Compositions and methods for treating cancer

Abstract

Described herein are compositions and methods for treating cancer by administering an enucleated cell, wherein the enucleated cell comprises exogenous messenger RNA (mRNA) encoding a cytokine polypeptide and organelles for expressing the cytokine polypeptide from the exogenous mRNA, wherein the cytokine polypeptide is expressed by the enucleated cell on a cell surface. The enucleated cell may also comprise exogenous mRNA encoding another cytokine or immune checkpoint inhibitor.

Description

WSGR Docket No.53712-720.601 COMPOSITIONS AND METHODS FOR TREATING CANCER CROSS-REFERENCE

[0001] This application claims the benefit of US Provisional Application Serial Number 63 / 477,464 filed on December 28, 2022, and US Provisional Application Serial Number 63 / 591,857 filed on October 20, 2023, each of which is hereby incorporated by reference in its entirety. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under CA250887-01 awarded by National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 9, 2023, is named 53712-720_601_SL and is 71,568 bytes in size. BACKGROUND

[0004] The systemic spread of tumor cells and occurrence of metastases are the underlying cause of death from cancer, yet few successful therapies exist that specifically target metastasis. Moreover, many patients already have undetectable micrometastases at the time of diagnosis. Further, many tumors are ineffectively controlled by systemically administered therapeutics, which perform sub optimally in the tumor microenvironment (TME). To overcome these challenges, effective antimetastatic therapies are required to deliver therapeutic agents into the anatomical niche(s) where metastatic tumor cells can thrive. SUMMARY

[0005] Described herein, in some aspects, are methods for treating a disease or condition in a subject, the method comprising: administering to the subject an enucleated cell, wherein the enucleated cell comprises (a) an exogenous messenger RNA (mRNA) encoding an interleukin 12 (IL-12) polypeptide; and (b) one or more organelles for expressing the IL-12 polypeptide from the exogenous mRNA, wherein the IL-12 polypeptide is expressed by the enucleated cell on a cell surface of the enucleated cell for at least 24 hours in vivo. In some embodiments, the IL-12 polypeptide comprises a membrane-bound IL-12 polypeptide. In some embodiments, the IL-12 polypeptide is complexed to the cell surface of the enucleated cell. In some embodiments, the enucleated cell induces an endogenous immune response at a site associated with the disease or condition of the subject. In some embodiments, the method further comprises treating theWSGR Docket No.53712-720.601 disease or condition of the subject, at least in part, by inducing an endogenous immune response in the subject. In some embodiments, the site associated with the disease or condition comprises a microenvironment. In some embodiments, the microenvironment is a tumor microenvironment. In some embodiments, the IL-12 polypeptide is not delivered to the microenvironment. In some embodiments, the site associated with the disease or condition comprises a lymphatic microenvironment, a hepatic microenvironment, a spleen microenvironment, a pancreatic microenvironment, an epidermis microenvironment, or a combination thereof. In some embodiments, the site associated with the disease or condition comprises a cell associated with the disease or condition. In some embodiments, the cell associated with the disease or condition is a cancer cell. In some embodiments, the cell associated with the disease or condition is an immune cell. In some embodiments, the immune cell is a leukocyte. In some embodiments, the IL-12 polypeptide is not delivered to the cell associated with the disease or condition. In some embodiments, the disease or condition comprises cancer. In some embodiments, the IL-12 polypeptide is covalently connected to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, the IL-12 polypeptide is complexed with the enucleated cell for the at least 24 hours. In some embodiments, the IL-12 polypeptide is not secreted by the enucleated cell. In some embodiments, the IL-12 polypeptide encoded by the exogenous mRNA results in a decreased toxicity in the subject compared to a toxicity caused by direct administration of an IL-12 therapy. In some embodiments, the administering further comprises administering to the subject a plurality of the enucleated cell formulated in a pharmaceutical formulation, wherein the pharmaceutical formulation comprises (a) the plurality of the enucleated cell, and (b) a pharmaceutically acceptable: excipient, diluent, or a combination thereof. In some embodiments, the IL-12 polypeptide comprises IL-12 or antigen-binding fragment of the IL-12.

[0006] Described herein, in some aspects, is a method for inducing endogenous immune response in a subject, the method comprising: administering to the subject an enucleated cell comprising (a) an exogenous mRNA encoding a cytokine polypeptide; and (b) one or more organelles for expressing the cytokine polypeptide on a cell surface of the enucleated cell, wherein the cytokine polypeptide induces the endogenous immune response in the subject. In some embodiments, the cytokine polypeptide comprises a membrane-bound cytokine polypeptide. In some embodiments the cytokine polypeptide is complexed to the cell surface of the enucleated cell. In some embodiments, the enucleated cell induces the endogenous immune response at a microenvironment. In some embodiments, the microenvironment is a tumor microenvironment. In some embodiments, the microenvironment is a lymphatic microenvironment, a hepatic microenvironment, a spleen microenvironment, a pancreaticWSGR Docket No.53712-720.601 microenvironment, an epidermis microenvironment, or a combination thereof. In some embodiments, the enucleated cell induces the endogenous immune response in proximity of a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a leukocyte. In some embodiments, the cytokine polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours. In some embodiments, the cytokine polypeptide is complexed with the enucleated cell for the at least 24 hours. In some embodiments, the cytokine polypeptide is not secreted by the enucleated cell. In some embodiments, the cytokine polypeptide is not delivered to the microenvironment. In some embodiments, the cytokine polypeptide is not delivered to the cell. In some embodiments, the cytokine polypeptide comprises a full-length cytokine or antigen binding fragment thereof of the cytokine. In some embodiments, the cytokine polypeptide encoded by the exogenous mRNA results in a decreased toxicity in the subject compared to a toxicity caused by direct delivery of the cytokine polypeptide. In some embodiments, the cytokine polypeptide comprises an interleukin polypeptide. In some embodiments, the interleukin polypeptide is an IL-12 polypeptide. In some embodiments, the cytokine polypeptide comprises an interleukin antibody or antigen binding fragment thereof. In some embodiments, the cytokine polypeptide comprises IL-12 or antigen binding fragment thereof. In some embodiments, the endogenous immune response comprises an innate immune response or adaptive immune response. In some embodiments, the enucleated cell further comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. In some embodiments, the immune checkpoint inhibitor comprises the PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell. In some embodiments, the enucleated cell is depleted of an immune recognition molecule. In some embodiments, the immune recognition molecule comprises HLA antigen, proteoglycan, sugar moiety, embryonic antigen, or any combination thereof. In some embodiments, the enucleated cell comprises a targeting moiety. In some embodiments, targeting moiety comprises CXCR2, CCR2, PSGL-1, or a combination thereof.

[0007] Described herein, in some aspects, is a composition, comprising: an enucleated cell comprising: an exogenous mRNA that encodes a cytokine polypeptide; and organelles for expressing the cytokine polypeptide, wherein the cytokine is expressed on a cell surface of the enucleated cell. In some embodiments, the enucleated cell further comprises a transmembrane domain coupled to the cytokine polypeptide. In some embodiments, the transmembrane and the cytokine polypeptides are covalently connected. In some embodiments, the cytokine polypeptideWSGR Docket No.53712-720.601 is expressed extracellularly on the cell surface of the enucleated cell. In some embodiments, the cytokine polypeptide is complexed to a cell surface of the enucleated cell. In some embodiments, the cytokine polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours. In some embodiments, the enucleated cell expresses an abundance of the cytokine polypeptide that is at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 1.0 fold, at least 2.0 fold, or at least 5.0 fold higher than the enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the abundance of the cytokine polypeptide and the abundance of the comparable naturally-occurring cytokine polypeptide is determined by FACS. In some embodiments, the cytokine polypeptide is not secreted by the enucleated cell. In some embodiments, the cytokine polypeptide comprises an interleukin polypeptide. In some embodiments, the interleukin polypeptide is IL-12 polypeptide. In some embodiments, the enucleated cell further comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. In some embodiments, the immune checkpoint inhibitor comprises the PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell. In some embodiments, the enucleated cell is depleted of an immune recognition molecule. In some embodiments, the immune recognition molecule comprises HLA antigen, proteoglycan, sugar moiety, embryonic antigen, or any combination thereof. In some embodiments, the enucleated cell comprises a targeting moiety. In some embodiments, the targeting moiety comprises CXCR2, CCR2, PSGL- 1, or a combination thereof. In some embodiments, the composition further comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. In some embodiments, the immune checkpoint inhibitor comprises the PD- 1 inhibitor. In some embodiments, the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell. In some embodiments, the immune checkpoint inhibitor is encoded by a second enucleated cell. INCORPORATION BY REFERENCE

[0008] All publications, patents, and patent applications mentioned in this specification are herein 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 publications and patents or patent applications incorporated by referenceWSGR Docket No.53712-720.601 contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] Fig.1A illustrates a non-limiting example of IL-12 mRNA design.

[0011] Fig.1B illustrates mesenchymal stem cells (MSCs), MSC-derived enucleated cells transfected with IL-12 mRNA (MSC IL-12 and Enucleated cell IL-12 respectively), or conditioned medium (CM) collected at the indicated time points.

[0012] Fig.1C illustrates CM from cells / enucleated cells as shown in Fig.1B assessed in mouse splenocytes for the activation of the phosphorylated / activated form of Stat4 (p-Stat4) by western blot.

[0013] Fig.1D illustrates a schematic for using the enucleated cell to treat a triple negative breast cancer (TNBC) mouse model.

[0014] Fig.1E illustrates measurements of IL-12 secreted by the enucleated cell within tumor microenvironment.

[0015] Fig.1F illustrates serum IL-12 measurement.

[0016] Fig.1G illustrates measurements of IL-12 mediated inflammatory biomarkers.

[0017] Fig.1H illustrates mice bearing SQ E0771 tumors injected with a total of three doses of either PBS, MSCs secreting IL-12, or CA-IL-12.

[0018] Fig.1I illustrates FACS analysis for detecting leukocyte activation.

[0019] Fig.2A illustrates a schematic of enucleated cell that secreted IL-12 alone or in combination with the checkpoint inhibitor anti-PD-1 antibody for treating cancer mouse model.

[0020] Fig.2B illustrates a survival curve for animals injected as described in Fig.2A.

[0021] Fig.2C illustrates a graph of tumor size over time post-injection.

[0022] Fig.2D illustrates a graph of fold change in animal weight during the treatment phase of the survival experiments shown in Fig.2B.

[0023] Fig.2E illustrates fold change in tumor size for each side (injected and contralateral / noninjected) for animals that were bilaterally injected with E0771 cells and then were intratumorally injected unilaterally with 3 doses of CA-IL-12 or PBS.

