Compositions and methods for treating cancer

Abstract

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

Description

[Background technology]

[0001] cross reference This application claims the benefit of U.S. Provisional Application Serial No. 63 / 477,464, filed December 28, 2022, and U.S. Provisional Application Serial No. 63 / 591,857, filed October 20, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under CA250887-01 awarded by the National Institutes of Health. The government has certain rights in this invention.

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

[0004] The systemic spread of tumor cells and the development of metastases are fundamental causes of cancer death, yet few effective therapies specifically target metastases. Furthermore, many patients already have undetectable micrometastases at the time of diagnosis. Furthermore, many tumors are not effectively controlled by systemically administered therapeutic agents, which function suboptimally in the tumor microenvironment (TME). To overcome these challenges, effective anti-metastatic therapies are needed to deliver therapeutic agents to the anatomical niche(s) where metastatic tumor cells can grow. Summary of the Invention

[0005] In some aspects, described herein are methods for treating a disease or condition in a subject, the methods comprising administering to the subject enucleated cells comprising: (a) 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 cells on their cell surface in vivo for at least 24 hours. 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 cells. In some embodiments, the enucleated cells induce an endogenous immune response at a site associated with the disease or condition in the subject. In some embodiments, the method further comprises treating the disease or condition in the subject, at least in part, by inducing an endogenous immune response in the subject. In some embodiments, the site associated with a 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 a disease or condition comprises a lymphatic microenvironment, a liver microenvironment, a splenic microenvironment, a pancreatic microenvironment, an epidermal microenvironment, or a combination thereof. In some embodiments, the site associated with a 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 a 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 attached to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, the IL-12 polypeptide is complexed to the enucleated cell for at least 24 hours.In some embodiments, the IL-12 polypeptide is not secreted by the enucleated cells. In some embodiments, the IL-12 polypeptide encoded by the exogenous mRNA results in reduced toxicity in the subject compared to toxicity caused by direct administration of IL-12 therapy. In some embodiments, the administering further comprises administering to the subject a plurality of enucleated cells formulated in a pharmaceutical formulation, the pharmaceutical formulation comprising (a) the plurality of enucleated cells and (b) a pharmaceutically acceptable excipient, diluent, or combination thereof. In some embodiments, the IL-12 polypeptide comprises IL-12 or an antigen-binding fragment of IL-12.

[0006] Described herein, in some aspects, are methods of inducing an endogenous immune response in a subject, the methods comprising administering to the subject enucleated cells comprising: (a) exogenous mRNA encoding a cytokine polypeptide; and (b) one or more organelles for expressing the cytokine polypeptide on the cell surface of the enucleated cell, wherein the cytokine polypeptide induces an 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 an endogenous immune response in a microenvironment. In some embodiments, the microenvironment is a tumor microenvironment. In some embodiments, the microenvironment is a lymphatic microenvironment, a liver microenvironment, a spleen microenvironment, a pancreatic microenvironment, an epidermal microenvironment, or a combination thereof. In some embodiments, the enucleated cell induces an endogenous immune response in the vicinity of the 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 the cell surface of the enucleated cell for at least 24 hours. In some embodiments, the cytokine polypeptide is complexed with the enucleated cell for 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 an antigen-binding fragment thereof. In some embodiments, the cytokine polypeptide encoded by exogenous mRNA results in reduced toxicity in a subject compared to 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 an antigen-binding fragment thereof. In some embodiments, the cytokine polypeptide comprises IL-12 or an antigen-binding fragment thereof. In some embodiments, the endogenous immune response comprises an innate immune response or an adaptive immune response. In some embodiments, the enucleated cells further comprise 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 CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cells. In some embodiments, the enucleated cells are depleted of immune recognition molecules. In some embodiments, the immune recognition molecules comprise an HLA antigen, a proteoglycan, a sugar moiety, a fetal 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.

[0007] Described herein, in some aspects, are compositions comprising an enucleated cell comprising exogenous mRNA encoding a cytokine polypeptide and an organelle for expressing the cytokine polypeptide, wherein the cytokine is expressed on the cell surface of the enucleated cell. In some embodiments, the enucleated cell further comprises a transmembrane domain linked to the cytokine polypeptide. In some embodiments, the transmembrane and cytokine polypeptide are covalently linked. In some embodiments, the cytokine polypeptide is extracellularly expressed on the cell surface of the enucleated cell. In some embodiments, the cytokine polypeptide is complexed to the cell surface of the enucleated cell. In some embodiments, the cytokine polypeptide has been expressed on the 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 an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the abundance of the cytokine polypeptide and the abundance of the equivalent naturally occurring cytokine polypeptide are 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 an 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 CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. In some embodiments, the immune checkpoint inhibitor comprises a 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 cells are depleted of immune recognition molecules. In some embodiments, the immune recognition molecules comprise HLA antigens, proteoglycans, sugar moieties, fetal antigens, or any combination thereof. In some embodiments, the enucleated cells comprise 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 CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cells. In some embodiments, the immune checkpoint inhibitor is encoded by a second enucleated cell.

[0008] Incorporation by Reference 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. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification shall supersede and / or take precedence over such conflicting material.

[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. [Brief explanation of the drawings]

