response for a particular subject, composition, and mode of administration. The dosage level selected may depend on a variety of pharmacokinetic factors, including the activity of the particular compositions used herein, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0171] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. Unit dosage form as used herein refers to physically discrete units suitable as unitary doses for subjects (e.g., subjects for immunization or subjects for treatment), each unit containing a predetermined amount of active agent calculated to produce a desired therapeutic effect, together with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present disclosure can be determined by and directly depend on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active agents for the treatment of susceptibility in individuals. The dose can be determined with reference to the plasma or local concentration of the cyclic polyribonucleotide or antibody or antigen-binding fragment thereof. Dosage can be determined with reference to plasma or local concentrations of the linear polyribonucleotide or antibody or antigen-binding fragment thereof.

[0172] The compositions described herein may be in unit dosage form suitable for single administration of precise dosage amounts. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of the composition. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of one or more linear polyribonucleotides, antibodies or antigen-binding fragments thereof, and / or therapeutic agents. The unit dosage may be in the form of a package containing a discrete amount of the formulation. Non-limiting examples are packaged injections, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, in combination with or without preservatives. The injectable formulations disclosed herein may be in unit dosage form, for example, in ampoules, or in multi-dose containers containing preservatives.

[0173] Dosages may be based on the amount of agent per kilogram of subject (e.g., subject to be vaccinated or treated) body weight. Doses of agent (e.g., antibody) may range from 10-3000 mg / kg, e.g., 100-2000 mg / kg, e.g., 300-500 mg / kg / day for 1-10 or 1-5 days, e.g., 400 mg / kg / day for 3-6 days, e.g., 1 g / kg / day for 2-3 days. In some embodiments, dosages may be based on the number of enucleated cells per kilogram of subject body weight. In some embodiments, dosages may be administered in amounts between about 1,000 cells / kg body weight and about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose is from about 1,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 10,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 100,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 1,000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 10,000,000 cells / kg body weight. / kg body weight, approximately 1,000 cells / kg body weight ~ approximately 100,000,000 cells / kg body weight, approximately 1,000 cells / kg body weight ~ approximately 1,000,000,000 cells / kg body weight, approximately 1,000 cells kg Body weight ~ approx. 10,000,000,000 cells / kg body weight, approx. 1,000 cells / kg body weight ~ approx. 100,000,000,000 cells / kg body weight, approx. 1,000 cells / kg body weight ~ approx. 1,000,0 00,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 100,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 1,000,000 cells / kg body weight, approximately 10, 000 cells / kg body weight ~ approx. 10,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 100,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 1,000 cells / kg body weight ,000,000 cells / kg body weight, approximately 10,000 cells kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 100,000,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 1,000,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 1,000,000 cells / kg body weight, approximately 100,0 cells / kg body weight to approximately 10,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight to approximately 100,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight to approximately 1,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight to approximately 10,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight to approximately 100,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 10,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 100,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 1,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 10,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 100,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight to approximately 100,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight to approximately 1,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight to approximately 10,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight to approximately 100,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight to approximately 1,000,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight to approximately 10,000,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight to approximately 100,000,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, approximately 1,000,000,000 cells / kg body weight to approximately 10,000,000,000 cells / kg body weight, approximately 1,000,000,000 cells / kg body weight to approximately 100,000,000,000 cells / kg body weight, approximately 1,000,000,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, approximately 10,000,000,In some cases, the cells may be administered in a dosage of between about 1,000,000,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, or between about 100,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose may be administered in an amount between about 1,000 cells / kg body weight to about 1000000000000 cells / kg body weight, about 1,000 cells / kg body weight, about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, about 100,000,000,000 cells / kg body weight, or about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose may be administered in an amount between about 1,000 cells / kg body weight and about 1000,000,00000 cells / kg body weight, at least about 1,000 cells / kg body weight, about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, or about 100,000,000,000 cells / kg body weight. In some embodiments, the dose is from about 1000 cells / kg body weight to about 1000000000000 cells / kg body weight, up to about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, about 100,000,000,000 cells / kg body weight, or about 1,000,000,000 cells / kg body weight.In some embodiments, the nucleated cells are administered to a subject twice within at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, or 4 years.

[0174] Disclosed herein are methods of repeatedly administering compositions or pharmaceutical compositions to a subject in need thereof. In some embodiments, a first administration of a composition or pharmaceutical composition comprising enucleated cells normalizes blood or lymphatic vessels. In some embodiments, the same composition or pharmaceutical composition comprising enucleated cells can be subsequently administered to the subject to maintain normalization of blood or lymphatic vessels and for delivery of exogenous agents to treat a disease or condition described herein.

[0175] kit In some aspects, disclosed herein are kits for using the compositions described herein. In some embodiments, the kits disclosed herein may be used to treat a disease or condition in a subject. In some embodiments, the kits include a collection of materials or components separate from the composition. In some embodiments, the kits include nucleated cells (e.g., nucleated cells engineered to express a targeting moiety (e.g., an antibody or antigen-binding fragment thereof)) described herein, a therapeutic agent, a transmembrane moiety, an immune evasion moiety, a heterologous gene product, or a combination thereof. In some embodiments, the kits can include enucleated cells obtained from nucleated cells. In some embodiments, the kits can include a mixed population of nucleated cells and enucleated cells obtained from nucleated cells. In some embodiments, the kits can include a substantially pure population of enucleated cells. In some embodiments, the kits include nucleated cells, enucleated cells, or a combination thereof suspended in at least one density gradient.

[0176] In some embodiments, the kit comprises a pharmaceutical formulation disclosed herein comprising an enucleated cell engineered to express (and optionally secrete) a targeting moiety (e.g., an antibody or antigen-binding fragment thereof), a therapeutic agent, a transmembrane moiety, an immune evasion moiety, a heterologous gene product, or a combination thereof. In some embodiments, the enucleated cell expresses or secretes a therapeutic agent, such as an immune checkpoint molecule or an immune checkpoint inhibitor, for treating a disease or condition in a subject. In some embodiments, the enucleated cell is further engineered to express a targeting moiety, such as a chemokine receptor, an integrin signaling molecule, or an antibody or antigen-binding fragment thereof, which allows the enucleated cell, upon administration, to efficiently migrate to a target tissue in a subject. In some embodiments, the kit further comprises an additional therapeutic agent, such as those disclosed herein. In some embodiments, the kit further comprises instructions for administering to the subject the pharmaceutical formulation and / or the additional therapeutic agent to treat a disease or condition in the subject, such as cancer. In some embodiments, the cancer comprises a cancer of lung tissue. In some embodiments, the cancer is lung cancer.