[0024] Fig.3A illustrates scRNA-seq datasets generated from two human TNBC primary tumors were integrated with eight normal mammary datasets. VCAM1-positive vascular endothelial cells from clusters 0 and 4 (Top panel of Fig.3A) were subsetted for differentialWSGR Docket No.53712-720.601 expression analysis. Dot plots confirmed vasculature within TNBC tumors overexpressing all CCP enucleated cell ligands relative to normal mammary controls (bottom panel of Fig.3A).

[0025] Fig.3B illustrates Boyden Chamber migration assay showing mesenchymal stem cells or enucleated cells engineered for expressing CCR2, CXCR4, and leukocyte adhesion molecule (PSGL-1; termed CCP MSCs or CCP enucleated cells respectively) for robustly homing towards CCL2, SDF-1a, and EO771 conditioned medium relative to a no-chemoattractant negative control.

[0026] Fig.3C illustrates RNA-seq analysis of the EO771 and 4T1 TNBC cell lines highly expressing Ccl2 and Cxcl12 (encoding SDF-1a).

[0027] Fig.3D illustrates RNA-seq of the 4T1 cell line revealing that the enucleated cell adhesion molecules / chemoattractants (encoding P-selectin), Ccl2, and Cxcl12 were upregulated with tumorigenesis (relative to tissue culture) and further upregulated in lung metastases. RNA- Seq data.

[0028] Fig.4A illustrates a schematic showing irradiation mouse model with RT and untreated bilateral tumors.

[0029] Fig.4B illustrates homing of the enucleated cells to tumors.

[0030] Fig.4C illustrates FACS for determining the abundance of enucleated cells homed to tumors.

[0031] Fig.5A illustrate infiltration of the enucleated cell infiltration.

[0032] Fig.5B illustrates optical sectioning and 3D reconstruction of the center of macrometastasis revealed that clusters of CCP enucleated cells.

[0033] Fig.5C illustrates single channel acquisition of DAPI stained sections showing normal lung architecture and no tumor mass.

[0034] Fig.6A illustrates compiled BLI data of E0771-LUC metastases that were untreated or treated with: empty CA + PD-1 mAB, CA-IL-12 + IgG, or CA-IL-12 + PD-1 mAB.

[0035] Fig.6B illustrates representative images of resected lungs with metastases treated as indicated (top) and representative IHC images of CD8+ cytotoxic T cells (bottom) infiltrating EO771 lung metastases from the resected lungs shown in the top row.

[0036] Fig.7 illustrates FACS analysis of indicated leukocyte populations in EO771 lung metastases harvested at day 21 as in Fig.6 relative to total CD45+ immune cells).

[0037] Fig.8A illustrates an exemplary design of the enucleated cell expressing cell membrane tethered IL-12.

[0038] Fig.8B illustrates non-limiting example of a vector map of a vector encoding IL-12.

[0039] Fig.8C illustrates engineering and screening for the enucleated cell expressing membrane-bound scIL-12.WSGR Docket No.53712-720.601

[0040] Fig.8D illustrates an exemplary mechanism of immune cell activation by CA-scIL-12.

[0041] Fig.9A illustrates MSCs or enucleated cells engineered with or without scIL-12 that were co-cultured with primary murine splenocytes and concanavalin A for 48 hours.

[0042] Fig.9B illustrates enucleated cells (1 x106) engineered to secrete IL-12 (CA-IL-12).

[0043] Fig.9C illustrates enucleated cells engineered with cell surface IL-12 (CA-scIL-12) and were injected i.v. in mice (n = 3) and the indicated organs removed 24 hours later and processed for PCR quantification of the IL-12 biomarkers CXCL9 and IFN-γ.

[0044] Fig.9D illustrates serum cytokine levels in the CA-scIL-12 or CA-IL-12 treated animals.

[0045] Fig.10A illustrates an exemplary design of the enucleated cell expressing cell membrane tethered IL-15.

[0046] Fig.10B illustrates non-limiting example of a vector map of a vector encoding IL-15.

[0047] Fig.10C illustrates hTERT MSCs infected with lentivirus construct encoding membrane-bound IL-15 receptor alpha.

[0048] Fig.11A illustrates a schematic for engineering the hTERT-MSC for expression both scIL-12 and IL-15Rα and the subsequent enucleation of the hTERT-MSC.

[0049] Fig.11B illustrates hTERT MSCs infected with two lentiviruses encoding constructs for either scIL-12 or IL-15Rα. Following drug selection, cells were FACS sorted for single cell clones. Clone 4 was enucleated to generate enucleated cells expressing both scIL-12 and IL- 15Rα, and receptor expression was analyzed by FACS at the indicated time points.

[0050] Fig.11C illustrates functionality of expressing membrane-bound IL-12, IL-15, or a combination of both IL-12 and IL-15.

[0051] Fig.12A illustrates biodistribution of the enucleated cells by bioluminescence.

[0052] Fig.12B illustrates quantification of biodistribution of the enucleated cells of Fig.12A.

[0053] Fig.13A illustrates increased homing of the enucleated cell described herein compared to nucleated mesenchymal stem cell (MSC).

[0054] Fig.13B illustrates quantification of homing of the enucleated cell of Fig.13A.

[0055] Fig.14A illustrates homing of the enucleated cell based on increased chemoattractants in the TNBC tumor metastatic sites.

[0056] Fig.14B illustrates dot plots of expression of Cxcl (Top) and Ccl (Bottom) chemokines within Vcam1-positive cells in Cluster 1 confirming Cxcl12 (encoding SDF-1α) and Ccl2 were the highest expressed Cxcl and Ccl chemokines, respectively.

[0057] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.WSGR Docket No.53712-720.601 DETAILED DESCRIPTION

[0058] Described herein, in some aspects, are enucleated cells engineered to express at least one cytokine on a cell surface of the enucleated cells. In some embodiments, the expressing of the at least one cytokine on the cell surface of the enucleated cells confers therapeutic effects. In some embodiments, the enucleated cell comprises messenger RNA (mRNA) that encodes the cytokine. For examples, an enucleated cell described herein can be engineered to express a cell membrane bound cytokine for triggering or inducing endogenous immunity response in a subject administered with the enucleated cell. In some embodiments, the enucleated cell expressing the membrane bound cytokine can be utilized to treat a disease or condition in the subject. In some embodiments, the disease or condition can be cancer. In some embodiments, the cancer can be attributed from manifestation of a cancer cell, a tumor microenvironment (TME), a metastatic cell, or a combination thereof. In some embodiments, the membrane bound cytokine can be a membrane bound interleukin. In some embodiments, the enucleated cell can be engineered to express at least one additional therapeutic. In some embodiments, the enucleated cell can be engineered to express at least one additional therapeutic such as an immune checkpoint inhibitor or an immune checkpoint blocker (ICB). COMPOSITIONS

[0059] Provided herein are cells engineered to express at least one active agent on the cell surface of the enucleated cell. The active agent may be a protein. The active agent may be an antibody, antigen, or a fragment of said antibody or antigen. Provided herein are cells comprising mRNA to express a protein, an antibody, an antigen, or portion thereof, and compositions containing such cells. In some embodiments, the cells are enucleated. In some embodiments, the protein, antibody, antigen, or portion thereof is a therapeutic agent. In some embodiments, the enucleated cells are capable of expressing the protein, antibody, antigen, or portion thereof (e.g., therapeutic agent) in the absence of a nucleus using one or more intracellular organelles retained by the enucleated cells from parent cells. In some embodiments, the protein, antibody, antigen, or portion thereof (e.g., therapeutic agent) is exogenous to the enucleated cell or parent cell thereof. In some embodiments, the enucleated cell expresses the protein, antibody, antigen, or portion thereof (e.g., therapeutic agent) at the surface of the enucleated cell. In some aspects, the composition comprises an enucleated cell described herein. In some aspects, the enucleated cell is obtained or derived from a nucleated cell (e.g., a parent cell). In some aspects, the enucleated cell comprises a transmembrane moiety. In some embodiments, the enucleated cells comprises a targeting moiety. In some embodiments, the enucleated cell comprises a therapeutic agent. In some embodiments, the enucleated cell comprises a protein. In some embodiments, the enucleated cell comprises a cytokine. In someWSGR Docket No.53712-720.601 embodiments, the enucleated cell comprises an antibody or antigen binding fragment thereof or a single-domain antibody or an antigen binding fragment thereof. In some aspects, the targeting moiety comprises an antibody or antigen binding fragment thereof or a single-domain antibody or an antigen binding fragment thereof. In some embodiments, the therapeutic agent comprises an antibody or antigen binding fragment thereof. In some embodiments, the enucleated cell is formulated into a pharmaceutical formulation described herein. Enucleated cells

[0060] 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, an enucleated cell may be derived from an adult stem cell, a mesenchymal stromal cell (MSC), a natural killer (NK) cell, a macrophage, a myoblast, a neutrophil, endothelial cell, endothelial precursor cell, and / or a fibroblast. In some embodiments, an enucleated cell is derived from a mesenchymal stromal cell. In some embodiments, the enucleated cell is derived from an inducible pluripotent stem cell (iPSC). In some embodiments, the parent cell is derived from a cell is immortalized using suitable methods. For example the, parent cell is immortalized by expressing human telomerase reverse transcriptase (hTERT), an oncogene, or a viral gene such as simian virus 40 (SV40). In some embodiments, the cytoplast is derived from a parent cell using suitable methods provided in United States Patent No.10,927,349, which is hereby incorporated by reference in its entirety. In some embodiments, the enucleated cell retains one or more intracellular organelles for synthesis of an exogenous protein. In some embodiments, the enucleated cell retains one or more intracellular organelles for synthesis of an exogenous cytokine.

[0061] In some embodiments, the enucleated cell does not require differentiation of the parent cell. In some embodiments, the parent cell containing a nucleus is engineered to express the single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and / or targeting moiety described herein; and subsequently, the nucleus of the parent cell is removed. In some embodiments, the parent cell containing the nucleus is enucleated, and the enucleated cell is engineered to express single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and / or targeting moiety described herein. In some embodiments, the parent cell is engineered to express one or more of the biomolecules above (e.g., immune-evading moiety and / or targeting moiety), and the resulting enucleated cell (e.g., already expressing the immune- evading moiety and / or targeting moiety) is further engineered to express a second of the biomolecules above (e.g., a therapeutic agent). In this manner, the enucleated cells of the presentWSGR Docket No.53712-720.601 disclosure can be extensively engineered prior to enucleation, stored for long periods of time as needed (through for e.g., lyophilization, cryohibernation, cryopreservation), and quickly engineered to express a therapeutic agent closer to the time of need.