[0010] [Figure 1A] A non-limiting example of an IL-12 mRNA design is shown. [Figure 1B]Shown are mesenchymal stem cells (MSCs), enucleated cells derived from MSCs transfected with IL-12 mRNA (MSC IL-12 and enucleated IL-12, respectively), or conditioned medium (CM) collected at the indicated time points. [Figure 1C] Shown is CM from the cells / enucleated cells shown in Figure 1B assessed in mouse splenocytes for activation of the phosphorylated / activated form of Stat4 (p-Stat4) by Western blot. [Figure 1D] A schematic diagram for using enucleated cells to treat a triple-negative breast cancer (TNBC) mouse model is shown. [Figure 1E] 1 shows the measurement of IL-12 secreted by enucleated cells within the tumor microenvironment. [Figure 1F] Measurement of serum IL-12 is shown. [Figure 1G] Measurement of IL-12 mediated inflammatory biomarkers. [Figure 1H] Shown are SQ E0771 tumor-bearing mice injected with a total of three doses of either PBS, IL-12-secreting MSCs, or CA-IL-12. [Figure 1I] 1 shows FACS analysis for detecting leukocyte activation. [Figure 2A] Schematic diagram of enucleated cells secreting IL-12 alone or in combination with a checkpoint inhibitor, anti-PD-1 antibody, to treat a mouse model of cancer. [Figure 2B] Survival curves for animals injected as described in Figure 2A are shown. [Figure 2C] A graph of tumor size over time after injection is shown. [Figure 2D] 2B shows a graph of the fold change in animal body weight during the treatment phase of the survival experiment shown in FIG. 2B. [Figure 2E] Shown is the fold change in tumor size on each side (injected and contralateral / uninjected side) of animals injected bilaterally with E0771 cells followed by unilateral intratumoral injection of three doses of CA-IL-12 or PBS. [Figure 3A]This figure shows scRNA-seq datasets generated from two human TNBC primary tumors merged with eight normal breast datasets. VCAM1-positive vascular endothelial cells from clusters 0 and 4 (upper panel of Figure 3A) were subset for differential expression analysis. Dot plots confirmed that the vasculature in TNBC tumors overexpressed all CCP enucleating cell ligands compared to normal breast controls (lower panel of Figure 3A). [Figure 3B] 1 shows Boyden chamber migration assays demonstrating that mesenchymal stem cells or enucleated cells engineered to express CCR2, CXCR4, and leukocyte adhesion molecule (PSGL-1, referred to as CCP MSCs or CCP enucleated cells, respectively) robustly home to CCL2, SDF-1a, and EO771-conditioned medium compared to negative controls without chemoattractant. [Figure 3C] RNA-seq analysis of EO771 and 4T1 TNBC cell lines, which highly express Ccl2 and Cxcl12 (encoding SDF-1a). [Figure 3D] We present RNA-seq of the 4T1 cell line, revealing that the enucleating cell adhesion molecule / chemoattractant (encoding P-selectin), Ccl2, and Cxcl12, are upregulated with tumorigenesis (compared to tissue culture) and further upregulated in lung metastases. RNA-seq data. [Figure 4A] Schematic diagram showing RT and untreated irradiated mouse model with bilateral tumors. [Figure 4B] 1 shows homing of enucleated cells to tumors. [Figure 4C] FACS to determine the abundance of enucleated cells homing to the tumor is shown. [Figure 5A] Shows enucleated cell infiltration. [Figure 5B] Optical sectioning and 3D reconstruction of the center of a macrometastasis revealing a cluster of CCP enucleated cells are shown. [Figure 5C] Shown is a single channel acquisition of a DAPI stained section showing normal lung architecture and no tumor mass. [Figure 6A]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 are shown. [Figure 6B] Representative images of resected lungs with metastases treated as indicated (top) and representative IHC images of CD8+ cytotoxic T cells infiltrating EO771 lung metastases from the resected lungs shown in the top row (bottom) are shown. [Figure 7] FACS analysis of the indicated leukocyte populations compared to total CD45+ immune cells in EO771 lung metastases harvested on day 21 as in Figure 6 is shown. [Figure 8A] 1 shows an exemplary design of an enucleated cell expressing a plasma membrane anchored IL-12. [Figure 8B] A non-limiting example of a vector map of a vector encoding IL-12 is shown. [Figure 8C] 1 shows the engineering and screening of enucleated cells expressing membrane-bound scIL-12. [Figure 8D] 1 shows an exemplary mechanism of immune cell activation by CA-scIL-12. [Figure 9A] Modified MSCs or enucleated cells with or without scIL-12 co-cultured with primary mouse splenocytes and concanavalin A for 48 hours are shown. [Figure 9B] Shown are enucleated cells (1 x 106) engineered to secrete IL-12 (CA-IL-12). [Figure 9C] Enucleated cells modified with cell surface IL-12 (CA-scIL-12) were injected intravenously into mice (n=3), and 24 hours later, the indicated organs were harvested and processed for PCR quantification of the IL-12 biomarkers CXCL9 and IFN-γ. [Figure 9D] Serum cytokine levels in animals treated with CA-scIL-12 or CA-IL-12 are shown. [Figure 10A] 1 shows an exemplary design of an enucleated cell expressing plasma membrane tethered IL-15. [Figure 10B]A non-limiting example of a vector map of a vector encoding IL-15 is shown. [Figure 10C] 1 shows hTERT MSCs infected with a lentiviral construct encoding membrane-bound IL-15 receptor alpha. [Figure 11A] FIG. 1 shows a schematic diagram for modifying hTERT-MSCs to express both scIL-12 and IL-15Rα, followed by enucleation of the hTERT-MSCs. [Figure 11B] hTERT MSCs infected with two lentiviral constructs encoding either scIL-12 or IL-15Rα are shown. After 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. [Figure 11C] Functionality is demonstrated to express membrane-bound IL-12, IL-15, or a combination of both IL-12 and IL-15. [Figure 12A] 1 shows the biodistribution of enucleated cells by bioluminescence. [Figure 12B] Quantification of the biodistribution of enucleated cells from FIG. 12A is shown. [Figure 13A] 1 shows that the homing of the enucleated cells described herein is increased compared to nucleated mesenchymal stem cells (MSCs). [Figure 13B] Quantification of homing of enucleated cells from Figure 13A. [Figure 14A] 1 shows homing of enucleated cells based on increased chemoattractants at TNBC tumor metastasis sites. [Figure 14B] Dot plots of Cxcl (top) and Ccl (bottom) chemokine expression in Vcam1-positive cells in cluster 1 are shown, confirming that Cxcl12 (encoding SDF-1α) and Ccl2 are the most highly expressed Cxcl and Ccl chemokines, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0011] The novel features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments.

[0012] Described herein, in some aspects, are enucleated cells engineered to express at least one cytokine on their cell surface. In some embodiments, the expression of at least one cytokine on the cell surface of the enucleated cells confers a therapeutic effect. In some embodiments, the enucleated cells comprise messenger RNA (mRNA) encoding the cytokine. For example, the enucleated cells described herein can be engineered to express a cell membrane-bound cytokine to elicit or induce an endogenous immune response in a subject administered the enucleated cells. In some embodiments, the enucleated cells expressing the membrane-bound cytokine can be used to treat a disease or condition in a subject. In some embodiments, the disease or condition can be cancer. In some embodiments, the cancer can be due to expression in cancer cells, the tumor microenvironment (TME), metastatic cells, or a combination thereof. In some embodiments, the membrane-bound cytokine can be a membrane-bound interleukin. In some embodiments, the enucleated cells can be engineered to express at least one additional therapeutic agent. In some embodiments, the enucleated cells can be engineered to express at least one additional therapeutic agent, such as an immune checkpoint inhibitor or immune checkpoint blocker (ICB).

[0013] composition Provided herein are cells engineered to express at least one active agent on the cell surface of the enucleated cell. The active agent can be a protein. The active agent can be an antibody, an antigen, or a fragment of the antibody or antigen. Provided herein are cells comprising mRNA for expressing a protein, antibody, antigen, or portion thereof, and compositions comprising 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 cell can express the protein, antibody, antigen, or portion thereof (e.g., a therapeutic agent) in the absence of a nucleus using one or more intracellular organelles retained in the enucleated cell from the parent cell. In some embodiments, the protein, antibody, antigen, or portion thereof (e.g., a therapeutic agent) is exogenous to the enucleated cell or its parent cell. In some embodiments, the enucleated cell expresses the protein, antibody, antigen, or portion thereof (e.g., a therapeutic agent) on the surface of the enucleated cell. In some aspects, a composition comprises the enucleated cell described herein. In some aspects, the enucleated cells are obtained or derived from a nucleated cell (e.g., a parent cell). In some aspects, the enucleated cells comprise a transmembrane moiety. In some embodiments, the enucleated cells comprise a targeting moiety. In some embodiments, the enucleated cells comprise a therapeutic agent. In some embodiments, the enucleated cells comprise a protein. In some embodiments, the enucleated cells comprise a cytokine. In some embodiments, the enucleated cells comprise an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof. In some aspects, the targeting moiety comprises an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic agent comprises an antibody or antigen-binding fragment thereof. In some embodiments, the enucleated cells are formulated into a pharmaceutical formulation described herein.

[0014] enucleated cells The enucleated cells of the present disclosure are obtained or derived from a corresponding nucleated cell (referred to herein as a "parent cell"). The parent cell can be derived from a variety of different cell types, including eukaryotic cells. For example, the enucleated cells can be derived from adult stem cells, mesenchymal stromal cells (MSCs), natural killer (NK) cells, macrophages, myoblasts, neutrophils, endothelial cells, endothelial progenitor cells, and / or fibroblasts. In some embodiments, the enucleated cells are derived from mesenchymal stromal cells. In some embodiments, the enucleated cells are derived from induced pluripotent stem cells (iPSCs). In some embodiments, the parent cell is derived from a cell that has been immortalized using a suitable method. 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 cytoplasts are derived from the parent cell using suitable methods provided in U.S. Patent No. 10,927,349, which is incorporated herein by reference in its entirety. In some embodiments, the enucleated cells retain one or more organelles for synthesis of exogenous proteins. In some embodiments, the enucleated cells retain one or more organelles for synthesis of exogenous cytokines.

[0015] In some embodiments, enucleated cells do not require differentiation of the parent cell. In some embodiments, a parent cell containing a nucleus is engineered to express a single domain antibody or antigen-binding fragment thereof, a therapeutic agent, a transmembrane portion, an immune evasion moiety, and / or a targeting moiety described herein, and then the parent cell's nucleus is removed. In some embodiments, the parent cell containing the nucleus is enucleated, and the enucleated cell is engineered to express a single domain antibody or antigen-binding fragment thereof, a therapeutic agent, a transmembrane portion, an immune evasion moiety, and / or a targeting moiety described herein. In some embodiments, the parent cell is engineered to express one or more of the above biomolecules (e.g., an immune evasion moiety and / or a targeting moiety), and the resulting enucleated cell (e.g., already expressing an immune evasion moiety and / or a targeting moiety) is further engineered to express a second of the above biomolecules (e.g., a therapeutic agent). In this manner, the enucleated cells of the present disclosure can be extensively engineered before enucleation, stored for long periods of time as needed (e.g., via lyophilization, cryohibernation, cryopreservation), and rapidly engineered to express a therapeutic agent closer to the time of need.