[0177] In some embodiments, the kit includes components for purifying enucleated cells from nucleated cells or other cellular debris. For example, the kit can include filter membranes with different pore sizes for isolating and purifying enucleated cells. In some embodiments, the kit includes components for staining and selecting enucleated cells. For example, the kit can include fluorescent dyes for staining nuclei, and nucleated cells can be stained and selectively removed, leaving a population of enucleated cells. In some embodiments, the kit includes components for inducing cell death of nucleated cells. For example, the kit can include molecules for inducing expression of heterologous gene products described herein for inducing cell death of nucleated cells.

[0178] In some embodiments, the kits described herein include components for selecting a homogenous population of enucleated cells. In some embodiments, the kits described herein include components for selecting a heterogeneous population of enucleated cells. In some embodiments, the kits include components for assaying the number of units of a biomolecule (e.g., a therapeutic agent) synthesized by the enucleated cells and / or released or expressed on the surface. In some embodiments, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, and qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein (e.g., cancer) in a subject. In some embodiments, the kits are configured specifically for the purpose of treating a mammalian subject. In some embodiments, the kits are configured specifically for the purpose of treating a human subject.

[0179] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the composition to a subject in need of administration. In some embodiments, the kit includes instructions for further manipulating the composition to express a biomolecule (e.g., a therapeutic agent). In some embodiments, the kit includes instructions for thawing or otherwise restoring biological activity of a composition that may be cryopreserved, lyophilized, or freeze-quiescent during storage or transport. In some embodiments, the kit includes instructions for measuring the viability of the restored composition to ensure its effectiveness for its intended purpose (e.g., therapeutic effectiveness when used to treat a subject).

[0180] Optionally, the kit also includes other useful components such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful parafermas. The substances or components assembled in the kit can be stored and provided to the practitioner in a convenient and suitable manner that maintains their operability and usefulness. For example, the components can be in dissolved, dehydrated, or lyophilized form and can be provided at room, refrigerated, or frozen temperatures. The components are contained in suitable packaging materials.

[0181] definition The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "initially," "next," "sequently," "before," "after," "lastly," and "final," is intended to be illustrative and not limiting of the scope of the embodiments disclosed herein.

[0182] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be included in a manner similar to the term "comprising."

[0183] As used herein, the terms "at least one," "one or more," and "and / or" are open-ended terms that are both conjunctive and disjunctive in operation. For example, each of the terms "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0184] As used herein, "or" refers to "and," "or," or "and / or," and may be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a particular meaning.

[0185] Any systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.

[0186] The term "about" when referring to a number or numerical range means that the number or numerical range referred to is an estimate within experimental variability (or within statistical experimental error) and that the number or numerical range may vary, for example, by 1% to 15% of the stated number or numerical range. In the examples, the term "about" refers to ±10% of the stated number or value.

[0187] The terms "increased," "increasing," or "increase" are generally used herein to mean an increase of a statistically significant amount. In some embodiments, the term "increased" or "increase" refers to an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100%, or any increase between 10-100%, compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0188] The terms "decreased," "decreasing," or "decrease" are generally used herein to mean a statistically significant amount of decrease. In some embodiments, "decreased" or "decrease" means a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% decrease (non-existent or undetectable levels compared to a reference level), or any decrease between 10-100%. In the context of a marker or condition, these terms mean a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably decreased to a level recognized to be within the normal range for a given disease-free individual. Other examples of a "decrease" include a decrease of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a baseline level.

[0189] The terms "individual" or "subject" are used interchangeably and include mammals. Non-limiting examples of mammals include any member of the class of mammals, including humans, non-human primates such as chimpanzees, and other apes and monkeys, domestic animals such as cows, horses, sheep, goats, pigs, domestic animals such as rabbits, dogs, and cats, and laboratory animals including rodents such as rats, mice, and guinea pigs. A mammal may be a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, e.g., a rodent such as a rat or mouse. As used herein, a "patient" refers to a subject having or diagnosed with a disease or disorder as described herein.

[0190] As used herein, the term "immune evasion moiety" refers to a signaling peptide or portion thereof that reduces cell phagocytosis by its interaction with a signal receptor protein expressed by phagocytes such as macrophages and dendritic cells. In some embodiments, the immune evasion moiety blocks immune cell recognition or immune cell activation.

[0191] As used herein, the term "targeting moiety" refers to an entity that directs a cell, e.g., an enucleated cell, to a target tissue or cell. A targeting moiety can be virtually any biomolecule, including a protein, polypeptide, sugar, nucleic acid, or small molecule, or portion thereof.

[0192] As used herein, the term "transmembrane moiety" refers to an entity that spans (at least partially) the cell membrane of a cell (eg, an enucleated cell).

[0193] The term "expression" or "expressing" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. With respect to expression, "upregulated" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression level in the wild-type state, and "downregulated" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression in the wild-type state.

[0194] As used herein, "cell" generally refers to a biological cell.

[0195] As used herein, "nucleation" refers to the rendering of a cell non-replicating, such as, for example, through removal of the nucleus.

[0196] As used herein, the terms "cytoplast," "cell without nucleus," or "nucleated cell" are used interchangeably to refer to an enucleated cell obtained from a previously nucleated cell (e.g., any cell described herein). In some embodiments, the nucleated cell contains organelles, and the cytoplasm derived from the nucleated cell retains such organelles, which in some cases allows for cellular functions such as cell motility, protein synthesis, protein secretion, etc. In some aspects, "obtaining" does not include differentiating a nucleated cell into an enucleated cell using natural processes or other methods.