[0062] In some embodiments, the cell can originate from any organism having one or more cells. Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g. cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g. kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell). In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem cell or a progenitor cell. In some embodiments, the cell is a mesenchymal stem or progenitor cell. In some embodiments, 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 example of cells includes lymphoid cells such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell), Natural killer cell, cytokine induced killer (CIK) cells; myeloid cells such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte / Hypersegmented neutrophil), Monocyte / Macrophage, Red blood cell (Reticulocyte), Mast cell, Thrombocyte / Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell), parathyroid (Parathyroid chief cell, Oxyphil cell), adrenal (Chromaffin cell), pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia), Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph ); cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, Type II pneumocyte), Clara cell, Goblet cell, Dust cell; cells of the circulatory system, including Myocardiocyte, Pericyte; cells of the digestive system, including stomach (Gastric chief cell, Parietal cell), Goblet cell, Paneth cell, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffm cell, APUD cell, liver (Hepatocyte, Kupffer cell), Cartilage / bone / muscle; bone cells, including Osteoblast, Osteocyte, Osteoclast, teethWSGR Docket No.53712-720.601 (Cementoblast, Ameloblast); cartilage cells, including Chondroblast, Chondrocyte; skin cells, including Trichocyte, Keratinocyte, Melanocyte (Nevus cell); muscle cells, including Myocyte; urinary system cells, including Podocyte, Juxtaglomerular cell, Intraglomerular mesangial cell / Extraglomerular mesangial cell, Kidney proximal tubule brush border cell, Macula densa cell; reproductive system cells, including Spermatozoon, Sertoli cell, Leydig cell, Ovum; and other cells, including Adipocyte, Fibroblast, Tendon cell, Epidermal keratinocyte (differentiating epidermal cell), Epidermal basal cell (stem cell), Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell), Wet stratified barrier epithelial cells, Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining urinary bladder and urinary ducts), Exocrine secretory epithelial cells, Salivary gland mucous cell (polysaccharide-rich secretion), Salivary gland serous cell (glycoprotein enzyme - rich secretion), Von Ebner's gland cell in tongue (washes taste buds), Mammary gland cell (milk secretion), Lacrimal gland cell (tear secretion), Ceruminous gland cell in ear (wax secretion), Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweat gland clear cell (small molecule secretion). Apocrine sweat gland cell (odoriferous secretion, sex -hormone sensitive), Gland of Moll cell in eyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium), Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm), Prostate gland cell (secretes seminal fluid components), Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), Gland of Littre cell (mucus secretion), Uterus endometrium cell (carbohydrate secretion), Isolated goblet cell of respiratory and digestive tracts (mucus secretion), Stomach lining mucous cell (mucus secretion), Gastric gland zymogenic cell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloric acid secretion), Pancreatic acinar cell (bicarbonate and digestive enzyme secretion), Paneth cell of small intestine (lysozyme secretion), Type II pneumocyte of lung (surfactant secretion), Clara cell of lung, Hormone secreting cells, Anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cell, Magnocellular neurosecretory cells, Gut and respiratory tract cells, Thyroid gland cells, thyroid epithelial cell, parafollicular cell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells, Ley dig cell of testes, Theca interna cell of ovarian follicle, Corpus luteum cell of rupturedWSGR Docket No.53712-720.601 ovarian follicle, Granulosa lutein cells, Theca lutein cells, Juxtaglomerular cell (renin secretion), Macula densa cell of kidney, Metabolism and storage cells, Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract), Kidney, Type I pneumocyte (lining air space of lung), Pancreatic duct cell (centroacinar cell), Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.), Duct cell (of seminal vesicle, prostate gland, etc.), Epithelial cells lining closed internal body cavities, Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell), Megakaryocyte (platelet precursor), Monocyte, Connective tissue macrophage (various types), Epidermal Langerhans cell, Osteoclast (in bone), Dendritic cell (in lymphoid tissues), Microglial cell (in central nervous system), Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types), Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sense organ and peripheral neuron supporting cells, Central nervous system neurons and glial cells, Lens cells, Pigment cells, Melanocyte, Retinal pigmented epithelial cell, Germ cells, Oogonium / Oocyte, Spermatid, Spermatocyte, Spermatogonium cell (stem cell for spermatocyte), Spermatozoon, Nurse cells, Ovarian follicle cell, Sertoli cell (in testis), Thymus epithelial cell, Interstitial cells, and Interstitial kidney cells.

[0063] 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 animal (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 mammalian, avian, plant, or insect cells. In some embodiments, the nucleated cell is a primary cell. In some embodiments, the nucleated cell is an immune cell (e.g., a lymphocyte (e.g., a T cell, a B cell), a macrophage, a natural killer cell, a neutrophil, a mast cell, a basophil, a dendritic cell, a monocyte, a myeloid- derived suppressor cell, an eosinophil). In some embodiments, the nucleated cell is a phagocyte or a leukocyte. In some embodiments, the nucleated cell is a stem cell (e.g., an adult stem cell (e.g., a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, mesenchymal stem cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a testicular cell), an embryonic stem cell, an inducible pluripotent stem cell (iPS)). In some embodiments, the nucleated cell is a progenitor cell. In some embodiments, the nucleated cell is from a cell line. In some embodiments, the nucleated cell is a suspension cell. In someWSGR Docket No.53712-720.601 embodiments, the nucleated cell is an adherent cell. In some embodiments, the nucleated cell is a cell that has been immortalized by expression of an oncogene. In some embodiments, the nucleated cell is immortalized by the expression of human telomerase reverse transcriptase (hTERT) or any oncogene. In some embodiments, the nucleated cell is immortalized by the expression of a viral gene such as simian virus 40 (SV40). In some embodiments, the nucleated cell is a patient or subject derived cell (e.g., an autologous patient-derived cell, or an allogenic patient-derived cell).

[0064] In some embodiments, the enucleated cell is derived from an immune cell. In some embodiments, the enucleated cell is derived from a natural killer (NK) cell, a neutrophil, a macrophage, a lymphocyte, a fibroblast, an adult stem cell (e.g., 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 precursor cell, or an inducible pluripotent stem cell.

[0065] In some embodiments, the parent cell is not an erythrocyte or erythroid precursor cell. In some embodiments, the parent cell is a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial precursor cell. In some embodiments, the enucleated cell is not an erythrocyte or erythroid precursor cell. In some embodiments, the enucleated cell is not a platelet cell. In some embodiments, the enucleated cell is not an endothelial cell. In some embodiments, the enucleated cell is not an endothelial precursor cell. In some embodiments, the enucleated cell does not express complement receptor one (CR1). In some embodiments, the enucleated cell does not express CD44. In some embodiments, the enucleated cell does not express VLA-4. In some embodiments, the enucleated cell does not express BCAM. In some embodiments, the enucleated cell does not express ICAM. In some embodiments, the enucleated cell does not express a receptor for collagen. In some embodiments, the enucleated cell does not express a receptor for thrombopoietin. In some embodiments, the enucleated cell does not express a receptor for collagen. In some embodiments, the enucleated cell does not express a receptor for von Willebrand factor (VWF). In some embodiments, the enucleated cell does not express a receptor for fibrinogen. In some embodiments, the enucleated cell does not express GP1b-IX-V receptor. In some embodiments, the enucleated cell does not express GPIIb / IIIa receptor. In some embodiments, the enucleated cell does not express prostanoid receptor. In some embodiments, the enucleated cell does not express purinergic receptor. In some embodiments, the enucleated cell does not express thromboxane receptor.WSGR Docket No.53712-720.601

[0066] In some embodiments, the parent cell may be enucleated and engineered for therapeutic use. In some embodiments, the parent cell is any one of the nucleated cells described herein. 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 inducible pluripotent stem cell (iPSC). In some embodiments, the parent cell is not an erythrocyte or erythroid precursor cell. In some embodiments, the parent cell is a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial precursor cell. A parent cell may be treated with cytochalasin to soften the cortical actin cytoskeleton. The nucleus may then physically extracted from the cell body by high-speed centrifugation in gradients of Ficoll to generate an enucleated cell. Because enucleate cells and intact nucleated cells sediment to different layers in the Ficoll gradient, enucleated cells may be easily isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated). The enucleation process is clinically scalable to process tens of millions of cells. In some embodiments, enucleated cells may be used as a disease-homing vehicle to express clinically relevant cargos / payloads to treat various diseases.

[0067] Following enucleation of the parent cell, the enucleated cells described herein retain one or more intracellular organelles that are endogenous to the parent cell. In some embodiments, all of the one or more intracellular organelles are retained. In some embodiments, fewer than all of the one or more intracellular organelles are retained. In some embodiments, the Golgi apparatus and / or the endoplasmic reticulum are retained, which are involved in protein synthesis and secretion. Retention of the one or more intracellular organelles at least partially enables the enucleated cells to synthesize or release the biomolecule disclosed herein (e.g., single-domain antibody, or portion thereof, targeting moiety, immune-evading moiety, etc.) in the absence of the nucleus.

[0068] Enucleated cells may be smaller than their nucleated counterparts (e.g., the nucleated parent cells), and for this reason may migrate better through small openings in the vasculature and tissue parenchyma. In addition, removing the large dense nucleus alleviates a major physical barrier allowing the cell to move freely through small openings in the vessels and tissue parenchyma. Therefore, enucleated cells have improved bio-distribution in the body and movement into target tissues. In some embodiments, an enucleated cell comprises at least 1 μm in diameter. In some embodiments, an enucleated cell is greater than 1 μm in diameter. In some embodiments, an enucleated cell is 1-100 μm in diameter (e.g., 1- 90 μm, 1-80 μm, 1-70 μm, 1- 60 μm, 1-50 μm, 1-40 μm, 1-30 μm, 1-20 μm, 1-10 μm, 1-5 μm, 5- 90 μm, 5-80 μm, 5-70 μm, 5- 60 μm, 5-50 μm, 5-40 μm, 5-30 μm, 5-20 μm, 5-10 μm, 10-90 μm, 10-80 μm, 10-70 μm, 10-60 μm, 10-50 μm, 10-40 μm, 10-30 μm, 10-20 μm, 10-15 μm 15-90 μm, 15-80 μm, 15-70 μm, 15-WSGR Docket No.53712-720.601 60 μm, 15-50 μm, 15-40 μm, 15-30 μm, 15-20 μm). In some embodiments, an enucleated cell is 10-30 μm in diameter. In some embodiments, the diameter of an enucleated cell is 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 cell has a diameter that is about 8 μm. In some embodiments, some enucleated cells may advantageously be small enough to allow for better homing to a target site. For examples, the enucleated cells described herein may pass through passages in narrow lung tissues or lung structures such as alveolar duct or microcapillary that most cells such as the parent cells may not pass through.