[0016] In some embodiments, the cell can be from any organism having one or more cells. Non-limiting examples of cells include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, Marchantia polymorpha, mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii), and the like. Cells include cells from organisms such as: Solanum reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, and Yatsumata sargassum, seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), and others. The cells may not be derived from a naturally occurring organism (e.g., the cells may be synthetically produced cells, sometimes referred to as artificial cells). In some embodiments, the cells are somatic cells. In some embodiments, the cells are stem or progenitor cells. In some embodiments, the cells are mesenchymal stem or progenitor cells. In some embodiments, the cells are hematopoietic stem or progenitor cells. In some embodiments, the cell is a muscle cell, a skin cell, a blood cell, or an immune cell. Other non-limiting examples of cells include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, and cytokine-induced killer (CIK) cells; myeloid cells such as granulocytes (basophilic granulocytes, eosinophils, neutrophilic granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, and dendritic cells; cells of the endocrine system (including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophilic cells), adrenal gland (chromaffin cells), and pineal gland (pineocyte) cells); cells of the nervous system (glial cells (astrocytes, microglia),Giant neurosecretory cells, stellate cells, Boettcher cells, and pituitary gland (including gonadotropic cells, adrenocorticotropic cells, thyrotropic cells, somatotropic cells, and lactogenic cells)), cells of the respiratory system (including alveolar epithelial cells (type I alveolar epithelial cells, type II alveolar epithelial cells), Clara cells, goblet cells, and dust cells), cells of the circulatory system (including cardiac muscle cells and pericytes), cells of the digestive system (including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells), enteroendocrine cells (enterochromaffin cells, APUD cells), liver (hepatocytes, Cook cells), fur cells), cartilage / bone / muscle), bone cells (including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts)), chondrocytes (including chondroblasts, chondrocytes), skin cells (including hair follicles, keratinocytes, melanocytes (nevus cells)), muscle cells (including myocytes), urinary tract cells (including podocytes, juxtaglomerular cells, intraglomerular / extraglomerular mesangial cells, kidney proximal tubule brush border cells, macula densa cells), reproductive system cells (including sperm, Sertoli cells, Leydig cells, oocytes), and other cells (adipocytes, fibroblasts, Tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, cuticle hair stem cells, cuticle root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, outer root sheath cells, hair matrix cells (stem cells), moist stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urothelial cells (lining of the bladder and ureters), exocrine secretory epithelial cells , salivary gland mucous cells (secreting polysaccharides), salivary gland serous cells (secreting glycoprotein enzymes), von Ebner's gland cells of the tongue (moistening taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), ear canal gland cells (secreting earwax), eccrine sweat gland dark cells (secreting glycoproteins), eccrine sweat gland clear cells (secreting small molecules), apocrine sweat gland cells (scent-producing secretions, sex hormone sensitive), Molar gland cells of the eyelid (specialized sweat glands), sebaceous gland cells (secreting lipid-rich sebum), Bowman's gland cells of the nose (moistening the olfactory epithelium), Brunner's gland cells of the duodenum (enzymes and alkaline mucus),Seminal vesicle cells (secrete components of seminal fluid, including fructose for swimming sperm), prostate cells (secrete components of seminal fluid), bulbourethral gland cells (secrete mucus), Bartholin's gland cells (secrete vaginal fluid), Little gland cells (secrete mucus), endometrial cells (secrete carbohydrates), isolated goblet cells of the respiratory and digestive tract (secrete mucus), mucous cells of the stomach lining (secrete mucus), gastric gland enzyme-secreting cells (secrete pepsinogen), gastric gland acid-secreting cells (secrete hydrochloric acid), pancreatic acinar cells (secrete bicarbonate and digestive enzymes), Paneth cells of the small intestine (secrete lysozyme), type II alveolar epithelial cells of the lungs (secrete surfactant), Clara cells of the lungs, hormonal Monotrophic cells, anterior pituitary cells, somatotrophs, prolactinocytes, thyrotrophs, gonadotrophs, corticotrophs, intermediate pituitary cells, giant neurosecretory cells, intestinal and airway cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, eosinophilic cells, adrenal gland cells, chromaffin cells, Leydig cells of the testis, theca interna cells of the follicle, lutein cells of the ruptured follicle, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells alveoli (lungs, intestines, exocrine glands and genitourinary tract), kidney, type I alveolar epithelial cells (lining the air pockets of the lungs), pancreatic duct cells (central acinar cells), non-striated duct cells (such as those in sweat glands, salivary glands and mammary glands), duct cells (such as those in seminal vesicles and the prostate), epithelial cells lining closed body cavities, ciliated cells with propulsive functions, extracellular matrix-secreting cells, contractile cells, skeletal muscle cells, stem cells, cardiac muscle cells, blood cells and cells of the immune system, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue) , microglial cells (in the central nervous system), neutrophilic granulocytes, eosinophilic granulocytes, basophilic granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitor cells (various types) of the blood and immune system, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic neuronal cells, sensory and peripheral neuronal supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells,Germ cells include oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), spermatozoa, nurse cells, follicle cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0017] In some embodiments, the cell is a eukaryotic cell. Non-limiting examples of eukaryotic cells include mammalian (e.g., rodent, non-human primate, or human), non-mammalian (e.g., fish, avian, reptile, or amphibian), invertebrate, insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as a mammalian, avian, plant, or insect cell. In some embodiments, the nucleated cell is a primary cell. In some embodiments, the nucleated cell is an immune cell (e.g., lymphocyte (e.g., T cell, B cell), macrophage, natural killer cell, neutrophil, mast cell, basophil, dendritic cell, monocyte, myeloid-derived suppressor cell, eosinophil). In some embodiments, the nucleated cell is a phagocyte or leukocyte. In some embodiments, the nucleated cells are stem cells (e.g., adult stem cells (e.g., hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, testicular cells), embryonic stem cells, induced pluripotent stem cells (iPS)). In some embodiments, the nucleated cells are progenitor cells. In some embodiments, the nucleated cells are from a cell line. In some embodiments, the nucleated cells are suspension cells. In some embodiments, the nucleated cells are adherent cells. In some embodiments, the nucleated cells are cells immortalized by expression of an oncogene. In some embodiments, the nucleated cells are immortalized by expression of human telomerase reverse transcriptase (hTERT) or any oncogene. In some embodiments, the nucleated cells are immortalized by expression of a viral gene, such as Simian Virus 40 (SV40). In some embodiments, the nucleated cells are cells derived from a patient or subject (e.g., cells derived from an autologous patient or cells derived from an allogeneic patient).

[0018] In some embodiments, the enucleated cells are derived from immune cells, ie, natural killer (NK) cells, neutrophils, macrophages, lymphocytes, fibroblasts, adult stem cells (e.g., hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, mesenchymal stromal cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, skin stem cells, or testicular cells), mast cells, basophils, eosinophils, endothelial cells, endothelial cell progenitor cells, or induced pluripotent stem cells.

[0019] In some embodiments, the parent cell is not an erythrocyte or an erythroid progenitor 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 progenitor cell. In some embodiments, the enucleated cell is not an erythrocyte or an erythroid progenitor 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 progenitor cell. In some embodiments, the enucleated cell does not express complement receptor 1 (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 cells do not express a von Willebrand factor (VWF) receptor. In some embodiments, the enucleated cells do not express a fibrinogen receptor. In some embodiments, the enucleated cells do not express a GP1b-IX-V receptor. In some embodiments, the enucleated cells do not express a GPIIb / IIIa receptor. In some embodiments, the enucleated cells do not express a prostanoid receptor. In some embodiments, the enucleated cells do not express a purinergic receptor. In some embodiments, the enucleated cells do not express a thromboxane receptor.