[0197] As used herein, the term "gene" refers to a segment of nucleic acid that encodes a particular protein or RNA (also called a "coding sequence" or "coding region"), optionally with associated regulatory regions such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence. The term "gene" should be interpreted broadly and may encompass mRNA, cDNA, cRNA and genomic DNA forms of a gene.

[0198] In some uses, the term "gene" encompasses transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region may include an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequences (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some embodiments, a gene does not encode a polypeptide, e.g., a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some embodiments, the term "gene" includes not only the transcribed sequence, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. The term "gene" may encompass mRNA, cDNA and genomic forms of a gene.

[0199] The term "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as compositions. The packaging material is preferably constructed in a well-known manner to provide a sterile and contaminant-free environment. The packaging material utilized in the kit is one that is conventionally utilized in gene expression assays and administration of treatments.

[0200] As used herein, the term "packaging" refers to a suitable solid matrix or solid material, such as glass, plastic, paper, foil, etc., that can hold individual kit components. For example, the packaging can be a glass vial or a pre-filled syringe that is used to contain an appropriate amount of pharmaceutical agent. The packaging material has an external label that indicates the contents and / or purpose of the kit and its components.

[0201] The terms "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either single-stranded, double-stranded, or multi-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any unknown or known function. A polynucleotide contains one or more analogs (e.g., altered backbones, sugars, or nucleobases). Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, a locus (or loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0202] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein in reference to a polymer of amino acid residues. A protein can refer to a full-length polypeptide as translated from a coding open reading frame or processed into its mature form, while a polypeptide or peptide can refer to degradation or processing fragments of a protein that still uniquely or identifiably maps to a particular protein. A polypeptide can be a single linear polymeric chain of amino acids that are bound together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides can be modified, for example, by the addition of carbohydrates, phosphorylation, etc.

[0203] As used herein, the term "fragment," "portion," or equivalent terms may refer to a portion of an entity that has less than the full length of the entity, and optionally maintains a function of the entity. In some embodiments, the entity is a protein.

[0204] The terms "complementary", "complementary", "complementary" and "complementarity" as used herein generally refer to a sequence that is completely complementary to and can hybridize to a given sequence. In some cases, a sequence hybridized to a given nucleic acid is called the "complement" or "reverse complement" of a given molecule if its sequence of bases on a given region can be complementarily bound to that of its binding partner, for example, such that AT, AU, GC and GU base pairs are formed. In general, a first sequence that can hybridize to a second sequence can specifically or selectively hybridize to the second sequence (e.g., is thermodynamically more stable under a given set of conditions, such as stringent conditions used in the relevant art) such that hybridization to the second sequence or set of second sequences is preferred over hybridization to non-target sequences during a hybridization reaction. Typically, hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, such as 25% to 100% complementarity, including at least about 25% or more, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity. Sequence identity can be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, for example, the EMBOSS needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html), the BLAST algorithm, for example, to assess percent complementarity. Optimal alignment may be assessed using any suitable parameters of the selected algorithm, including default parameters.

[0205] The term "percent (%) identity" as used herein generally refers to the percent of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment can be achieved in a variety of ways known in the relevant art for purposes of determining percent identity. The percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0206] As used herein, the term "in vivo" may be used to describe events that take place in a living organism, such as the body of a subject.

[0207] As used herein, the term "ex vivo" may be used to describe events that occur outside of an organism, such as a subject's body. An "ex vivo" assay may not be performed on a subject. Rather, it may be performed on a sample that is separate from the subject. Ex vivo may be used to describe events that occur in intact cells outside of a subject's body.

[0208] As used herein, the term "in vitro" may be used to describe events that occur in a container for holding a laboratory reagent such that the laboratory reagent is separated from the living organism of the biological source from which the material is obtained. In vitro assays may include cell-based assays in which living or dead cells are utilized. In vitro assays may also include cell-free assays in which no intact cells are utilized.

[0209] "Treat", "treating" or "treatment" as used herein refers to alleviating or abrogating a disorder, disease or condition, or is meant to include alleviating or abrogating one or more symptoms associated with a disorder, disease or condition, or alleviating or eradicating the cause of the disorder, disease or condition itself. Desirable effects of treatment may include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastasis, reduction in the rate of disease progression, improvement or amelioration of the disease state, and remission or improved prognosis.

[0210] The terms "effective amount" and "therapeutically effective amount", when used interchangeably herein, generally refer to an amount of a composition, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells) comprising the system of the present disclosure, sufficient to produce a desired activity upon administration to a subject in need thereof. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition sufficient to delay the onset, halt the progression, and reduce or alleviate at least one symptom of a disorder treated by the method of the present disclosure.

[0211] The terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component may be "pharmaceutical acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It may also be suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit-risk ratio.

[0212] As used herein, the terms "administration", "administering" and variations thereof refer to the introduction of a composition or agent into a subject, including simultaneous and sequential introduction of the composition or agent. The introduction of the composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, or topical. Administration includes self-administration and administration by another. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein. Administration can be by any suitable route. In some embodiments, the administration is intravenous administration. In some embodiments, the administration is pulmonary administration. In some embodiments, the administration is inhalation.

[0213] The term "pharmaceutical composition" refers to a mixture of a composition disclosed herein with a diluent or carrier (e.g., a pharma- ceutically acceptable inactive ingredient), such as a carrier, excipient, binder, filler, suspending agent, flavoring agent, sweetener, disintegrant, dispersant, surfactant, lubricant, colorant, diluent, solubilizer, wetting agent, plasticizer, stabilizer, permeation enhancer, wetting agent, antifoaming agent, antioxidant, preservative, or one or more combinations thereof. A pharmaceutical composition may facilitate administration of the composition to an organism. Multiple techniques of administering a compound exist in the art, including, but not limited to, oral administration, injection, aerosol administration, parenteral administration, and topical administration.

[0214] As used herein, the term "fusion protein" refers to a polypeptide that, when expressed on the surface of a cell, such as an enucleated cell, promotes fusion of the intercellular membrane of the cell expressing the fusion protein with a target cell.