[0069] In some embodiments, enucleated cells possess significant therapeutic value, because they remain viable, do not differentiate into other cell types, secrete bioactive molecules, and may physically migrate / home for fewer than or equal to about 5 days, may be extensively enucleated ex vivo to perform specific therapeutic functions, and may be fused to the same or other cell types to transfer desirable production, natural or enucleated. Therefore, enucleated cells have wide utility as a cellular vehicle to express therapeutically important biomolecules and disease-targeting cargos including genes, viruses, bacteria, mRNAs, shRNAs, siRNA, polypeptides (including antibodies and antigen binding fragments), plasmids, gene-editing machinery, or nanoparticles. The present disclosure enables the generation of safe (e.g., no unwanted DNA is transferred to the subject), and controllable (e.g., cell death occurs in 3-4 days) cell-based carrier that may be genetically enucleated to express specific disease-fighting and health promoting cargos to humans. In some embodiments, the enucleated cell remains viable and retain the function to migrate or home for greater than or equal to about 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 5 days after being administered to the subject in need thereof.

[0070] In some embodiments, the enucleated cell is engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous protein, gene-editing machinery or combinations thereof. In some embodiments, the exogenous DNA molecule is a single-stranded DNA, a double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or combinations thereof. In some embodiments, the exogenous RNA molecule is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), a RNA virus, or combinations thereof. In some embodiments, the exogenous protein is a cytokine, a growth factor, a hormone, an antibody or the antigen-binding fragment thereof, an enzyme, or combinations thereof. In some embodiments, the antibody is a single-domain antibody or antigen-binding fragment thereof. In some embodiments, parental cells (e.g., nucleated cells) areWSGR Docket No.53712-720.601 genetically enucleated before enucleation (e.g., pre-enucleation). In some embodiments, the parent cell is genetically enucleated after enucleation (e.g., post-enucleation).

[0071] In some embodiments, the enucleated cell described herein comprises a transmembrane moiety. In some embodiments, the enucleated cell comprises an immune-evading moiety. In some embodiments, the immune-evading comprises a “don’t eat me” signaling peptide such as CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof. In some embodiments, the enucleated cell is depleted of an immune recognition molecule. Non-limiting examples of immune recognition molecules include an HLA antigen, a proteoglycan, a sugar moiety, and an embryonic antigen.

[0072] In some embodiments, the enucleated cell or the composition comprising the enucleated cell may be cryopreserved (e.g., storing the enucleated cell or the composition comprising the enucleated cell at freezing temperature) or cryohibernated (e.g., storing the enucleated cell or the composition comprising the enucleated cell at a temperature that is between the ambient temperature and freezing temperature). The duration of cryopreservation or cryohibernation may be greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the enucleated cell exhibits a viability after cryopreservation or cryohibernation that is greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been cryopreserved or cryo-hibernated) after same the period of time of cryopreservation or cryohibernation. For example, in some embodiments, the viability is reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared with the comparable cell. In another example, in some embodiments, the viability is increased by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared with the comparable cell. Viability in this context may be measured by Trypan blue dye exclusion as described herein. In some embodiments, the Trypan blue dye exclusion is performed by: (a) centrifuging an aliquot of a plurality of the cell without the nucleus in a suspension to create a cell pellet; (b) resuspending the cell pellet in serum-free medium to produce a serum-free cell suspension; (c) mixing 1 part Trypan blue dye and 1 part of the serum-free cell suspension; (d) counting the plurality of the cells without the nucleus within 3-5 minutes of (c), wherein at least some of the plurality of cells without the nucleus are unstained with the Trypan blue dye, which is indicative of viability. In some embodiments, the viability is measured using Annexin-V cell surface staining. In some embodiments, the viability is measured by expression of the exogenous polypeptide. For example, the viability of the enucleated cell can be determined by the expression of theWSGR Docket No.53712-720.601 exogenous antibody or single-domain antibody expressed by the enucleated cell. In some embodiments, the viability is measured by expression of cell surface markers of any one of the cell surface markers described herein such as CD105, CD90, CD45, CXCR4, PSGL-1, or CCR2. In some embodiments, the viability is measured by the cell activity of the enucleated cell. In some embodiments, the viability is measured by the homing capability of the enucleated cell as determined by the chemosensing or chemokine homing activity described herein.

[0073] In some embodiments, the enucleated cell or the composition comprising the enucleated cell may be lyophilized. In some embodiments, the enucleated cell exhibits a viability after being reconstituted from lyophilization that is greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been lyophilized).

[0074] In some embodiments, the enucleated cell or the composition comprising the enucleated cell may be dehydrated. In some embodiments, the enucleated cell exhibits a viability after being rehydrated from lyophilization that is greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been dehydrated).

[0075] In some embodiments, the enucleated cell or the composition comprising the enucleated cell is stable at 4°C for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the composition is stable at room temperature for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the composition is stable at 37°C for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the enucleated cell or the composition comprising the enucleated cell may remain viable after being administered to a subject in need thereof for treating the disease or condition described herein. In some embodiments, the enucleated cell or the composition comprising the enucleated cell may remain viable after being administered to the subject for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.WSGR Docket No.53712-720.601

[0076] In some embodiments, the enucleated cell may be obtained from a parent cell that is autologous to the subject, who is in need of the treatment by the enucleated cell described herein. In some embodiments, the enucleated cell may be obtained from a parent cell that is allogenic to the subject, who is in need of the treatment by the enucleated cell described herein. Active Agents

[0077] An enucleated cell may comprise an active agent. The active agent may be encoded by an exogenous messenger RNA (mRNA). The active agent comprises a polypeptide, a sugar, a metabolite, a nucleic acid (e.g., RNA, DNA), a small molecule, or any combination thereof. The polypeptide may be a cytokine, cytokine receptor, integrin, or an adhesion protein, or a catalytically active fragment thereof. The adhesion protein may be PSGL-1. The cytokine may be a chemokine, for example, SDF-1α, CCL2, IL-12, and IL-15. The cytokine may be a chemokine receptor, for example, CXCR2, CCR2, and CXCR4. The small molecule may be a chemosensor. The polypeptide may be expressed by the enucleated cell (in absence of the nucleus). In some embodiments, the polypeptide may be expressed by the nucleated parent cell prior to the enucleation to produce an enucleated cell comprising the polypeptide. In some embodiments, the active agent is expressed on the surface of the enucleated cell. In some embodiments, the active agent is or comprises a therapeutic agent of the present disclosure.

[0078] The active agent may be a targeting moiety of the present disclosure. In some embodiments, the active agent is or comprises a transmembrane moiety. In some embodiments, the active agent is covalently coupled to the enucleated cell surface. In some embodiments, the active agent is non-covalently coupled. In some embodiments, the amount of active agent is about 0.10 to about 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell. In some embodiments, the amount of the active agent is about 0.10 to about 0.50 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 1.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 2.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 3.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 0.10 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 135.00 foldWSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 1.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 2.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 3.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 80.00 fold increase over endogenous amount of the activeWSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 0.50 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 2.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 3.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 1.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 150.00 foldWSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 3.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 4.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 2.00 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 10.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 2.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 55.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 2.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 105.00 fold increase over endogenous amount of the activeWSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 2.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 3.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 35.00 foldWSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 150.00 fold increase over endogenous amount of the activeWSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 5.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 5.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 100.00 foldWSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 80.00 fold increase over endogenous amount of the activeWSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 15.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 20.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 60.00 foldWSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 65.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 25.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 55.00 fold increase over endogenous amount of the activeWSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 30.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 95.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 30.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 35.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 65.00 foldWSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 75.00 fold increase over endogenous amount of the activeWSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 45.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 150.00.In some embodiments, the amount of the active agent is about 50.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 95.00 fold increaseWSGR Docket No.53712-720.601 over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 150.00.In some embodiments, the amount of the active agent is about 55.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 55.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 145.00 foldWSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 95.00 fold increase over endogenous amount of the activeWSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 70.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of theWSGR Docket No.53712-720.601 active agent is about 75.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 135.00.In some embodiments, the amount of the active agent is about 80.00 to about 140.00.In some embodiments, the amount of the active agent is about 80.00 to about 145.00.In some embodiments, the amount of the active agent is about 80.00 to about 150.00.In some embodiments, the amount of the active agent is about 85.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 125.00.In some embodiments, the amount of the active agent is about 85.00WSGR Docket No.53712-720.601 to about 130.00.In some embodiments, the amount of the active agent is about 85.00 to about 135.00.In some embodiments, the amount of the active agent is about 85.00 to about 140.00.In some embodiments, the amount of the active agent is about 85.00 to about 145.00.In some embodiments, the amount of the active agent is about 85.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent isWSGR Docket No.53712-720.601 about 95.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 120.00 fold increase overWSGR Docket No.53712-720.601 endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 140.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 115.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 145.00 fold increase over endogenous amount of the active agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the active agent is about 125.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 140.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 140.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 145.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell. In some embodiments, the amount of the active agent is greater than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell. In some embodiments, the amount of the active agent is less than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell. In some embodiments, theWSGR Docket No.53712-720.601 amount of the active agent and the amount of the comparable endogenous active agent is determined by flow cytometry (FACS).

[0079] The active agent may be a cytokine. The cytokine may be expressed by the mRNA in the enucleated cell. The cytokine may be expressed by the enucleated cell on the surface of the enucleated cell. In some embodiments, the cytokine polypeptide encoded by the exogenous mRNA has decreased toxicity in the subject as compared to direct delivery of the cytokine polypeptide. The cytokine may comprise a transmembrane domain. The mRNA may encode the cytokine and the transmembrane domain. The cytokine may be attached to the cell surface of the enucleated cells. The cytokine may be attached, complexed, tethered, connected, coupled, linked, or any of the like to the cell surface of the enucleated cell. The cytokine may be attached to the cell surface of an enucleated cell exogenously. In some embodiments, the cytokine is not delivered to a target tissue or microenvironment by an enucleated cell. In some embodiments, the cytokine is not secreted to a target tissue or microenvironment by an enucleated cell. In some embodiments, the mRNA encodes a cytokine or an antigen-binding fragment of a cytokine. In some embodiments, the cytokine is an antigen-binding fragment of said cytokine. In some embodiments, the cytokine is a full-length cytokine.