[0020] In some embodiments, the parent cell can be enucleated and manipulated 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 induced pluripotent stem cell (iPSC). In some embodiments, the parent cell is not an erythrocyte or an erythroid progenitor 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 progenitor cell. The parent cell can be treated with cytochalasin to soften the cortical actin cytoskeleton. The nucleus can then be physically extracted from the cell body by high-speed centrifugation on a Ficoll gradient to generate enucleated cells. Because enucleated cells and intact nucleated cells sediment in different layers on a Ficoll gradient, the enucleated cells can be easily isolated and prepared for therapeutic purposes or for fusion with other cells (nucleated or enucleated). The enucleation process is clinically scalable to process tens of millions of cells. In some embodiments, enucleated cells can be used as disease-homing vehicles to express clinically relevant cargo / payloads to treat various diseases.

[0021] After enucleation of the parent cell, the enucleated cells described herein retain one or more organelles endogenous to the parent cell. In some embodiments, all of one or more organelles are retained. In some embodiments, less than all of one or more organelles are retained. In some embodiments, the Golgi apparatus and / or the endoplasmic reticulum, which are involved in protein synthesis and secretion, are retained. Retention of one or more organelles at least partially enables the enucleated cell to synthesize or release a biomolecule disclosed herein (e.g., a single domain antibody or portion thereof, a targeting moiety, an immune evasion moiety, etc.) in the absence of a nucleus.

[0022] Enucleated cells may be smaller than their nucleated counterparts (e.g., nucleated parent cells) and thus may be better able to migrate through small openings in blood vessels and tissue parenchyma. Furthermore, removing the dense nucleus alleviates a major physical barrier, allowing the cells to migrate freely through small openings in blood vessels and tissue parenchyma. Thus, enucleated cells have improved biodistribution within the body and migration to target tissues. In some embodiments, the diameter of the enucleated cells is at least 1 μm. In some embodiments, the diameter of the enucleated cells is greater than 1 μm. In some embodiments, the diameter of the enucleated cells is 1 to 100 μm (e.g., 1 to 90 μm, 1 to 80 μm, 1 to 70 μm, 1 to 60 μm, 1 to 50 μm, 1 to 40 μm, 1 to 30 μm, 1 to 20 μm, 1 to 10 μm, 1 to 5 μm, 5 to 90 μm, 5 to 80 μm, 5 to 70 μm, 5 to 60 μm, 5 to 50 μm, 5 to 40 μm, 5 to 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, 15-60 μm, 15-50 μm, 15-40 μm, 15-30 μm, 15-20 μm). In some embodiments, the enucleated cells have a diameter of 10-30 μm. In some embodiments, the enucleated cells have a diameter of 5-25 μm (e.g., 5-20 μm, 5-15 μm, 5-10 μm, 10-25 μm, 10-20 μm, 10-15 μm, 15-25 μm, 15-20 μm, or 20-25 μm). In some embodiments, the enucleated cells have a diameter of approximately 8 μm. In some embodiments, some enucleated cells may advantageously be small enough to allow for better homing to the target site. For example, the enucleated cells described herein may pass through narrow passages in lung tissue or structures, such as alveolar ducts or microcapillaries, that most cells, such as parent cells, cannot pass through.

[0023] In some embodiments, enucleated cells have significant therapeutic value because they maintain viability, do not differentiate into other cell types, secrete bioactive molecules, are capable of physical migration / homing within approximately 5 days, can be widely enucleated ex vivo to perform specific therapeutic functions, and can be fused with the same or other cell types to transfer desired products (natural or enucleated). Thus, enucleated cells have broad utility as cellular vehicles for expressing therapeutically important biomolecules and disease-targeting cargoes, including genes, viruses, bacteria, mRNA, shRNA, siRNA, polypeptides (including antibodies and antigen-binding fragments), plasmids, gene editing mechanisms, or nanoparticles. The present disclosure enables the generation of safe (e.g., no unwanted DNA is transferred to the subject) and controllable (e.g., cell death occurs within 3-4 days) cell-based carriers that can be genetically enucleated to express cargoes for specific disease treatment and health promotion in humans. In some embodiments, the enucleated cells survive and retain migration or homing function for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 5 days or more after administration to a subject in need thereof.

[0024] In some embodiments, the enucleated cells are engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous protein, a gene editing mechanism, or a combination thereof. In some embodiments, the exogenous DNA molecule is single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or a combination thereof. In some embodiments, the exogenous RNA molecule is messenger RNA (mRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an RNA virus, or a combination thereof. In some embodiments, the exogenous protein is a cytokine, a growth factor, a hormone, an antibody or an antigen-binding fragment thereof, an enzyme, or a combination thereof. In some embodiments, the antibody is a single-domain antibody or an antigen-binding fragment thereof. In some embodiments, the parent cell (e.g., a nucleated cell) is genetically enucleated before enucleation (e.g., pre-enucleation). In some embodiments, the parent cell is genetically enucleated after enucleation (e.g., post-enucleation).

[0025] In some embodiments, the enucleated cells described herein comprise a transmembrane moiety. In some embodiments, the enucleated cells comprise an immune evasion moiety. In some embodiments, the immune evasion comprises a "don't eat me" signaling peptide, such as CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof. In some embodiments, the enucleated cells are depleted of immune recognition molecules. Non-limiting examples of immune recognition molecules include HLA antigens, proteoglycans, sugar moieties, and embryonic antigens.

[0026] In some embodiments, enucleated cells or compositions comprising enucleated cells may be cryopreserved (e.g., storing enucleated cells or compositions comprising enucleated cells at freezing temperatures) or cryohiberinated (e.g., storing enucleated cells or compositions comprising enucleated cells at temperatures between ambient and freezing temperatures). The duration of cryopreservation or cryohibernation can be about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, enucleated cells exhibit a post-cryopreservation or cryohibernation viability of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or greater after cryopreservation or cryohibernation, similar to comparable cells (e.g., parental cells or enucleated cells described herein that have not been cryopreserved or cryohiberinated) for the same period of time. For example, in some embodiments, viability is decreased by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to comparable cells. In another example, in some embodiments, viability is increased by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to comparable cells. Viability in this context may be measured by trypan blue exclusion as described herein. In some embodiments, trypan blue exclusion is performed by (a) centrifuging an aliquot of nucleated cells in suspension to produce a cell pellet; (b) resuspending the cell pellet in serum-free medium to generate a serum-free cell suspension; (c) mixing one part trypan blue and one part serum-free cell suspension; and (d) counting the nucleated cells within 3-5 minutes of (c), wherein at least some of the nucleated cells are not stained with trypan blue (which is an indicator of viability). In some embodiments, viability is measured using Annexin V cell surface staining. In some embodiments, viability is measured by expression of an exogenous polypeptide. For example, the viability of enucleated cells can be determined by expression of an exogenous antibody or single domain antibody expressed by the enucleated cells.In some embodiments, viability is measured by expression of any one of the cell surface markers described herein, such as CD105, CD90, CD45, CXCR4, PSGL-1, or CCR2. In some embodiments, viability is measured by cellular activity of the enucleated cells. In some embodiments, viability is measured by the homing ability of the enucleated cells, as determined by chemosensing activity or chemokine homing activity described herein.

[0027] In some embodiments, enucleated cells or compositions comprising enucleated cells may be lyophilized, hi some embodiments, the enucleated cells, after reconstitution from lyophilization, exhibit viability similar to or greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to comparable cells (e.g., parent cells or enucleated cells described herein that have not been lyophilized).

[0028] In some embodiments, enucleated cells or compositions comprising enucleated cells may be dehydrated, hi some embodiments, the enucleated cells, after rehydration from lyophilization, exhibit viability similar to or greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to comparable cells (e.g., parent cells or enucleated cells described herein that have not been dehydrated).