[0215] While preferred embodiments of the inventive concept have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the inventive concept be limited by the specific examples provided within the specification. Although the inventive concept has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the inventive concept. Furthermore, it will be understood that all aspects of the inventive concept are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It will be understood that various alternatives of the embodiments of the inventive concept described herein may be utilized in the practice of the inventive concept. It is therefore contemplated that the inventive concept shall cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the inventive concept, and it is intended that methods and structures within the scope of the claims, and equivalents thereof, be covered thereby.

[0216] Embodiment Embodiment 1: a) providing a composition comprising nucleated cells; b) enucleating a portion of the nucleated cells using continuous flow centrifugation to produce an enucleated cell fraction, wherein the portion of the nucleated cells comprises about 70% or more of the nucleated cells.

[0217] Embodiment 2: The method of embodiment 1, wherein the composition provided in a) herein has a volume comprised between about 10 milliliters (mL) or more and about 10,000 mL.

[0218] Embodiment 3: The method of embodiment 2, wherein the composition has a volume comprising at least about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 80 mL, about 100 mL, about 200 mL, about 300 mL, about 500 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, about 9000 mL, or about 10000 mL.

[0219] Embodiment 4: The method of embodiment 1, wherein continuous flow centrifugation creates a density gradient that separates the enucleated cell fraction from nucleated cells in the composition.

[0220] Embodiment 5: The method of embodiment 4, wherein the density gradient comprises a polysaccharide density gradient.

[0221] Embodiment 6: The method of embodiment 5, wherein the polysaccharide density gradient comprises a Ficoll density gradient.

[0222] Embodiment 7: The method of embodiment 6, wherein the Ficoll density gradient comprises at least two, at least three, at least four, at least five, at least six, or at least seven range density gradients.

[0223] Embodiment 8: The method of embodiment 7, wherein the Ficoll density gradient comprises about 25% Ficoll, about 17% Ficoll, about 16% Ficoll, about 15% Ficoll, or about 12.5% ​​Ficoll.

[0224] Embodiment 9: The method of any one of embodiments 1 to 8, wherein the portion of nucleated cells comprises about 75% or more of nucleated cells.

[0225] Embodiment 10: The method of any one of embodiments 1 to 9, wherein the portion of nucleated cells comprises about 80% or more of enucleated cells.

[0226] Embodiment 11: The method of any one of embodiments 1 to 10, wherein the portion of nucleated cells comprises about 90% or more of enucleated cells.

[0227] Embodiment 12: The enucleated cell fraction generated by a single continuous flow centrifugation comprises about (i) 6×10 7 of enucleated cells, (ii) 7 × 10 7 enucleated cells, (iii) 8 × 10 7 enucleated cells, (iv) 9 × 10 7 of enucleated cells (v) 10 × 10 7 enucleated cells, (vi) 15 × 10 7 of enucleated cells, (vii) 20 × 10 7 of enucleated cells, (viii) 50 × 10 7 of enucleated cells, (ix) 100 × 10 7 of enucleated cells, (x) 150 × 10 7 of enucleated cells, (xi) 200 × 10 7 of enucleated cells, or (xii) 250 × 10 7 12. The method according to any one of embodiments 1 to 11, comprising at least one enucleated cell.

[0228] Embodiment 13: The method of embodiment 4, further comprising generating a density gradient comprising centrifuging the polysaccharide at an acceleration ranging from 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.

[0229] Embodiment 14: The method of embodiment 4, further comprising generating a density gradient comprising centrifuging the polysaccharide at an acceleration ranging from about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, or about 50 minutes.

[0230] Embodiment 15: The method of embodiment 4, further comprising generating a density gradient comprising centrifuging the polysaccharide at an acceleration for about 30 minutes.

[0231] The method of embodiment 16, wherein the enucleating step of b) further comprises generating a density gradient comprising centrifuging the polysaccharides with minimal deceleration.

[0232] The method of embodiment 6, wherein the enucleating step of embodiment 17:b) further comprises creating a density gradient comprising centrifuging the polysaccharides at a maximum centrifugal force of about 30,000 relative centrifugal force (RCF) to about 200,000 RCF.

[0233] The method of embodiment 17, wherein the enucleation step of embodiment 18: b) further comprises creating a density gradient, comprising centrifuging the polysaccharides at a maximum centrifugal force of about 50,000 RCF to about 120,000 RCF.

[0234] The method according to any one of embodiments 1 to 18, wherein in embodiment 19:b), the step of enucleating a portion of the nucleated cells using continuous flow centrifugation to generate an enucleated cell fraction is carried out using an ultracentrifuge.

[0235] The method according to any one of the preceding embodiments, wherein the step of enucleating a portion of the nucleated cells using continuous flow centrifugation to generate an enucleated cell fraction in embodiment 20:b) is carried out using a fixed angle centrifuge or a swinging bucket centrifuge.

[0236] Embodiment 21: The method of any one of embodiments 1 to 20, wherein the nucleated cells comprise a heterologous polynucleotide.

[0237] Embodiment 22: The method comprises inducing cell death of nucleated cells that are not enucleated after b), wherein cell death is induced by expressing at least one heterologous gene encoded by a heterologous polynucleotide.

[0238] Embodiment 23: The method according to any one of the previous embodiments, wherein continuous flow centrifugation creates a zonal centrifugation for separating at least one enucleated cell from nucleated cells.

[0239] Embodiment 24: The method of embodiment 23, wherein the at least one enucleated cell is separated from the nucleated cells based on the size of the at least one enucleated cell by zonal centrifugation.

[0240] Embodiment 25: The method of embodiment 23, wherein the at least one enucleated cell is separated from the nucleated cells based on the mass of the at least one enucleated cell by zonal centrifugation.

[0241] Embodiment 26: The method of embodiment 23, wherein the at least one enucleated cell is separated from the nucleated cells based on the size and mass of the at least one enucleated cell by zonal centrifugation.

[0242] Embodiment 27: The method of embodiment 4, wherein at least one density fraction is obtained from the density gradient, and wherein at least one density fraction comprises a mixed population of nucleated cells and a subset of enucleated cells of the enucleated cell fraction.

[0243] Embodiment 28: The method of embodiment 27, wherein the mixed population comprises at least 70% enucleated cells.

[0244] Embodiment 29: The method of embodiment 27, wherein the mixed population comprises at least 99% enucleated cells.