[0080] In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.1 fold to at least 5.0 fold higher than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.1 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.3 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.4 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.5 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.7 fold than an enucleated cell expressing an abundance of aWSGR Docket No.53712-720.601 comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.8 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.9 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokineWSGR Docket No.53712-720.601 polypeptide that is at least 3.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 5.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the abundance of the cytokine polypeptide and the abundance of the comparable naturally- occurring cytokine polypeptide is determined by flow cytometry (FACS).

[0081] In some embodiments, the cytokine polypeptide is coupled to the enucleated cell surface for a certain length of time. In some embodiments, the length of time is following delivery to a subject (e.g., administration) disclosed herein. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 36 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 72 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, aWSGR Docket No.53712-720.601 cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 36 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 72 hours. In some embodiments, a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 12 hours. In some embodiments, a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 24 hours. In some embodiments, a cytokine polypeptide can be expressed on a cell surface of an enucleated cell for at least 36 hours. In some embodiments, a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide can be expressed on a cell surface of an enucleated cell for at least 72 hours.

[0082] The enucleated cell disclosed herein may comprise an mRNA encoding a polypeptide. The polypeptide may be a cytokine. The cytokine may comprise an interleukin. The interleukin may comprise IL-12. The IL-12 may comprise IL-12α. In some embodiments, IL-12α comprises SEQ ID NO: 35. In some embodiments, IL-12α comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. The IL-12 may comprise IL-12β. In some embodiments, IL-12 β comprises SEQ ID NO: 36. In some embodiments, IL-12β comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36.The IL-12 may comprise IL-12α and IL-12β. In some embodiments, the IL-12α and IL-12β interact with a disulfide bond connecting the IL-12α and IL-12β. The IL-12 may comprise a transmembrane domain, such as a transmembrane from a B7 cell surface ligand. In some embodiments, the transmembrane from the B7 cell surface ligand is from a B7-2 cell surface ligand. In some embodiments, B7-2 cell surface ligand comprises SEQ ID NO: 37. In some embodiments, B7-2 cell surface ligand comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37. In some embodiments, the IL-12 comprises IL-12α, IL-12β, and a transmembrane B7-2 domain. In some embodiments, IL-12α, IL-12β, and the transmembrane B7-2 domain comprises SEQ ID NO: 33. In some embodiments, IL-12α, IL-12β, and the transmembrane B7-2 domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. The transmembrane domain, e.g., transmembrane B7-2, can allow the IL-12 to be tethered to the cell surface. The interleukin may be IL-15. In some embodiments, IL-15 comprises SEQ ID NO: 38. In some embodiments, IL-15 comprises an amino acidWSGR Docket No.53712-720.601 sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38. The IL-15 may further comprise IL-15 receptor alpha (RA). In some embodiments, IL-15 RA comprises SEQ ID NO: 40. In some embodiments, IL-15 RA comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 40. The IL-15 RA may comprise a sushi domain. In some embodiments, the sushi domain comprises SEQ ID NO: 41. In some embodiments, sushi domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41. The sushi domain can allow the IL-15RA to interact with the IL-15. The sushi domain can allow the IL-15RA to bind to the IL-15. The IL-15 RA may comprise a transmembrane domain In some embodiments, the transmembrane domain comprises SEQ ID NO: 42. In some embodiments, transmembrane domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42. The IL-15 RA may comprise a sushi domain and a transmembrane domain. In some embodiments, the sushi domain comprises cysteine residues that form disulfide bonds, such as cysteine residues at position 3, 29, 45, and 63 of SEQ ID NO: 41. The IL-15 and IL-15 RA may be connected. The IL-15 and IL-15 RA may be connected by a linker. In some embodiments, the linker comprises SEQ ID NO: 39. In some embodiments, linker comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39. The polypeptide may comprise the IL-15, the IL-15 RA, and the linker. In some embodiments, the IL-15, the IL-15 RA, and the linker comprises SEQ ID NO: 34. In some embodiments, the IL- 15, the IL-15 RA, and the linker comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. The sequences for these regions may be found in Table 9. Table 9: Amino acid sequences for IL-12 and IL-15 complexesWSGR Docket No.53712-720.601

[0083] The enucleated cell disclosed herein may comprise more than one mRNAs encoding more than one polypeptides. The polypeptides may comprise cytokines. The cytokines may comprise interleukins. The interleukins may comprise IL-12 and IL-15. In some embodiments, IL-12 and IL-15 are expressed on the surface of the enucleated cell. In some embodiments, the polypeptides comprise a cytokine and an immune checkpoint inhibitor. The enucleated cell disclosed herein may comprise an immune checkpoint inhibitor. The immune checkpoint inhibitor may comprise a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. The immune checkpoint inhibitor may be encoded by a second exogenous mRNA in the enucleated cell.WSGR Docket No.53712-720.601 Therapeutic Agent

[0084] In some embodiments, the cells of the present disclosure comprise at least one therapeutic agent. In some embodiments, the cells are enucleated, such as with the methods of enucleation disclosed herein. In some embodiments, the therapeutic agent is or comprises an active agent of the present disclosure. In some embodiments, an active agent comprises at least one of a DNA molecule, a RNA molecule, a protein (e.g., an enzyme, an antibody, an antigen, a toxin, 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, a colchicine, a taxol, a mitomycin, or emtansine), a gene editing factor, a nanoparticle, or another active agent (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses (e.g., oncolytic viruses), exosomes, lipids, or ions). Non-limiting examples of RNA molecules include messenger RNA (mRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA, long non-coding RNA (lncRNA) and a RNA virus. Non-limiting examples of DNA molecules include a single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule and a DNA virus. Non-limiting examples of proteins include a cytokine, a growth factor, a hormone, an antibody or an antigen-binding fragment thereof, a single-domain antibody or antigen binding fragment thereof, a small-peptide based drug, and an enzyme. Non-limiting examples of oncolytic viruses include Talimogene laherparepvec, Onyx-015, GL-ONC1, CV706, Voyager- V1, and HSV-1716. Some wild-type viruses also show oncolytic behavior such as Vaccinia virus, Vesicular stomatitis virus, Poliovirus, Reovirus, Senecavirus, ECHO-7, or Semliki Forest virus.

[0085] In some embodiments, an enucleated cell is engineered to produce (e.g., express, and in some cases have tethered) the therapeutic agent. In some embodiments, the parent cell from which the enucleated cell was obtained may be engineered to produce the therapeutic agent prior to enucleation to produce the enucleated cell. In some embodiments, the enucleated cell is engineered to produce the therapeutic agent after enucleation (in absence of the nucleus). In some embodiments, the therapeutic agent is exogenous to the enucleated cell or parent cell thereof. In some embodiments, the therapeutic agent is endogenous to the enucleated cell or parent cell thereof. In some embodiments, the enucleated cell of the present disclosure comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more therapeutic agents.

[0086] In some embodiments, the therapeutic agent comprises modified version of the DNA molecule, the RNA molecule, the protein, the peptide, the small molecule active agent, and / or the gene-editing factor as compared to a naturally occurring version. In some embodiments, theWSGR Docket No.53712-720.601 therapeutic agent is a corrected, a truncated, or a non-mutated version and / or copy of the DNA molecule, the RNA molecule, the protein, the peptide, the small molecule active agent, and / or the gene-editing factor. For example, the therapeutic agent can correct a mutated tumor protein p53 (p53) or epidermal growth factor receptor (EGFR) in the target cell or target environment as part of the treatment for lung cancer.

[0087] The therapeutic agent may be coupled to the surface of the enucleated cell. The therapeutic agent may be, or include, a targeting moiety described herein. Non-limiting example of the targeting moieties that may be produced by or contained in an enucleated cell includes chemokine receptors, adhesion molecules, and antigen-binding polypeptides (e.g., single-domain antibodies and antigen-binding fragments thereof), or a portion thereof. In some embodiments, the therapeutic agent may be, or include, a transmembrane moiety described herein. In some embodiments, the therapeutic agent is covalently coupled to the enucleated cell surface. In some embodiments, the therapeutic agent is non-covalently coupled. In some embodiments, the amount of therapeutic agent is about 0.10 to about 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or a nucleated parent cell. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 0.50 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 1.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 2.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 3.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 0.10 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 0.10 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 1.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 2.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 3.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 0.50 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 2.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 3.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 1.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 1.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 3.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 4.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 2.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 2.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 2.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 2.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 3.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 3.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 4.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 5.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 5.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 10.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 15.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 20.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 25.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 25.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 25.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 30.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 30.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 35.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 40.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 45.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 150.00.In some embodiments, the amount of the therapeutic agent is about 50.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 115.00 fold increaseWSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 150.00.In some embodiments, the amount of the therapeutic agent is about 55.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 55.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 60.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 70.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In someWSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 75.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 135.00.In some embodiments, the amount of the therapeutic agent is about 80.00 to about 140.00.In some embodiments, the amount of the therapeutic agent is about 80.00 to about 145.00.In some embodiments, the amount of the therapeutic agent is about 80.00 to about 150.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 90.00 fold increaseWSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 125.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 130.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 135.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 140.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 145.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 145.00 fold increaseWSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 145.00 fold increaseWSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 130.00 fold increaseWSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 115.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 135.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 135.00 to about 145.00 fold increaseWSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 135.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 140.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 140.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 145.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or a nucleated parent cell. In some embodiments, the amount of the therapeutic agent is greater than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or nucleated parent cell. In some embodiments, the amount of the therapeutic agent is less than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or nucleated parent cell In some embodiments, the amount of the therapeutic agent and the amount of the comparable endogenous therapeutic agent (if any) is determined by flow cytometry (FACS).

[0088] In some embodiments, the therapeutic agent is recombinantly expressed by the enucleated cell or 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, 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 theWSGR Docket No.53712-720.601 enucleated cell to recombinantly express the therapeutic agent. In some embodiments, the parent cell is engineered to recombinantly express the therapeutic agent prior to enucleation.

[0089] In some embodiments, the therapeutic agent is not naturally expressed (e.g., in the absence of engineering) in the cell from which the enucleated cell was 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 therapy (e.g., a tumor, or a particular tissue such as the brain, the intestine, the lungs, the heart, the liver, the spleen, the pancreas, muscles, eyes, and the like) (e.g., the therapeutic agent is exogenous to the intended site of therapy). In some embodiments, the level of the therapeutic agent is not naturally occurring in the enucleated cell of the parent cell, such as over expression or under expression of the therapeutic agent.

[0090] 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 prior to or after enucleation of the cell. Alternatively, the therapeutic agent may be synthesized by the cell.

[0091] In some embodiments, the enucleated cell comprises a plurality of therapeutic agents. In some embodiments, the enucleated cell comprises at least 1 (e.g., at least 1, 2, 3, 4, 5, or more) different DNA molecules, RNA molecules, proteins, peptides, small molecule active agents, or gene-editing factors, in any combination. For example, in some embodiments, a therapeutic agent comprises a 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.