[0029] In some embodiments, the enucleated cells or compositions comprising the enucleated cells are stable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more at 4° C. In some embodiments, the compositions are stable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more at room temperature. In some embodiments, the compositions are stable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more at 37° C. In some embodiments, the enucleated cells or compositions comprising the enucleated cells may remain viable after being administered to a subject in need thereof to treat a disease or condition described herein. In some embodiments, the enucleated cells or compositions comprising the enucleated cells may remain viable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more after administration to a subject.

[0030] In some embodiments, the enucleated cells may be obtained from parent cells that are autologous to a subject in need of treatment with the enucleated cells described herein, hi some embodiments, the enucleated cells may be obtained from parent cells that are allogeneic to a subject in need of treatment with the enucleated cells described herein.

[0031] activator The enucleated cells may comprise an active agent. The active agent may be encoded by exogenous messenger RNA (mRNA). The active agent may include 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 adhesion protein, or a catalytically active fragment thereof. The adhesion protein may be PSGL-1. The cytokine may be a chemokine, such as SDF-1α, CCL2, IL-12, and IL-15. The cytokine may be a chemokine receptor, such as CXCR2, CCR2, and CXCR4. The small molecule may be a chemical sensor. The polypeptide may be expressed by the enucleated cells (in the absence of a nucleus). In some embodiments, the polypeptide is expressed by a nucleated parent cell prior to enucleation to produce enucleated cells containing the polypeptide. In some embodiments, the active agent is expressed on the surface of the enucleated cells. In some embodiments, the active agent is or comprises a therapeutic agent of the present disclosure.

[0032] The active agent can be a targeting moiety of the present disclosure. In some embodiments, the active agent is or includes a transmembrane moiety. In some embodiments, the active agent is covalently attached to the enucleated cell surface. In some embodiments, the active agent is non-covalently attached. In some embodiments, the amount of active agent is increased by about 0.10 to about 150.0 fold over the endogenous amount of active agent in an otherwise identical cell or nucleated parent cell. In some embodiments, the amount of active agent is increased by about 0.10 to about 0.50 fold over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 1.00 fold over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 2.00 fold over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 3.00 fold over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 4.00 fold over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 5.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 10.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 15.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 20.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 25.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 30.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 35.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 40.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 45.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 50.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 55.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 60.00 times the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 0.10 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 135.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 140.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 145.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.10 to about 150.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 1.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 2.00 times the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 0.50 to about 3.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 4.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 5.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 10.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 15.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 20.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 25.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 30.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 35.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 40.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 45.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 50.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 55.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 60.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 65.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 70.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 75.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 80.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 85.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 90.00 times the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 0.50 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 0.50 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 2.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 3.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 4.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 5.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 10.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 15.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 20.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 25.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 1.00 to about 30.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 35.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 40.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 45.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 50.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 55.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 60.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 65.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 70.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is. The amount of active agent is increased by about 1.00 to about 110.00 times over the endogenous amount. In some embodiments, the amount of active agent is increased by about 1.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 1.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 3.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 4.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 5.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 10.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 15.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 20.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 25.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 30.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 35.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 40.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 45.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 50.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 2.00 to about 55.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 60.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 65.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 70.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 2.00 to about 150.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 3.00 to about 4.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 5.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 10.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 15.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 20.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 25.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 30.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 35.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 40.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 45.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 50.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 55.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 60.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 65.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 70.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 95.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 3.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 3.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 5.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 10.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 15.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 20.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 25.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 30.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 35.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 40.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 45.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 4.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, The amount of active agent is increased by about 4.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 4.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 10.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 15.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 20.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 25.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 30.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 35.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 40.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 45.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 50.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 85.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 5.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 5.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 15.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 20.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 25.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 30.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 35.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 40.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 45.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 10.00 to about 50.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 55.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 60.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 65.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 70.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 10.00 to about 145.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 10.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 20.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 25.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 30.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 35.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 40.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 45.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 110.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 15.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 15.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 25.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 30.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 35.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 40.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 45.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 80.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is. The amount of active agent is increased by about 20.00 to about 85.00 times over the endogenous amount. In some embodiments, the amount of active agent is increased by about 20.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 20.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 30.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 35.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 40.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 45.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 55.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 25.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 100.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 105.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 110.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 115.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 25.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 35.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 30.00 to about 40.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 45.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 100.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 105.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of the active agent is increased by about 30.00 to about 110.00 times over the endogenous amount of the active agent. The amount of active agent is increased by about 30.00 to about 115.00 times over the endogenous amount. In some embodiments, the amount of active agent is increased by about 30.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 30.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 40.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 45.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 95.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 35.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 35.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 45.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 85.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 40.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, The amount of active agent is increased by about 40.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 40.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 50.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 100.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 105.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 135.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 45.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 45.00 to about 150.00 times. In some embodiments, the amount of active agent is increased by about 50.00 to about 55.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 100.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 105.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 135.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 50.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 50.00 to about 150.00 times. In some embodiments, the amount of active agent is increased by about 55.00 to about 60.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 100.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 105.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 110.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 140.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 55.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 55.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 65.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 70.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 75.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 100.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 105.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 110.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 115.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 60.00 to about 150.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 65.00 to about 70.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 80.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 85.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 90.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 95.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 100.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 65.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 75.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is. The amount of active agent is increased by about 70.00 to about 80.00 times over the endogenous amount. In some embodiments, the amount of active agent is increased by about 70.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 70.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 80.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 100.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 75.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 75.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 85.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 90.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 80.00 to about 130.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is about 80.00 to about 135.00. In some embodiments, the amount of active agent is about 80.00 to about 140.00. In some embodiments, the amount of active agent is about 80.00 to about 145.00. In some embodiments, the amount of active agent is about 80.00 to about 150.00. In some embodiments, the amount of active agent is increased by about 85.00 to about 90.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 85.00 to about 95.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 85.00 to about 100.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 85.00 to about 105.00 times the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 85.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 85.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 85.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is about 85.00 to about 125.00. In some embodiments, the amount of active agent is about 85.00 to about 130.00. In some embodiments, the amount of active agent is about 85.00 to about 135.00. In some embodiments, the amount of active agent is about 85.00 to about 140.00. In some embodiments, the amount of active agent is about 85.00 to about 145.00. In some embodiments, the amount of active agent is increased by about 85.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 95.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 115.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 90.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 90.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 100.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 105.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 110.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 115.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 120.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 125.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 130.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 135.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 140.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 95.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 105.00 times over the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 100.00 to about 110.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 115.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 145.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 100.00 to about 150.00 times over the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 110.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 115.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 105.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 115.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 110.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 115.00 to about 120.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 115.00 to about 125.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 115.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 115.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 115.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 115.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 115.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 120.00 to about 125.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 120.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 120.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 120.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 120.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 120.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 125.00 to about 130.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 125.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 125.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 125.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 125.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 130.00 to about 135.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 130.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 130.00 to about 145.00 times greater than the endogenous amount of active agent.In some embodiments, the amount of active agent is increased by about 130.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 135.00 to about 140.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 135.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 135.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 140.00 to about 145.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 140.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of active agent is increased by about 145.00 to about 150.00 times greater than the endogenous amount of active agent. In some embodiments, the amount of 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, 4.00, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.00, 8.10, 8.20, 8.30, 8.40 Increases by 0.10, 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 times.In some embodiments, the amount of 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, 3.00, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.00, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.00, 8.10, 8.20, 8.30, 8.4 Increases by 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 times or more. In some embodiments, the amount of 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, 3.00, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.00, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.00, 8.10, 8.20, 8.30, 8.4 In some embodiments, the amount of active agent and the amount of equivalent endogenous active agent are determined by flow cytometry (FACS).