[0245] Embodiment 30: a) providing a composition comprising enucleated cells derived from (i) a first subset of nucleated cells and (ii) a second subset of nucleated cells, wherein the first subset of nucleated cells comprises a heterologous polynucleotide encoding a heterologous gene product; b) expressing a heterologous gene product, thereby inducing cell death of at least one nucleated cell of the first subset of nucleated cells; A method for treating cells comprising:

[0246] Embodiment 31: The method of embodiment 30, wherein the heterologous polynucleotide comprises a promoter.

[0247] Embodiment 32: The method of embodiment 31, wherein the promoter comprises an inducible promoter.

[0248] Embodiment 33: The method of embodiment 32, wherein the inducible promoter is induced by contacting the nucleated cell with a temperature of less than 37° C.

[0249] Embodiment 34: The method of embodiment 33, wherein the inducible promoter comprises dsrA or CIRP.

[0250] Embodiment 35: The method of embodiment 32, wherein the inducible promoter is induced by contacting the nucleated cell with a temperature greater than 37° C.

[0251] Embodiment 36: The method of embodiment 35, wherein the inducible promoter comprises HSP70, HSP90, GADD153, MDR1, or HSE-CMV.

[0252] Embodiment 37: The method of embodiment 32, wherein the inducible promoter is induced by contacting a nucleated cell with a molecule.

[0253] Embodiment 38: The method of embodiment 37, wherein the molecule comprises rtTA, TRE, TetR, Cumate, rapamycin, abscisic acid, IPTG, or metallothionein.

[0254] Embodiment 39: The method of embodiment 32, wherein the inducible promoter is induced by contacting a nucleated cell with light.

[0255] Embodiment 40: The method of embodiment 39, wherein the inducible promoter comprises CIB1-CRY2 or GAL4-VVD.

[0256] Embodiment 41: The method of embodiment 32, wherein the inducible promoter is induced by contacting the nucleated cell with a hormone.

[0257] Embodiment 42: The method of embodiment 41, wherein the inducible promoter comprises estradiol-Gal4.

[0258] Embodiment 43: The method of embodiment 31, wherein the promoter comprises a constitutively active promoter.

[0259] Embodiment 44: The composition of embodiment 43, wherein the constitutively active promoter is configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product.

[0260] Embodiment 45: The method of embodiment 44, wherein the heterologous gene product comprises herpes simplex virus-thymidine kinase (HSV-TK), cytosine deaminase (CD), varicella zoster TK (VZV-TK), nitroreductase, carboxypeptidase G2 (CPG2), cytochrome P450, or purine nucleoside phosphorylase.

[0261] Embodiment 46: The method of embodiment 44, wherein the heterologous gene product comprises an FKBP or a caspase.

[0262] Embodiment 47: The method of embodiment 44, wherein the heterologous gene product comprises an antigen.

[0263] Embodiment 48: The method according to any one of embodiments 30 to 47, wherein the heterologous polynucleotide is integrated into a chromosome of a nucleated cell.

[0264] Embodiment 49: The method of any one of embodiments 30 to 48, wherein the heterologous polynucleotide comprises a vector.

[0265] Embodiment 50: The method of any one of embodiments 1 to 49, further comprising cryopreserving the enucleated cell fraction to generate a cryopreserved enucleated cell fraction.

[0266] Embodiment 51: The method according to any one of embodiments 1 to 50, further comprising a step of thawing the cryopreserved enucleated cell fraction, wherein after thawing, the enucleated cells of the cryopreserved enucleated cell fraction are as viable as other equivalent enucleated cells that are not cryopreserved.

[0267] Embodiment 52: The method of embodiment 1 or embodiment 30, wherein the nucleated cells comprise stem cells.

[0268] Embodiment 53: The method of embodiment 52, wherein the stem cells comprise induced pluripotent stem cells (iPSCs) from a cell line, adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells, or a combination thereof.

[0269] Embodiment 54: The method of embodiment 53, wherein the nucleated cells comprise mesenchymal stromal cells.

[0270] Embodiment 55: The method according to any one of embodiments 1 to 54, wherein the enucleated cell lacks a nucleus and comprises one or more organelles for the synthesis or secretion of an exogenous polypeptide in the absence of a nucleus.

[0271] Embodiment 56: The method of embodiment 55, wherein the exogenous polypeptide comprises a therapeutic agent.

[0272] Embodiment 57: The method of any one of embodiments 1 to 56, wherein the enucleated cell comprises at least one targeting moiety.

[0273] Embodiment 58: The method of any one of embodiments 1 to 57, wherein the enucleated cell comprises at least one fusion moiety.

[0274] Embodiment 59: The method of any one of embodiments 1 to 58, wherein the enucleated cell comprises at least one immune evasion moiety.

[0275] Embodiment 60: The method of any one of embodiments 1 to 59, wherein the enucleated cells comprise at least one therapeutic moiety.

[0276] Embodiment 61: The method of any one of embodiments 1 to 60, wherein the enucleated cells of the enucleated cell fraction have a diameter that is 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 provided in a).

[0277] Embodiment 62: The method according to any one of embodiments 1 to 60, wherein the enucleated cells of the enucleated cell fraction have a diameter that comprises about 70% or less of the average diameter of the nucleated cells provided in a).

[0278] Embodiment 63: The method of any one of embodiments 1 to 62, wherein the enucleated cells of the enucleated cell fraction have a diameter that is 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.

[0279] Embodiment 64: The method of any one of embodiments 1 to 62, wherein the enucleated cells of the enucleated cell fraction comprise a diameter comprised between about 10 micrometers (μm) and about 100 μm.

[0280] Embodiment 65: The method of embodiment 64, wherein the diameter comprises about 8 μm.

[0281] Embodiment 66: a) an enucleated cell obtained from a first subset of the plurality of nucleated cells; b) a second subset of the plurality of nucleated cells, wherein the nucleated cells of the second subset of the plurality of nucleated cells comprise a heterologous polynucleotide encoding a heterologous gene product configured to induce cell death of the nucleated cells; and A composition comprising:

[0282] Embodiment 67: The composition of embodiment 66, wherein the heterologous polynucleotide comprises a promoter configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express a heterologous gene product.