[0092] In some embodiments, the therapeutic agent comprises a polypeptide. In some embodiments, the polypeptide is exogenous. In some embodiments, the polypeptide is encoded by an exogenous polynucleotide delivered into the parent cell or the enucleated cell. In some embodiments, the polypeptide is synthesized by at least one intracellular organelle of the enucleated cell. In some embodiments, the polypeptide is tethered to the enucleated cell. In some embodiments, the polypeptide is expressed on the cell surface or the enucleated cell. In some embodiments, the enucleated cell expresses the polypeptide in a target environment. In some embodiments, the target environment is a microenvironment. In some embodiments, the microenvironment is a tumor microenvironment. In some embodiments, the enucleated cell expresses the polypeptide at a target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell expresses the cancer biomarker of any cancer describedWSGR Docket No.53712-720.601 herein. In some embodiments, the target cell is an endothelial cell. In some embodiments, the endothelial cell expresses an endothelial biomarker described herein. In some embodiments, the endothelial cell is a blood vessel cell. In some embodiments, the endothelial cell is a lymphatic vessel cell.

[0093] In some embodiments, the exogenous polypeptide comprises a cytokine. An “exogenous” polypeptide provided herein may refer to a presence of the polypeptide that is exogenous to the cell or enucleated cell, or an amount or level of expression of the polypeptide that exogenous to the cell or enucleated cell. In some embodiments, the cytokine comprises a membrane binding domain. In some embodiments, the exogenous polypeptide comprises a soluble cytokine. For example, the exogenous polypeptide can comprise an extracellular domain or fragment of the cytokine. In some embodiments, the exogenous polypeptide comprises a solubility as determined by turbidimetric solubility assay or thermodynamic solubility assay by dissolving the exogenous polypeptide in solvent such as organic solvent, including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc., or 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. In some embodiments, the cytokine comprises an interleukin. In some embodiments, the cytokine comprises chemokines. In some embodiments, the cytokine comprises interferons. In some embodiments, the cytokine comprises a lymphokine. In some embodiments, the cytokine comprises a tumor necrosis factor. In some embodiments, the cytokine comprises a monokine. In some embodiments, the cytokine comprises a colony-stimulating factor. In some embodiments, the cytokine comprises a transforming growth factor. Non-limiting examples of cytokines include bFGF, TNF-α, IL-10, IL-12(p70), IL-1β, IL-2, IL-6, GM-CSF, IL-13, IFN-γ, TGF-β1, TGF-02, TGF-β3, and IL-15.

[0094] In some embodiments, the exogenous polypeptide comprises an interleukin. In some embodiments, the exogenous polypeptide comprises more than one interleukin. Non-limiting examples of interleukins include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36. Thus, in certain embodiments, a cytokine of the composition is an interleukin or a mutant thereof, including, but not limited to, wild-type and mutant forms of IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8. (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, Il29, IL30, IL31, IL32, IL33, IL35, or IL36.WSGR Docket No.53712-720.601

[0095] In some embodiments, the cytokine comprises IL-12. In some embodiments, the IL-12 comprises an IL-12 complex. In some embodiments, the IL-12 complex comprises SEQ ID NO: 33. In some embodiments, the IL-12 complex comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the IL*12 complex comprises IL-12α, IL-12β, and a transmembrane domain. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 33. In some embodiments, the exogenous polypeptide comprises IL-12α or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 35. In some embodiments, the exogenous polypeptide comprises IL-12β or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 36. In some embodiments, the exogenous polypeptide comprises a transmembrane region or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 37.

[0096] In some embodiments, the cytokine comprises IL-15. In some embodiments, the IL-15 is an IL-15 complex. In some embodiments, the IL-15 complex comprises SEQ ID NO: 34. In some embodiments, the IL-15 complex comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments, the IL-15 complex is formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor α (SuIL-15 RA). In some embodiments, the complex is a fusion protein, e.g., with IgG4 Fc. In some embodiments, the IL-15 complex comprises IL-15 RA with a sushi domain and a transmembrane domain connected to IL-15 by a linker. In some embodiments, the cytokine comprises IL-15 and IL-12 in the same enucleated cell.

[0097] In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 34. In some embodiments, the exogenous polypeptide comprises IL-15 or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 38. In some embodiments, the exogenous polypeptide comprises a linker. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 39. In some embodiments, the exogenousWSGR Docket No.53712-720.601 polypeptide comprises IL-15 RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 40. In some embodiments, the exogenous polypeptide comprises a sushi domain of IL-15 RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 41. In some embodiments, the exogenous polypeptide comprises a transmembrane domain of IL-15 RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 42. In some embodiments, the exogenous polypeptide comprises a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof. Non-limiting examples of the TNF superfamily member polypeptide 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 (4-1 BBL), TNF-related apoptosis-inducing ligand (CD253 or APO-2L), Receptor activator of nuclear factor kappa-Β ligand (CD254, OPGL, TRANCE, or ODF), TNF-related weak inducer of apoptosis (APO-3L or DR3L), a 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).

[0098] In some embodiments, the therapeutic agent comprises any one of the immune checkpoint molecule described herein or an immune checkpoint molecule inhibitor for inhibiting any one of the immune checkpoint molecule described herein. Non-limiting examples of the immune checkpoint molecule include PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), 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, CSF-1R, IL-3, IL-1RAP, IL-8, SEMA4D, Angiopoietin-2, CLEVER-1, Axl, phosphatidylserine or a fragment thereof.

[0099] In some embodiments, the therapeutic agent comprises an antibody. In some embodiments, the antibody is a single-domain antibody described herein. In some embodiments, the antibody or the single-domain antibody binds to an immune checkpoint molecule. In some embodiments, the single-domain antibody bind to, and modulates the expression or the activity of the immune checkpoint molecule. In some embodiments, the single-domain antibody is an inhibitor of the activity or expression of the immune checkpoint molecule. In someWSGR Docket No.53712-720.601 embodiments, the single-domain antibody is an activator of the activity or expression of the immune checkpoint molecule. In some embodiments, the antibody or single-domain antibody binds to PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), 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, CSF-1R, IL-3, IL-1RAP, IL-8, SEMA4D, Angiopoietin-2, CLEVER-1, Axl, phosphatidylserine or a fragment thereof. In some embodiments, the therapeutic agent comprising the antibody or the single-domain antibody binds to PD-L1. In some embodiments, the therapeutic agent comprising the antibody or the single-domain antibody binds to CTLA-4. Targeting moiety

[0100] Described herein, in some embodiments, are cell or enucleated cells comprising a targeting moiety. In some embodiments, the cells are enucleated, such as with the methods of enucleation disclosed herein. The targeting moiety described herein is designed to guide the enucleated cell to a target cell or target environment (e.g., tissue) in a subject following 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. The enucleated cells comprising the targeting moiety localizes at the target cell or target environment in a subject with a 2 fold, 5 fold, 10 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1,000 fold, 5,000 fold, or 10,000 fold increase as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 2 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 5 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 10 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 20 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 50 fold increased as compared toWSGR Docket No.53712-720.601 localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 5% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 10% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 20% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 30% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 40% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 50% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 60% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 70% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 80% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 90% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 100% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety.

[0101] 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-WSGR Docket No.53712-720.601 binding fragment for targeting a chemokine receptor or a chemokine ligand, or portion thereof, involved in chemokine signaling. In some embodiments, the exogenous antibody is an exogenous single-domain antibody or fragment thereof.

[0102] In some embodiments, the targeting moiety targets the biomarker expressed by, or associated with, a target cell or with a microenvironment. In some embodiments, the biomarker may be released by the target cell. The biomarker may indicate the presence of the disease or the condition. In some embodiments, the biomarker is expressed by immune cells responding to the target cell or the microenvironment associated with the disease or the condition. In some embodiments, the biomarker may be an epitope or antigen. In some embodiments, the biomarker comprising the epitope may be bound by an antibody that is different from the antibody or the antigen-binding fragment thereof that confers therapeutic property (e.g., the therapeutic agent).

[0103] In some embodiments, the targeting moiety targets a biomarker expressed or released by a cancer cell that has metastasized. For example, the cancer cell may arise from one tissue and subsequently metastasizes to a different location. In some embodiments, the targeting moiety targets a biomarker expressed or released by an endothelial cell. In some embodiments, the endothelial cells are a blood vessel cell. In some embodiments, the 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.

[0104] In some embodiments, the enucleated cells may further include (e.g., by engineering or from the cell from which they were obtained) a surface marker that aids in their evasion of the subject immune system. For example, in some embodiments, the enucleated cells may include a CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof. Without being bound by any particular theory, it is believed that a CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof helps to 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 immunomodulatory peptides, polyamines, and ATP. In some embodiments, the therapeutic molecules can be recognized by immune cells and can induce immune response. For example, the therapeutic molecules can be 4-1BB or any one of the cytokine described herein to induce immune response. In some embodiments, the enucleated cell is depleted of an immune recognition molecule. Non-limiting examples of immune recognition molecules include an HLA antigen, a proteoglycan, a sugar moiety, and an embryonic antigen.WSGR Docket No.53712-720.601 Transmembrane moiety

[0105] Described herein, in some embodiments, are cells or compositions comprising the enucleated cell comprising at least one transmembrane moiety. In some embodiments, the cells are enucleated, such as with the methods of enucleation disclosed herein. In some embodiments, transmembrane moiety is coupled to a polypeptide. In some embodiments, the polypeptide is a cytokine. In some embodiments, the polypeptide is an interleukin. In some embodiments, the polypeptide is a single-domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed here, or a combination thereof. In some embodiments, the transmembrane moiety is coupled by way of a covalent bond. In some embodiments, the transmembrane moiety is a fusion protein comprising the single-domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed here, or a combination thereof. In some embodiments, the exogenous polypeptide is complexed to the transmembrane moiety. In some embodiments, the transmembrane moiety comprises a full length protein or a variation thereof or a fragment thereof. In some embodiments, the transmembrane moiety is endogenous to the parent cell that is being enucleated for obtaining the enucleated cell. In some embodiments, the transmembrane moiety may be an exogenous transmembrane moiety to the parent cell or to the enucleated cell. In some embodiments, the transmembrane moiety comprises a single transmembrane α-helix (bitopic membrane protein. The transmembrane moiety comprises a polytopic transmembrane α- helical protein. In some embodiments, the transmembrane moiety comprises a polytopic transmembrane β-sheet protein. In some embodiments, the transmembrane moiety comprises a Type I, II, III, or IV transmembrane protein. Non-limiting examples of transmembrane protein may include CD4, CD14, glycophorin a (GPA), or any combination of integrins.