[0033] The active agent can be a cytokine. The cytokine can be expressed by mRNA in the enucleated cell. The cytokine can be expressed by the enucleated cell on its surface. In some embodiments, a cytokine polypeptide encoded by exogenous mRNA has reduced toxicity in a subject compared to direct delivery of the cytokine polypeptide. The cytokine can include a transmembrane domain. The mRNA can encode the cytokine and the transmembrane domain. The cytokine can be attached to the cell surface of the enucleated cell. The cytokine can be attached, complexed, tethered, connected, bound, linked, or the like to the cell surface of the enucleated cell. The cytokine can be exogenously attached to the cell surface of the enucleated cell. In some embodiments, the cytokine is not delivered by the enucleated cell to a target tissue or microenvironment. In some embodiments, the cytokine is not secreted by the enucleated cell into a target tissue or microenvironment. In some embodiments, the mRNA encodes a cytokine or an antigen-binding fragment of the cytokine. In some embodiments, the cytokine is an antigen-binding fragment of the cytokine. In some embodiments, the cytokine is a full-length cytokine.

[0034] In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 0.1-fold to at least 5.0-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 0.1-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 0.2-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 0.3-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 0.4-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 0.5-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 0.6-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 0.7-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 0.8-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 0.9-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 1.0-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance.In some embodiments, the enucleated cells express at least 1.2-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 1.4-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 1.6-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 1.8-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 2.0-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 2.2-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 2.4-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 2.6-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 2.8-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 3.0-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 3.2-fold greater abundance of cytokine polypeptide than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance.In some embodiments, the enucleated cells express at least 3.4-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 3.6-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 3.8-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 4.0-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 4.2-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 4.4-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express at least 4.6-fold greater abundance of cytokine polypeptides than enucleated cells expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 4.8-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the enucleated cells express an abundance of a cytokine polypeptide that is at least 5.0-fold greater than an enucleated cell expressing an equivalent naturally occurring cytokine polypeptide abundance. In some embodiments, the cytokine polypeptide abundance and the equivalent naturally occurring cytokine polypeptide abundance are determined by flow cytometry (FACS).

[0035] In some embodiments, the cytokine polypeptide is bound to the enucleated cell surface for a period of time. In some embodiments, this period of time is after delivery (e.g., administration) to a subject as disclosed herein. In some embodiments, the cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, the cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, the cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 36 hours. In some embodiments, the cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 48 hours. In some embodiments, the cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 72 hours. In some embodiments, the cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, the cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, the cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 36 hours. In some embodiments, the cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 48 hours. In some embodiments, the cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 72 hours. In some embodiments, the cytokine polypeptide is expressed on the cell surface of the enucleated cell for at least 12 hours. In some embodiments, the cytokine polypeptide is expressed on the cell surface of the enucleated cell for at least 24 hours. In some embodiments, the cytokine polypeptide may be expressed on the cell surface of the enucleated cell for at least 36 hours. In some embodiments, the cytokine polypeptide is expressed on the cell surface of the enucleated cell for at least 48 hours. In some embodiments, the cytokine polypeptide may be expressed on the cell surface of the enucleated cell for at least 72 hours.

[0036] The enucleated cells disclosed herein may comprise mRNA encoding a polypeptide. The polypeptide may be a cytokine. The cytokine may include an interleukin. The interleukin may include IL-12. The IL-12 may include IL-12α. In some embodiments, the IL-12α comprises SEQ ID NO: 35. In some embodiments, the IL-12α comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 35. The IL-12 may include IL-12β. In some embodiments, the IL-12β comprises SEQ ID NO: 36. In some embodiments, IL-12β comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 36. IL-12 can comprise IL-12α and IL-12β. In some embodiments, IL-12α and IL-12β interact through a disulfide bond connecting IL-12α and IL-12β. IL-12 can comprise a transmembrane domain, such as a transmembrane derived from a B7 cell surface ligand. In some embodiments, the transmembrane derived from a B7 cell surface ligand is derived from a B7-2 cell surface ligand. In some embodiments, the B7-2 cell surface ligand comprises SEQ ID NO: 37. In some embodiments, the B7-2 cell surface ligand comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 37. In some embodiments, the IL-12 comprises IL-12α, IL-12β, and the transmembrane B7-2 domain. In some embodiments, the IL-12α, IL-12β, and the transmembrane B7-2 domain comprise SEQ ID NO: 33. In some embodiments, the IL-12α, IL-12β, and the transmembrane B7-2 domain comprise an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 33.The transmembrane domain, e.g., transmembrane B7-2, allows IL-12 to be anchored to the cell surface. The interleukin can be IL-15. In some embodiments, the IL-15 comprises SEQ ID NO: 38. In some embodiments, the IL-15 comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 38. The IL-15 can further comprise an IL-15 receptor alpha (RA). In some embodiments, the IL-15RA comprises SEQ ID NO: 40. In some embodiments, the IL-15RA comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 40. IL-15RA may comprise a sushi domain. In some embodiments, the sushi domain comprises SEQ ID NO:41. In some embodiments, the sushi domain comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:41. The sushi domain can enable IL-15RA to interact with IL-15. The sushi domain can enable IL-15RA to bind to IL-15. IL-15RA may comprise a transmembrane domain. In some embodiments, the transmembrane domain comprises SEQ ID NO:42. In some embodiments, the transmembrane domain comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:42. IL-15RA can comprise a sushi domain and a transmembrane domain. In some embodiments, the sushi domain comprises cysteine ​​residues that form disulfide bonds, such as the cysteine ​​residues at positions 3, 29, 45, and 63 of SEQ ID NO: 41. IL-15 and IL-15RA can be connected. IL-15 and IL-15RA can be connected by a linker. In some embodiments, the linker comprises SEQ ID NO: 39.In some embodiments, the linker comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 39. The polypeptide may comprise IL-15, IL-15RA, and a linker. In some embodiments, the IL-15, IL-15RA, and linker comprise SEQ ID NO: 34. In some embodiments, the IL-15, IL-15RA, and linker comprise an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 34. The sequences of these regions can be found in Table 9. [Table 1-1] [Table 1-2]

[0037] The enucleated cells disclosed herein may comprise two or more mRNAs encoding two or more polypeptides. The polypeptides may include cytokines. The cytokines may include interleukins. The interleukins may include IL-12 and IL-15. In some embodiments, IL-12 and IL-15 are expressed on the surface of the enucleated cells. In some embodiments, the polypeptides include a cytokine and an immune checkpoint inhibitor.

[0038] The enucleated cells disclosed herein may comprise an immune checkpoint inhibitor. The immune checkpoint inhibitor may include 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 CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof. The immune checkpoint inhibitor may be encoded by a second exogenous mRNA in the enucleated cells.

[0039] therapeutic agent In some embodiments, the cells of the present disclosure comprise at least one therapeutic agent. In some embodiments, the cells are enucleated, such as using the enucleation methods disclosed herein. In some embodiments, the therapeutic agent is or comprises an active agent of the present disclosure. In some embodiments, the active agent comprises at least one of a DNA molecule, an RNA molecule, a protein (e.g., an enzyme, an antibody, an antigen, a toxin, a cytokine, a protein hormone, a growth factor, a cell surface receptor, or a vaccine), a peptide (e.g., a peptide hormone or antigen), a small molecule (e.g., a steroid, a polyketide, an alkaloid, a toxin, an antibiotic, an antiviral, colchicine, taxol, mitomycin, or emtansine), a gene editing factor, a nanoparticle, or another active agent (e.g., a bacterium, a bacterial spore, a bacteriophage, a bacterial component, a virus (e.g., an oncolytic virus), an exosome, a lipid, or an ion). 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 RNA viruses. Non-limiting examples of DNA molecules include single-stranded DNA, double-stranded DNA, oligonucleotides, plasmids, bacterial DNA molecules, and DNA viruses. Non-limiting examples of proteins include cytokines, growth factors, hormones, antibodies or antigen-binding fragments thereof, single-domain antibodies or antigen-binding fragments thereof, small peptide-based drugs, and enzymes. Non-limiting examples of oncolytic viruses include talimogene laherparepvec, Onyx-015, GL-ONC1, CV706, Voyager-V1, and HSV-1716. Several wild-type viruses, such as vaccinia virus, vesicular stomatitis virus, poliovirus, reovirus, Seneca virus, ECHO-7, or Semliki Forest virus, also exhibit oncolytic behavior.