[0283] Embodiment 68: The composition of embodiment 67, wherein the promoter comprises an inducible promoter configured, upon induction, to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product.

[0284] Embodiment 69: The composition of embodiment 68, wherein the inducible promoter is induced by contacting the nucleated cell with a temperature of less than 37° C.

[0285] Embodiment 70: The composition of embodiment 69, wherein the inducible promoter comprises dsrA or CIRP.

[0286] Embodiment 71: The composition of embodiment 68, wherein the inducible promoter is induced by contacting a nucleated cell with a temperature greater than 37° C.

[0287] Embodiment 72: The composition of embodiment 71, wherein the inducible promoter comprises HSP70, HSP90, GADD153, MDR1, or HSE-CMV.

[0288] Embodiment 73: The composition of embodiment 68, wherein the inducible promoter is induced by contacting a nucleated cell with a molecule.

[0289] Embodiment 74: The composition of embodiment 73, wherein the molecule comprises rtTA, TRE, TetR, Cumate, rapamycin, abscisic acid, IPTG, or metallothionein.

[0290] Embodiment 75: The composition of embodiment 68, wherein the inducible promoter is induced by contacting a nucleated cell with light.

[0291] Embodiment 76: The composition of embodiment 75, wherein the inducible promoter comprises CIB1-CRY2 or GAL4-VVD.

[0292] Embodiment 77: The composition of embodiment 68, wherein the inducible promoter is induced by contacting the nucleated cell with a hormone.

[0293] Embodiment 78: The composition of embodiment 77, wherein the inducible promoter comprises estradiol-Gal4.

[0294] Embodiment 79: The composition of embodiment 67, wherein the promoter comprises a constitutively active promoter.

[0295] Embodiment 80: The composition of embodiment 79, wherein the constitutively active promoter is configured to activate transcription of the heterologous polynucleotide under conditions sufficient to express the heterologous gene product.

[0296] Embodiment 81: The composition of embodiment 80, wherein the heterologous gene product comprises herpes simplex virus-thymidine kinase (HSV-TK), cytosine deaminase (CD), varicella zoster TK (VZV-TK), nitroreductase, carboxypeptidase G2 (CPG2), cytochrome P450, or purine nucleoside phosphorylase.

[0297] Embodiment 82: The composition of embodiment 80, wherein the heterologous gene product comprises FKBP or caspase.

[0298] Embodiment 83: The composition of embodiment 80, wherein the heterologous gene product comprises an antigen.

[0299] Embodiment 84: The composition according to any one of embodiments 66 to 86, wherein the heterologous polynucleotide is integrated into the chromosome of a nucleated cell.

[0300] Embodiment 85: The composition of any one of embodiments 66 to 84, wherein the heterologous polynucleotide comprises a vector.

[0301] EMBODIMENT 86: a) an enucleated cell obtained from a first subset of the plurality of nucleated cells; b) a second subset of the plurality of nucleated cells, wherein about 0.1% or less by volume of the composition comprises the second subset of the plurality of nucleated cells; and A composition comprising:

[0302] Embodiment 87: The composition of any one of embodiments 66 to 86, wherein the plurality of nucleated cells comprises stem cells.

[0303] Embodiment 88: The composition of embodiment 87, wherein the stem cells comprise induced pluripotent stem cells (iPSCs) derived from a cell line, adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells, or a combination thereof.

[0304] Embodiment 89: The composition of embodiment 88, wherein the nucleated cells comprise mesenchymal stromal cells.

[0305] Embodiment 90: The composition of any one of embodiments 66 to 89, wherein the enucleated cells lack a nucleus and comprise one or more structural characteristics of a plurality of nucleated cells.

[0306] Embodiment 91: The composition of embodiment 90, wherein the one or more structural features include one or more intracellular organelles, one or more tunneling nanotubes, or a combination thereof.

[0307] Embodiment 92: The composition described in any one of embodiments 66 to 91, wherein the enucleated cell lacks a nucleus and comprises one or more intracellular organelles for the synthesis or secretion of an exogenous polypeptide in the absence of a nucleus.

[0308] Embodiment 93: The composition of embodiment 91 or embodiment 92, wherein the one or more intracellular organelles comprises a Golgi apparatus, an endoplasmic reticulum, or a combination thereof.

[0309] Embodiment 94: The composition of embodiment 92, wherein the exogenous polypeptide comprises a therapeutic agent.

[0310] Embodiment 95: The composition of any one of embodiments 66 to 94, wherein the enucleated cell comprises at least one targeting moiety.

[0311] Embodiment 96: The composition of any one of embodiments 66 to 95, wherein the enucleated cell comprises at least one fusion moiety.

[0312] Embodiment 97: The composition of any one of embodiments 66 to 96, wherein the enucleated cell comprises at least one immune evasion moiety.

[0313] Embodiment 98: The composition of any one of embodiments 66 to 97, wherein the enucleated cells comprise at least one therapeutic moiety.

[0314] Embodiment 99: A composition described in any one of embodiments 66 to 98, wherein the enucleated cells of the enucleated cell fraction have a diameter that is at least 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 provided in a).

[0315] Embodiment 100: A composition described in any one of embodiments 66 to 99, wherein the enucleated cells of the enucleated cell fraction have a diameter that comprises less than or equal to about 70% of the average diameter of the nucleated cells provided in a).

[0316] Embodiment 101: A composition described in any one of embodiments 66 to 100, wherein the enucleated cells of the enucleated cell fraction have a diameter comprised between about 10 μm and about 100 μm.

[0317] EMBODIMENT 102: The composition of embodiment 101, wherein the enucleated cells of the enucleated cell fraction have a diameter of about 1 μm or more, including about 5 μm, about 8 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm.

[0318] Embodiment 103: The composition of embodiment 102, wherein the diameter comprises about 8 μm.

[0319] Embodiment 104: The composition of any one of embodiments 66 to 103, wherein the composition is in a form suitable for intravenous administration.

[0320] Embodiment 105: The composition of embodiment 104, wherein the dosage form comprises a solid dosage form.

[0321] Embodiment 106: The composition of any one of embodiments 66 to 105, wherein the composition comprises a tablet, a pill, a powder, a capsule, a solid dispersion, a solid solution, a bioerodible dosage form, a controlled release formulation, a pulsatile release dosage form, a multiparticulate dosage form, beads, pellets, or granules.