[0106] In some embodiments, the transmembrane moiety is added to the exogenous polypeptide by way of a modification. For example, a transmembrane moiety may be added to the N 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 modifications that are made to the exogenous polypeptide to add the transmembrane moiety may include adding an anchor molecule. Anchor molecule can be any molecule (e.g., a glycolipid) that can be inserted and remain in the cellular membrane. In some embodiments, the anchor molecules comprises a glycosylphosphatidylinositol, a farnesyl, a palmitate, a myristate, or a combination thereof.

[0107] In some embodiments, the enucleated cell comprises a cytokine. In some embodiments, the cytokine comprises an interleukin. In some embodiments, the cytokine comprises an IL-12 (interleukin 12). In some embodiments, the cytokine comprises an IL-15 (interleukin 15). In some embodiments, the exogenous cytokine or fragment thereof is complexed with a transmembrane moiety. In some embodiments, the transmembrane moiety comprises aWSGR Docket No.53712-720.601 transmembrane polypeptide. In some embodiments, the exogenous cytokine or fragment thereof is complexed with N-terminus of the transmembrane polypeptide. In some embodiments, the exogenous cytokine or fragment thereof is complexed with C-terminus of the transmembrane polypeptide. In some embodiments, the exogenous cytokine or fragment thereof comprises a modification relative to an otherwise identical reference cytokine or fragment thereof, wherein the modification anchors the exogenous cytokine or fragment thereof to an exoplasmic or a cytosolic side of a cell membrane of the enucleated cell. In some embodiments, the modification comprises complexing the exogenous single-domain antibody or fragment thereof to glycosylphosphatidylinositol, famesyl, palmitate, myristate, or a combination thereof.

[0108] In some embodiments, the transmembrane domain comprises IL-12. In some embodiments, IL-12 is coupled to a cell surface protein ligand. In some embodiments, the cell surface protein ligand is from the B7 family of cell surface ligand proteins. Non-limiting examples of B7 family members includes but is not limited to B7.1, B7.2, B7-H4, and B7-H3. In some embodiments, Il-12 is coupled to a portion of a cell surface protein ligand. In some embodiments, IL-12 is coupled to only a transmembrane portion of a cell surface protein ligand. In some embodiments, IL-12 is coupled to the transmembrane portion of a B7 family member. In some embodiments, IL-12 is coupled to the transmembrane portion of B7.1. As depicted in FIG.8A, the transmembrane (TM) region of B7.1 (B7) is linked to IL-12. B7TM is depicted across cell membrane of the enucleated cell and the IL-12 portion is linked to the B7TM on the exoplasmic side of the cell membrane of the enucleated cell. In some embodiments, IL-12 comprises IL-12α. In some embodiments, IL-12 comprises IL-12β. In some embodiments, IL-12 comprises IL-12α and IL-12β. In some embodiments, IL-12α comprises a signal peptide and an IL-12 subunit alpha chain. In some embodiments, IL-12α comprises an IL-12 subunit alpha chain. In some embodiments, the IL-12 subunit alpha chain comprises a disulfide linkage. In some embodiments, the IL-12 subunit alpha chain comprises more than one disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises less than five disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises four disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises three disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises a glycosylation modification. In some embodiments, the IL-12 subunit alpha chain comprises more than one glycosylation modification. In some embodiments, the glycosylation modification is N-linked. In some embodiments, the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue. In some embodiments, the asparagine residue is located near the center of the amino acid sequence of the IL-12 subunit alpha chain. In some embodiments, the IL-12 subunit alpha chain has a disulfide bond thatWSGR Docket No.53712-720.601 connects to an IL-12 subunit beta chain. In some embodiments, IL-12β comprises a signal peptide and an interleukin-12 subunit beta. In some embodiments, IL-12β comprises an interleukin-12 subunit beta. In some embodiments, the IL-12 subunit beta chain comprises a disulfide linkage. In some embodiments, the IL-12 subunit beta chain comprises more than one disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises less than five disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises four disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises three disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises two disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises a glycosylation modification. In some embodiments, the IL-12 subunit beta chain comprises more than one glycosylation modification. In some embodiments, the glycosylation modification is N-linked. In some embodiments, the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue. In some embodiments, the glycosylation modification is C-linked. In some embodiments, the glycosylation modification is C- mannosylation. In some embodiments, the glycosylation modification takes place on a tryptophan residue.

[0109] In some embodiments, the transmembrane domain comprises IL-15. In some embodiments, IL-15 may be coupled to a transmembrane domain through a linker. In some embodiments, the transmembrane domain comprises IL-15 receptor-alpha (IL-15 Rα). In some embodiments, IL-15 may be coupled to IL-15 Rα to the exoplasmic side of the cell membrane of the enucleated cell. As depicted in FIG.10A, IL-15 Rα spans the cell membrane of an enucleated cell. The portion of IL-15 Rα on the exoplasmic surface is coupled to IL-15 by a linker. On the opposite end of IL-15 from the linker connecting to IL-15 Rα is a signal peptide. In some embodiments, IL-15 Rα comprises a Sushi domain and a transmembrane region. In some embodiments, the IL-15 Rα comprises a Sushi domain, linker, proline / threonine-rich region, a transmembrane region, and a cytoplasmic domain. In some embodiments, IL-15 Rα comprises the Sushi domain, a transmembrane region and any combination of a linker, proline / threonine-rich region, or a cytoplasmic domain. In some embodiments, the Sushi domain comprises two disulfide linkages. In some embodiments, the Sushi domain may bind to IL-15. In some embodiments, IL-15 Rα comprises a glycosylation modification. In some embodiments, the Sushi domain comprises a glycosylation modification. In some embodiments, the glycosylation modification is N-linked. In some embodiments, the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue. In some embodiments, IL-15 comprises a disulfide linkage. In some embodiments, IL-15 comprises two disulfide linkages. In some embodiments, IL-15 comprises aWSGR Docket No.53712-720.601 glycosylation modification. In some embodiments, IL-15 comprises two glycosylation modifications. In some embodiments, IL-15 comprises three glycosylation modifications. In some embodiments, IL-15 comprises one or more glycosylation modifications. In some embodiments, the glycosylation modification is N-linked. In some embodiments, the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue.

[0110] The enucleated cells described herein may be engineered to express one or more targeting moieties that, when expressed by the enucleated cell (e.g., on its surface), guide the enucleated cell to the target cell or tissue in vivo. In order to avoid unintended clearance of the enucleated cells in vivo, the enucleated cell may also include an immune system evading moiety (a “don't eat me” signaling polypeptide) such as CD47, PD-L1, HLA-E, or HLA-G. In some embodiments, the enucleated cell described herein comprises an additional active agent such as a therapeutic agent. In some embodiments, the enucleated cells may be engineered before or after enucleation to express targeting moieties (e.g., homing receptors), immune-evading moieties (e.g., “don’t eat me” signaling peptides), among other biomolecules sufficient to target the enucleated cell to a target tissue without risk of clearance by the immune system before they get there. The enucleated cells may be cryopreserved, cryo-hibernated, or cryodesiccated, and stored for long periods of time with their biological activity slowed or stopped. In some embodiments, the enucleated cells may be engineered with immune evading moieties (e.g., CD34+) to avoid an antigenic response in the host. Enucleated cells may also be engineered to express cell-surface receptors (e.g, adhesion molecules, chemokine receptors) used for cellular homing, chemokine sensing, and other biological functions that are essential to targeting damaged tissue. An immune-evading moiety may comprise a signaling peptide, or portion thereof, that reduces cellular phagocytosis through its interaction with a signal receptor protein expressed by phagocytic cells such as macrophages and dendritic cells. In some embodiments, the immune-evading moiety blocks immune cell recognition or immune cell activation.

[0111] The compositions, methods and systems of the present disclosure utilize an enucleated cell platform developed by the inventors of the present disclosure and initial described in United States Patent Application No.10,927,349, which is hereby incorporated by reference in its entirety. In addition, additional utility and advantages of the enucleated cells disclosed herein are discussed in U.S. Patent Application No.18 / 176,259, filed February 28, 2023; U.S. Patent Application No.17 / 885,867, filed August 11, 2022; and U.S. Patent Application No. 18 / 190,838, filed March 27, 2023, each of which is hereby incorporated by reference in its entirety. Nucleic AcidsWSGR Docket No.53712-720.601

[0112] Disclosed herein, in some embodiments, are nucleic acids encoding or comprising a biomolecule (e.g., the therapeutic agent, transmembrane moiety, immune-evading moiety, and / or targeting moiety described herein) of the present disclosure. In some embodiments, the nucleic acid may be introduced into the parent cell or the enucleated cell. Non-limiting examples for introducing a biomolecule into the parent cell or the enucleated cell include: liposome mediated transfer, an adenovirus, an adeno-associated virus, a herpes virus, a retroviral based vector, a lentiviral vector, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetofection, nanoparticle transfection, chemicalporation, or combinations thereof. In some embodiments of any of the compositions and methods provided herein, a therapeutic agent, a virus, an antibody, or a nanoparticle may be introduced into the enucleated cells.

[0113] In some embodiments, the nucleated cell is transfected with a vector (e.g., a viral vector (e.g., a retrovirus vector (e.g., a lentivirus vector), an adeno-associated virus (AAV) vector, a vesicular virus vector (e.g., vesicular stomatitis virus (VSV) vector), or a hybrid virus vector), a plasmid) before the nucleated cell is enucleated using any of the enucleation techniques described herein.

[0114] In some embodiments, IL-12 is encoded in a vector. In some embodiments, IL-12 and a transmembrane region are encoded in a vector. In some embodiments, IL-12, a transmembrane region, and a signal peptide are encoded in a vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a mammalian expression vector. As depicted in FIG.8B, IL-12 and the transmembrane region of B7 (B7TM) may be inserted in a vector such as the vector depicted (vector ID: VB190513-1151unt). In this depicted embodiment, the IL-12 B7TM coding region is preceded by a human eukaryotic translation elongation factor 1 α1 promoter (EF1A) and a signal peptide or Kozak. In some embodiments, the IL-12 B7TM coding region comprises SEQ ID NO: 10. In some embodiments, the IL-12 B7TM coding region comprises a nucleotide sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.