[0040] In some embodiments, the enucleated cells are engineered to produce (e.g., express, and optionally tether) a therapeutic agent. In some embodiments, the parent cell from which the enucleated cells are derived may be engineered to produce a therapeutic agent prior to enucleation to produce the enucleated cell. In some embodiments, the enucleated cells are engineered to produce a therapeutic agent after enucleation (in the absence of a nucleus). In some embodiments, the therapeutic agent is exogenous to the enucleated cell or its parent cell. In some embodiments, the therapeutic agent is endogenous to the enucleated cell or its parent cell. In some embodiments, the enucleated cells of the present disclosure comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more therapeutic agents.

[0041] In some embodiments, a therapeutic agent comprises a modified version of a DNA molecule, an RNA molecule, a protein, a peptide, a small molecule active agent, and / or a gene editing factor, compared to the naturally occurring version. In some embodiments, a therapeutic agent is a corrected, truncated, or non-mutated version and / or copy of a DNA molecule, an RNA molecule, a protein, a peptide, a small molecule active agent, and / or a gene editing factor. For example, a therapeutic agent can correct mutated tumor protein p53 (p53) or epidermal growth factor receptor (EGFR) in a target cell or target environment as part of a treatment for lung cancer.

[0042] The therapeutic agent may be bound to the surface of the enucleated cell. The therapeutic agent may be or may include a targeting moiety described herein. Non-limiting examples of targeting moieties that may be produced by or contained in the enucleated cell include chemokine receptors, adhesion molecules, and antigen-binding polypeptides (e.g., single domain antibodies and antigen-binding fragments thereof), or portions thereof. In some embodiments, the therapeutic agent may be or may include a transmembrane moiety described herein. In some embodiments, the therapeutic agent is covalently attached to the enucleated cell surface. In some embodiments, the therapeutic agent is non-covalently attached. In some embodiments, the amount of therapeutic agent is increased by about 0.10 to about 150.0-fold over the endogenous amount of therapeutic agent in an otherwise identical cell or nucleated parent cell. In some embodiments, the amount of therapeutic agent is increased by about 0.10 to about 0.50-fold over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 0.10 to about 1.00-fold over the endogenous amount of therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 2.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 3.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 4.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 5.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 10.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 15.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 20.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 25.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 30.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 35.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 40.00 times the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 45.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 50.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 55.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 60.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 65.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 70.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 75.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 80.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 85.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 90.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 95.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 100.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 105.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 110.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 115.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 120.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 125.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 130.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 135.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 140.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 145.00-fold above the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 0.10 to about 150.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 1.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 2.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 3.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 4.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 5.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 10.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 15.00 times above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 20.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 25.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 30.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 35.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 40.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 45.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 50.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 55.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 60.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 65.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 70.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 75.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 80.00 times the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 85.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 90.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 95.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 100.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 105.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 110.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 115.00 times the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 120.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 125.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 130.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 135.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 140.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 145.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 0.50 to about 150.00-fold above the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 2.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 3.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 4.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 5.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 10.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 15.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 20.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 1.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 95.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 100.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 105.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 110.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 115.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is. The amount of therapeutic agent is increased by about 1.00 to about 120.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 125.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 130.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 135.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 140.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 145.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 1.00 to about 150.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 3.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 4.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 5.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 10.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 15.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 20.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 2.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 4.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 5.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 10.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 15.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 20.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 3.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 5.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 10.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 15.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 20.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, The amount of the therapeutic agent is increased by about 4.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 4.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 10.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 15.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 20.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 5.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 15.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 20.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 10.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 20.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, The amount of the therapeutic agent is increased by about 15.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 15.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 25.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 20.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 30.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 35.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 45.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 25.00 to about 150.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 35.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 40.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 45.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 50.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 55.00 times over the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 30.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 40.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 45.00 times over the endogenous amount of the therapeutic agent. The amount of therapeutic agent is increased by about 35.00 to about 50.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 35.00 to about 55.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 35.00 to about 60.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 35.00 to about 65.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 35.00 to about 70.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 35.00 to about 75.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 35.00 to about 80.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 35.00 to about 150.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 45.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 50.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 55.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 60.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 65.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 70.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 40.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 50.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 45.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 55.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 50.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 60.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 55.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 65.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 60.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 70.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 65.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 75.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is. The amount of therapeutic agent is increased by about 70.00 to about 90.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 70.00 to about 95.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 70.00 to about 100.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 70.00 to about 105.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 70.00 to about 110.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 70.00 to about 115.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 70.00 to about 120.00 times over the endogenous amount of therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 70.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 80.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 75.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 80.00 to about 85.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 80.00 to about 90.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 80.00 to about 95.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 80.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 80.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 80.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 80.00 to about 115.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 80.00 to about 120.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 80.00 to about 125.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 80.00 to about 130.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is about 80.00 to about 135.00 times greater. In some embodiments, the amount of therapeutic agent is about 80.00 to about 140.00 times greater.In some embodiments, the amount of therapeutic agent is about 80.00 to about 145.00. In some embodiments, the amount of therapeutic agent is about 80.00 to about 150.00. In some embodiments, the amount of therapeutic agent is increased by about 85.00 to about 90.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 85.00 to about 95.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 85.00 to about 100.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 85.00 to about 105.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 85.00 to about 110.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 85.00 to about 115.00 times greater than the endogenous amount of therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 85.00 to about 120.00 times over the 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 increased by about 85.00 to about 150.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 95.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 90.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 100.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 105.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 110.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 115.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 120.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 125.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 130.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 135.00 times over the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 95.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 105.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 100.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 110.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 105.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 115.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 110.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 115.00 to about 120.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 115.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 115.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 115.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 115.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 115.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 115.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 120.00 to about 125.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 120.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 120.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 120.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 120.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 120.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 125.00 to about 130.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 125.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 125.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 125.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 125.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 130.00 to about 135.00 times greater than the endogenous amount of the therapeutic agent.In some embodiments, the amount of the therapeutic agent is increased by about 130.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 130.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 130.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 135.00 to about 140.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 135.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 135.00 to about 150.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is increased by about 140.00 to about 145.00 times greater than the endogenous amount of the therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 140.00 to about 150.00 fold over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by about 145.00 to about 150.00 fold over the endogenous amount of therapeutic agent. In some embodiments, the amount of therapeutic agent is increased by 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, 4.00, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00 Increases by 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 times.In some embodiments, the amount of 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, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.40, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 5.50, 5.51, 5.52, 5.53, 5.54, 5. Increases by 0, 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 times or more. In some embodiments, the amount of 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, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.40, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 5.50, 5.51, 5.52, 5.53, 5.54, 5. In some embodiments, the amount of therapeutic agent and the amount of equivalent endogenous therapeutic agent (if present) are determined by flow cytometry (FACS).

[0043] In some embodiments, the therapeutic agent is recombinantly expressed by the enucleated cell or its parent cell. 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, expression of the therapeutic agent by the parent cell is transient (e.g., non-persistent). In some embodiments, the parent cell is enucleated before engineering the 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.

[0044] In some embodiments, the therapeutic agent is not naturally expressed (e.g., in the absence of engineering) in the cells from which the enucleated cells are 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 at the intended treatment site in the subject (e.g., a tumor or a particular tissue such as the brain, intestine, lung, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is exogenous to the intended treatment site). In some embodiments, the level of therapeutic agent is not naturally present in the enucleated cells of the parent cell, such as overexpression or underexpression of the therapeutic agent.

[0045] 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 before or after enucleation of the cell. Alternatively, the therapeutic agent may be synthesized by the cell.

[0046] In some embodiments, the enucleated cells comprise multiple therapeutic agents. In some embodiments, the enucleated cells comprise at least one (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, the 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.

[0047] 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 to the parent cell or the enucleated cell. In some embodiments, the polypeptide is synthesized by at least one 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 on 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 in a target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell expresses a cancer biomarker for any cancer described 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 vascular cell. In some embodiments, the endothelial cell is a lymphatic cell.