[0322] Embodiment 107: The composition according to any one of embodiments 66 to 106, wherein the total number of enucleated cells in the composition is greater than or equal to about 10 million enucleated cells, about 20 million enucleated cells, about 30 million enucleated cells, about 40 million enucleated cells, about 45 million enucleated cells, about 50 million enucleated cells, about 55 million enucleated cells, about 60 million enucleated cells, about 65 million enucleated cells, about 70 million enucleated cells, about 75 million enucleated cells, about 80 million enucleated cells, about 90 million enucleated cells, or about 100 million enucleated cells.

[0323] Embodiment 108: The composition according to any one of embodiments 66 to 107, wherein the enucleated cells are further cryopreserved to generate cryopreserved enucleated cells.

[0324] Embodiment 109: The composition of embodiment 108, wherein the cryopreserved enucleated cell fraction is thawed, and after thawing, the enucleated cells of the cryopreserved enucleated cell fraction are as viable as other equivalent enucleated cells that are not cryopreserved.

[0325] Embodiment 110: The composition of any one of embodiments 66 to 107, wherein the enucleated cells exhibit viability after cryostasis.

[0326] Embodiment 111: The composition of embodiment 110, wherein the enucleated cells exhibit a viability after cryostasis measured 24 hours after cryostasis that is equal to or greater than the viability of comparable enucleated cells that have not been cryostasis.

[0327] Embodiment 112: The composition according to any one of embodiments 66 to 107, wherein the enucleated cells exhibit viability after cryopreservation.

[0328] Embodiment 113: The composition of embodiment 112, wherein the enucleated cells exhibit a post-cryopreservation viability measured 24 hours after cryopreservation that is equal to or greater than the viability of comparable enucleated cells that have not been cryopreserved.

[0329] Embodiment 114: The composition of any one of embodiments 66 to 113, wherein the composition is purified.

[0330] Embodiment 115: A composition described in any one of embodiments 66 to 113, wherein the composition is lyophilized.

[0331] Embodiment 116: The composition of any one of embodiments 66 to 115, wherein the enucleated cell and the plurality of nucleated cells are at the same stage of cell differentiation.

[0332] Embodiment 117: The composition according to any one of embodiments 66 to 116, wherein the enucleated cells are not obtained from a plurality of nucleated cells by cell differentiation.

[0333] Embodiment 118: The composition of any one of embodiments 66 to 116, wherein the enucleated cells are not terminally differentiated cells.

[0334] Embodiment 119: The composition of any one of embodiments 66 to 116, wherein the enucleated cells are not platelets.

[0335] Embodiment 120: The composition according to any one of embodiments 66 to 116, wherein the enucleated cells are not obtained from platelet lineage cells.

[0336] Embodiment 121: The composition of any one of embodiments 66 to 116, wherein the enucleated cells are not red blood cells.

[0337] Embodiment 122: The composition according to any one of embodiments 66 to 116, wherein the enucleated cells are not obtained from erythroid cells.

[0338] Embodiment 123: A plurality of enucleated cells, comprising a plurality of the enucleated cells according to any one of embodiments 66 to 122.

[0339] EMBODIMENT 124: a) an enucleated cell according to any one of embodiments 66 to 112; b) a pharma- ceutically acceptable excipient, carrier, or diluent; 23. A pharmaceutical composition comprising:

[0340] Embodiment 125: The pharmaceutical composition of embodiment 124, wherein the pharmaceutical composition is in a unit dosage form.

[0341] Embodiment 126: A pharmaceutical composition according to embodiment 124 or 125, wherein the pharmaceutical composition is formulated for administration to a subject intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intralateral cerebroventricularly, intraparenchymal, subcutaneously, intratumorally, intrapulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled spray form, by intraluminal-GI route, or a combination thereof.

[0342] Embodiment 127: The pharmaceutical composition of embodiment 126, wherein the pharmaceutical composition is formulated for intravenous administration.

[0343] Embodiment 128: A pharmaceutical composition according to any one of embodiments 124 to 127, comprising at least one additional active substance.

[0344] Embodiment 129: The pharmaceutical composition of embodiment 128, wherein the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or a combination thereof.

[0345] EMBODIMENT 130: a) a composition according to any one of embodiments 66 to 123 or a pharmaceutical composition according to embodiments 124 to 129, b) Container and A kit comprising: EXAMPLES

[0346] The following illustrative examples are representative of embodiments of the stimulation actions, systems, and methods described herein, and are not intended to be limiting in any way.

[0347] Example 1 - Successful enucleation and survival of mammalian cells The enucleation efficiency and recovery rate of various types of mammalian cells (e.g., mesenchymal stem cells, neutrophils, fibroblasts, and natural killer cells) were determined. After removal of mammalian cells from cell culture plates, mammalian cells were enucleated by density gradient centrifugation using a discontinuous Ficoll gradient, high-speed centrifugation (Figure 4A-Figure 4C). Table 1 summarizes the results of enucleation using the suspension protocol. Enucleation efficiency and cell viability were highest in both hTERT-transformed and primary mesenchymal stem cells (MSCs), and similarly high in fibroblasts and neutrophils. Table 2 summarizes the results of enucleation using the adhesion protocol. Enucleation efficiency was greater than 70% in both mesenchymal stem cells and macrophages. This experiment demonstrated that various types of mammalian cells can undergo enucleation using any of the methods described herein.

[0348] [Table 1]

[0349] [Table 2]

[0350] Next, the viability of cytoplasts was determined over a 96-hour period (Figure 4A). While MSCs proliferated over time, cytoplasts did not. Instead, the relative fold change of viable cytoplasts remained almost constant for 72 hours before decreasing at 96 hours. Thus, cytoplast survival spanned 3-4 days. As most cell-based therapies are not used immediately, the viability of cytoplasts after cryopreservation was determined. Surprisingly, the viability of cytoplasts after cryopreservation was higher than that of MSCs after cryopreservation (Figure 4B). Cytoplasts plated immediately after enucleation and cytoplasts recovered from cryopreservation showed similar relative cell viability after 24 hours (Figure 4C). This experiment demonstrated that cytoplast survival was not affected by cryopreservation. Furthermore, the viability of cytoplasts after cryopreservation was similar to that of MSCs after cryopreservation (Figure 5A). Cytoplasts recovered after cryostasis for various lengths of time were able to undergo directed migration in Boyden chamber assays similar to MSCs recovered after cryostasis (Figure 5B).