[0115] In some embodiments, IL-15 is encoded by a vector. In some embodiments, IL-15 and IL-15 Rα are encoded by a vector. In some embodiments, IL-15, IL-15 Rα, and a linker are encoded by a vector. In some embodiments, IL-15, IL-15 Rα, a linker, and a signal peptide are encoded by a vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a mammalian expression vector. As depicted in FIG.10B, IL-15 and IL-15 Rα may be inserted in a vector such as the vector depicted (vector ID: VB190220-WSGR Docket No.53712-720.601 1118ayw). In this depicted embodiment, the IL-15 and IL-15 Rα coding region is preceded by a human eukaryotic translation elongation factor 1 α1 promoter (EF1A) and a signal peptide or Kozak. In some embodiments, the IL-15 and IL-15 Rα coding region comprises SEQ ID NO: 25. In some embodiments, the IL-15 and IL-15 Rα coding region comprises a nucleotide sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25. Pharmaceutical Formulations

[0116] Described herein are pharmaceutical formulations comprising the enucleated cells or the compositions described herein. In some embodiments, the pharmaceutical formulations further comprise a pharmaceutically acceptable: carrier, excipient, diluent, or nebulized inhalant.

[0117] In some embodiments, the pharmaceutical formulations include two or more active agents, or two or more therapeutic agents as disclosed herein. In some embodiments, the two or more active agents are contained in a single dosage unit such as, for example, when the enucleated cell comprises two or more therapeutic agents. In embodiments, the two or more active agents are contained in separate dosage units such as when the enucleated cell is administered separately from an additional therapeutic agent or adjuvant. In some embod...

Claims

WSGR Docket No.53712-720.601 CLAIMS WHAT IS CLAIMED IS:

1. A method for treating a disease or condition in a subject, the method comprising: administering to the subject an enucleated cell, wherein the enucleated cell comprises (a) an exogenous messenger RNA (mRNA) encoding an interleukin 12 (IL-12) polypeptide; and (b) one or more organelles for expressing the IL-12 polypeptide from the exogenous mRNA, wherein the IL-12 polypeptide is expressed by the enucleated cell on a cell surface of the enucleated cell for at least 24 hours in vivo.

2. The method of claim 1, wherein the IL-12 polypeptide comprises a membrane-bound IL-12 polypeptide.

3. The method of claim 1, wherein the IL-12 polypeptide is complexed to the cell surface of the enucleated cell.

4. The method of any one of claims 1-3, wherein the enucleated cell induces an endogenous immune response at a site associated with the disease or condition of the subject.

5. The method of any one of claims 1-3, further comprising treating the disease or condition of the subject, at least in part, by inducing an endogenous immune response in the subject.

6. The method of claim 4, wherein the site associated with the disease or condition comprises a microenvironment.

7. The method of claim 6, wherein the microenvironment is a tumor microenvironment.

8. The method of claim 7, wherein the IL-12 polypeptide is not delivered to the microenvironment.

9. The method of claim 4, wherein the site associated with the disease or condition comprises a lymphatic microenvironment, a hepatic microenvironment, a spleen microenvironment, a pancreatic microenvironment, an epidermis microenvironment, or a combination thereof.

10. The method of claim 4, wherein the site associated with the disease or condition comprises a cell associated with the disease or condition.

11. The method of claim 10, wherein the cell associated with the disease or condition is a cancer cell.

12. The method of claim 10, wherein the cell associated with the disease or condition is an immune cell.

13. The method of claim 12, wherein the immune cell is a leukocyte.

14. The method of claim 10, wherein the IL-12 polypeptide is not delivered to the cell associated with the disease or condition.

15. The method of any one of claims 1-12, wherein the disease or condition comprises cancer.WSGR Docket No.53712-720.601 16. The method of any one of claims 1-15, wherein the IL-12 polypeptide is covalently connected to the cell surface of the enucleated cell for at least 24 hours.

17. The method of any one of claims 1-16, wherein the IL-12 polypeptide is complexed with the enucleated cell for the at least 24 hours.

18. The method of any one of claims 1-17, wherein the IL-12 polypeptide is not secreted by the enucleated cell.

19. The method of any one of claims 1-18, wherein the IL-12 polypeptide encoded by the exogenous mRNA results in a decreased toxicity in the subject compared to a toxicity caused by direct administration of an IL-12 therapy.

20. The method of any one of claims 1-19, wherein the administering further comprises administering to the subject a plurality of the enucleated cell formulated in a pharmaceutical formulation, wherein the pharmaceutical formulation comprises (a) the plurality of the enucleated cell, and (b) a pharmaceutically acceptable: excipient, diluent, or a combination thereof.

21. The method of any one of claims 1-20, wherein the IL-12 polypeptide comprises IL-12 or antigen-binding fragment of the IL-12.

22. A method for inducing endogenous immune response in a subject, the method comprising: administering to the subject an enucleated cell comprising (a) an exogenous mRNA encoding a cytokine polypeptide; and (b) one or more organelles for expressing the cytokine polypeptide on a cell surface of the enucleated cell, wherein the cytokine polypeptide induces the endogenous immune response in the subject.

23. The method of claim 22, wherein the cytokine polypeptide comprises a membrane-bound cytokine polypeptide.

24. The method of claim 22, wherein the cytokine polypeptide is complexed to the cell surface of the enucleated cell.

25. The method of any one of claims 22-24, wherein the enucleated cell induces the endogenous immune response at a microenvironment.

26. The method of claim 25, wherein the microenvironment is a tumor microenvironment.

27. The method of claim 25, wherein the microenvironment is a lymphatic microenvironment, a hepatic microenvironment, a spleen microenvironment, a pancreatic microenvironment, an epidermis microenvironment, or a combination thereof.

28. The method of any one of claims 22-24, wherein the enucleated cell induces the endogenous immune response in proximity of a cell.

29. The method of claim 28, wherein the cell is a cancer cell.

30. The method of claim 28, wherein the cell is an immune cell.WSGR Docket No.53712-720.601 31. The method of claim 30, wherein the immune cell is a leukocyte.

32. The method of any one of claims 22-31, wherein the cytokine polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours.

33. The method of claim 32, wherein the cytokine polypeptide is complexed with the enucleated cell for the at least 24 hours.

34. The method of any one of claims 22-33, wherein the cytokine polypeptide is not secreted by the enucleated cell.

35. The method of claim 25, wherein the cytokine polypeptide is not delivered to the microenvironment.

36. The method of claim 28, wherein the cytokine polypeptide is not delivered to the cell.

37. The method of any one of claims 22-36, wherein the cytokine polypeptide comprises a full- length cytokine or antigen binding fragment thereof of the cytokine.

38. The method of any one of claims 22-37, wherein the cytokine polypeptide encoded by the exogenous mRNA results in a decreased toxicity in the subject compared to a toxicity caused by direct delivery of the cytokine polypeptide.

39. The method of any one of claims 22-38, wherein the cytokine polypeptide comprises an interleukin polypeptide.

40. The method of claim 39, wherein the interleukin polypeptide is an IL-12 polypeptide.

41. The method of claim 37, wherein the cytokine polypeptide comprises an interleukin antibody or antigen binding fragment thereof.

42. The method of claim 41, wherein the cytokine polypeptide comprises IL-12 or antigen binding fragment thereof.

43. The method of any one of claims 1-42, wherein the endogenous immune response comprises an innate immune response or adaptive immune response.

44. The method of any one of claims 1-43, wherein the enucleated cell further comprises an immune checkpoint inhibitor.

45. The method of claim 44, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.

46. The method of claim 43, wherein the immune checkpoint inhibitor comprises the PD-1 inhibitor.

47. The method of any one of claims 44-46, wherein the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell.

48. The method of any one of claims 1-47, wherein the enucleated cell is depleted of an immune recognition molecule.WSGR Docket No.53712-720.601 49. The method of claim 48, wherein the immune recognition molecule comprises HLA antigen, proteoglycan, sugar moiety, embryonic antigen, or any combination thereof.

50. The method of any one of claims 1-49, wherein the enucleated cell comprises a targeting moiety.

51. The method of claim 50, wherein targeting moiety comprises CXCR2, CCR2, PSGL-1, or a combination thereof.

52. A composition, comprising: an enucleated cell comprising: an exogenous mRNA that encodes a cytokine polypeptide; and organelles for expressing the cytokine polypeptide, wherein the cytokine is expressed on a cell surface of the enucleated cell.

53. The composition of claim 52, wherein the enucleated cell further comprises a transmembrane domain coupled to the cytokine polypeptide.

54. The composition of claim 53, wherein the transmembrane and the cytokine polypeptides are covalently connected.

55. The composition of claim 52, wherein the cytokine polypeptide is expressed extracellularly on the cell surface of the enucleated cell.

56. The composition of claim 52, wherein the cytokine polypeptide is complexed to a cell surface of the enucleated cell.

57. The composition of any one of claims 52-56, wherein the cytokine polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours.

58. The composition of any one of claims 52-57, wherein the enucleated cell expresses an abundance of the cytokine polypeptide that is at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 1.0 fold, at least 2.0 fold, or at least 5.0 fold higher than the enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.

59. The composition of claim 58, wherein the abundance of the cytokine polypeptide and the abundance of the comparable naturally-occurring cytokine polypeptide is determined by FACS.

60. The composition of any one of claims 52-59, wherein the cytokine polypeptide is not secreted by the enucleated cell.

61. The composition of any one of claims 52-60, wherein the cytokine polypeptide comprises an interleukin polypeptide.

62. The composition of claim 58 wherein the interleukin polypeptide is IL-12 polypeptide.

63. The composition of any one of claims 52-62, wherein the enucleated cell further comprises an immune checkpoint inhibitor.WSGR Docket No.53712-720.601 64. The composition of claim 63, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.

65. The composition of claim 64, wherein the immune checkpoint inhibitor comprises the PD-1 inhibitor.

66. The composition of any one of claims 63-65, wherein the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell.

67. The composition of any one of claims 52-66, wherein the enucleated cell is depleted of an immune recognition molecule.

68. The composition of claim 67, wherein the immune recognition molecule comprises HLA antigen, proteoglycan, sugar moiety, embryonic antigen, or any combination thereof.

69. The composition of any one of claims 52-68, wherein the enucleated cell comprises a targeting moiety.

70. The composition of claim 69, wherein the targeting moiety comprises CXCR2, CCR2, PSGL-1, or a combination thereof.

71. The composition of claim 52, further comprising an immune checkpoint inhibitor.

72. The composition of claim 71, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.

73. The composition of claim 72, wherein the immune checkpoint inhibitor comprises the PD-1 inhibitor.

74. The composition of any one of claims 70-73, wherein the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell.

75. The composition of any one of claims 70-74, wherein the immune checkpoint inhibitor is encoded by a second enucleated cell.

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