[0351] Further viability testing of cytoplasts generated by the methods described herein. Figure 10 shows cell surface staining of FITC-labeled Annexin V on MSCs or cytoplasts analyzed by flow cytometry. Data was analyzed in Flowjo and normalized to mode. Parental MSCs = non-manipulated MSCs, isotype control = MSCs stained with isotype-matched IgG. 2h (hr) / 24h / 48h / 72h cytoplasts = MSC-derived cytoplasts analyzed at the indicated time points after enucleation, heat-shocked cells served as a positive control for apoptotic MSC cell death. Representative results from three independent experiments are shown. Three days after enucleation, cytoplasts showed apoptosis as shown by Annexin V staining and FACS.

[0352] Large-scale production of cells was then set up ex vivo, followed by large-volume density gradient centrifugation and enucleation, resulting in the generation of therapeutic cytoplasts. In one embodiment, the therapeutic cytoplasts are loaded with therapeutic cargo (e.g., mRNA, drugs, peptides, etc.) for disease treatment. In another embodiment, the therapeutic cytoplasts are prepared for immediate use (e.g., for intravenous injection (IV), intraperitoneal injection (IP), tissue, or in vitro application) for diagnostic applications.

[0353] Example 2. Enucleated cells retain intact and functional organs After determining whether the cytoplasts could retain viability after cryopreservation, flow cytometry analysis was performed to determine whether the cell surface marker profile of MSC-derived cytoplasts differed from bone marrow-derived MSCs. Both MSC-derived cytoplasts and bone marrow-derived MSCs maintained cell surface expression of CD45, CD90, CD44, CD146, and CD166. Cytoplasts rearranged their cytoskeleton and spread on matrix proteins in 2D and 3D culture systems, forming tunneling nanotubes that could transfer bioproducts between cells of the same or different origins. Organelle staining showed that the Golgi, ER, F-actin cytoskeleton, lysosomes, endosomes, microtubules, and mitochondria remained intact in the cytoplasts. Furthermore, the cytoplasts exhibited homing ability in vitro. The cytoplasts readily moved on extracellular matrix proteins and unidirectionally (e.g., via chemosensing) toward soluble chemokine gradients. In particular, cytoplasts exogenously transfected with purified mRNA produced functional intracellular proteins that could mimic therapeutic mRNA applications being developed for various clinical uses and disease conditions. This also demonstrates that the machinery for mRNA translation and protein synthesis operates normally in the cytoplasm in the absence of a nucleus and can therefore be used to produce bioactive molecules with therap...

Claims

1. a) providing a composition comprising nucleated cells; b) enucleating a portion of the nucleated cells using continuous flow centrifugation to produce an enucleated cell fraction; A method for treating cells, comprising:

2. 2. The method of claim 1, wherein the portion of nucleated cells comprises about 95% or more of the nucleated cells.

3. 10. The method of claim 1, wherein the composition provided in a) has a volume comprised between about 500 mL or more and about 10,000 mL.

4. 2. The method of claim 1, wherein the continuous flow centrifugation creates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition.

5. The method of claim 4 , wherein the density gradient comprises a polysaccharide density gradient.

6. 5. The method of claim 4, wherein the density gradient comprises at least two, at least three, at least four, at least five, at least six, or at least seven ranges of the density gradient.

7. 6. The method of claim 5, wherein the polysaccharide density gradient comprises about 25% polysaccharide, about 17% polysaccharide, about 16% polysaccharide, about 15% polysaccharide, or about 12.5% ​​polysaccharide.

8. The enucleated cell fraction generated by a single continuous flow centrifugation run was approximately 6 x 10 7 More than 250 x 10 enucleated cells 7 The method of claim 1, comprising enucleating cells of

9. 10. The method of claim 1, wherein the enucleating step of b) further comprises creating a density gradient comprising centrifuging the polysaccharide at a maximum centrifugal force of between about 30,000 RCF and about 200,000 RCF.

10. 10. The method of claim 1, wherein the continuous flow centrifugation creates a zonal centrifugation for separating at least one enucleated cell from the nucleated cells.

11. 11. The method of claim 10, wherein the zonal centrifugation separates the at least one enucleated cell from the nucleated cells based on the size of the at least one enucleated cell, or wherein the zonal centrifugation separates the at least one enucleated cell from the nucleated cells based on the mass of the at least one enucleated cell.

12. 5. The method of claim 4, wherein at least one density fraction is obtained from the density gradient, the at least one density fraction comprising a mixed population of the nucleated cells and a subset of enucleated cells of the enucleated cell fraction.

13. 13. The method of claim 12, wherein the mixed population comprises at least 70% of the enucleated cells.

14. 2. The method of claim 1, wherein the use of continuous flow centrifugation increases the yield of enucleated cells from nucleated cells by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, or more, compared to a method of obtaining enucleated cells from nucleated cells by a method that does not use continuous flow centrifugation.

15. The method of claim 1 , wherein the nucleated cells comprise a heterologous polynucleotide.

16. 16. The method of claim 15, wherein the method comprises inducing cell death of the nucleated cells that are not enucleated after b), wherein the cell death is induced by expressing a heterologous gene product encoded by the heterologous polynucleotide in the nucleated cells.

17. The method of claim 1 , wherein the nucleated cells comprise stem cells, mesenchymal stromal cells, or immune cells.

18. 2. The method of claim 1, wherein the enucleated cell lacks a nucleus and, in the absence of the nucleus, comprises one or more organelles for the synthesis or secretion of an exogenous polypeptide.

19. 20. The method of claim 18, wherein the exogenous polypeptide comprises a therapeutic agent.

20. 20. The method of any one of claims 1 to 19, wherein the enucleated cell comprises at least one targeting moiety, fusion moiety, and / or immune evasion moiety.

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