[0048] In some embodiments, the exogenous polypeptide comprises a cytokine. An "exogenous" polypeptide as provided herein can refer to the presence of a polypeptide exogenous to a cell or enucleated cell, or the amount or level of expression of a polypeptide exogenous to a cell or enucleated cell. In some embodiments, the cytokine comprises a membrane-bound domain. In some embodiments, the exogenous polypeptide comprises a soluble cytokine. For example, the exogenous polypeptide can comprise the extracellular domain or fragment of a cytokine. In some embodiments, the exogenous polypeptide comprises a solubility determined by a turbidimetric solubility assay or a thermodynamic solubility assay by dissolving the exogenous polypeptide in a solvent such as an organic solvent including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or acetonitrile, or an inorganic solvent including water or phosphate-buffered saline (PBS). In some embodiments, the exogenous polypeptide comprises a solubility of at least 0.0001 mg / ml, 0.0005 mg / ml, 0.001 mg / ml, 0.005 mg / ml, 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 5.0 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 500 mg / ml, 1,000 mg / ml, 5,000 mg / ml, 10,000 mg / ml, 50,000 mg / ml, or 100,000 mg / ml. In some embodiments, the cytokine comprises an interleukin. In some embodiments, the cytokine comprises a chemokine. In some embodiments, the cytokine comprises an interferon. In some embodiments, the cytokine comprises a lymphokine. In some embodiments, the cytokine comprises tumor necrosis factor. In some embodiments, the cytokine comprises a monokine. In some embodiments, the cytokine comprises a colony stimulating factor, hi 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.

[0049] In some embodiments, the exogenous polypeptide comprises an interleukin. In some embodiments, the exogenous polypeptide comprises two or more interleukins. 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, the cytokine of the composition is an interleukin or variant thereof, including, but not limited to, wild-type and variant 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.

[0050] 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 about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more 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 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 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 domain or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 37.

[0051] 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 about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 34. In some embodiments, the IL-15 complex is formed by non-covalent assembly of IL-15 with a dimeric or monomeric sushi domain of IL-15 receptor alpha (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 having 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.

[0052] In some embodiments, the exogenous polypeptide comprises a peptide sequence 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 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 at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 39. In some embodiments, the exogenous polypeptide comprises IL-15 RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 40. In some embodiments, the exogenous polypeptide comprises the sushi domain of IL-15RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 41. In some embodiments, the exogenous polypeptide comprises the transmembrane domain of IL-15RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence 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 TNF superfamily member polypeptides 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-B ligand (CD254, OPGL, TRANCE, or ODF), TNF-related weak inducer of apoptosis (APO-3L or DR3L), proliferation-inducing ligand (CD256, TALL-2, or TRDL1), B-cell activating factor (CD257, BLyS, TALL-1, or TNFSF20), LIGHT (CD258 or HVEML), vascular endothelial growth inhibitory factor (TL1 or TL-1A), TNF superfamily member 18 (GITRL, AITRL, or TL-6), or ectodysplasin A (ED1-A1 or ED1-A2).

[0053] In some embodiments, the Therapeutic Agent comprises any one of the immune checkpoint molecules described herein or an immune checkpoint molecule inhibitor for inhibiting any one of the immune checkpoint molecules described herein. Non-limiting examples of immune checkpoint molecules include PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), B7-H3 (also known as 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.

[0054] 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 single domain antibody binds to an immune checkpoint molecule. In some embodiments, the single domain antibody binds to an immune checkpoint molecule and modulates its expression or activity. In some embodiments, the single domain antibody is an inhibitor of immune checkpoint molecule activity or expression. In some embodiments, the single domain antibody is an activator of immune checkpoint molecule activity or expression. In some embodiments, the antibody or single domain antibody binds to PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), B7-H3 (also known as 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, a therapeutic agent comprising an antibody or single domain antibody binds to PD-L1. In some embodiments, a therapeutic agent comprising an antibody or single domain antibody binds to CTLA-4.

[0055] targeting part Described herein, in some embodiments, are cells or enucleated cells comprising a targeting moiety. In some embodiments, the cells are enucleated, such as using the enucleation methods disclosed herein. The targeting moieties described herein are designed to direct the enucleated cells to a target cell or target environment (e.g., tissue) in the subject after delivery (e.g., systemic delivery) of the enucleated cells to the subject. In some embodiments, the targeting moiety is expressed on the surface of the enucleated cells. In some embodiments, the targeting moiety is conjugated to a transmembrane moiety described herein. In some embodiments, the targeting moiety is secreted by the enucleated cells. Enucleated cells comprising a targeting moiety localize to a target cell or target environment within 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 compared to the localization of comparable enucleated cells lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a two-fold increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a five-fold increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a ten-fold increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a twenty-fold increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a fifty-fold increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 5% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety, hi some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 10% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety.In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 20% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 30% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 40% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 50% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 60% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 70% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least an 80% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 90% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment within a subject with at least a 100% increase compared to the localization of a comparable enucleated cell lacking the targeting moiety.

[0056] In some embodiments, the targeting moiety comprises an exogenous antibody or exogenous antigen-binding fragment for targeting a biomarker described herein. In some embodiments, the targeting moiety comprises an exogenous antibody or exogenous antigen-binding fragment for targeting a chemokine receptor or chemokine ligand, or portion thereof, involved in chemokine signaling. In some embodiments, the exogenous antibody is an exogenous single-domain antibody or fragment thereof.

[0057] In some embodiments, the targeting moiety targets a biomarker expressed by or associated with the target cell or microenvironment. In some embodiments, the biomarker may be released by the target cell. The biomarker may indicate the presence of a disease or condition. In some embodiments, the biomarker is expressed by an immune cell responsive to the target cell or microenvironment associated with the disease or condition. In some embodiments, the biomarker may be an epitope or antigen. In some embodiments, a biomarker that includes an epitope may be bound by an antibody that is different from the antibody or antigen-binding fragment thereof that confers the therapeutic property (e.g., a therapeutic agent).

[0058] In some embodiments, the targeting moiety targets a biomarker expressed or released by metastatic cancer cells. For example, cancer cells may originate from one tissue and then metastasize to a different location. In some embodiments, the targeting moiety targets a biomarker expressed or released by endothelial cells. In some embodiments, the endothelial cells are vascular cells. In some embodiments, the endothelial cells are lymphatic cells. In some embodiments, the biomarker is expressed or released by vascular cells. In some embodiments, the biomarker is expressed or released by lymphatic cells.

[0059] In some embodiments, enucleated cells may further comprise (e.g., by manipulation or from the cells from which they are obtained) surface markers that aid in evasion from a subject's immune system. For example, in some embodiments, enucleated cells may comprise CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof. Without being bound by theory, it is believed that CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof help prevent enucleated cells from being phagocytosed by macrophages. Non-limiting examples of cell-matrix receptors and intercellular adhesion molecules include integrins, cadherins, glycoproteins, or heparin sulfate proteoglycans. In some embodiments, the cell-matrix receptor or intercellular adhesion molecule comprises PD-L1, HLA-E, or HLA-G. Non-limiting examples of therapeutic molecules include tumor antigens and immunomodulatory peptides, polyamines, and ATP. In some embodiments, the therapeutic molecule can be recognized by immune cells and induce an immune response. For example, the therapeutic molecule can be 4-1BB or any of the cytokines that induce an immune response described herein. In some embodiments, the enucleated cells are depleted of immune recognition molecules. Non-limiting examples of immune recognition molecules include HLA antigens, proteoglycans, sugar moieties, and embryonic antigens.

[0060] Transmembrane part Described herein, in some embodiments, are cells or compositions comprising enucleated cells comprising at least one transmembrane portion. In some embodiments, the cells are enucleated, such as using the enucleation methods disclosed herein. In some embodiments, the transmembrane portion is attached 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 herein, or a combination thereof. In some embodiments, the transmembrane portion is covalently attached. In some embodiments, the transmembrane portion is a fusion protein comprising a single domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed herein, or a combination thereof. In some embodiments, an exogenous polypeptide is complexed to the transmembrane portion. In some embodiments, the transmembrane portion comprises a full-length protein, a variant thereof, or a fragment thereof. In some embodiments, the transmembrane portion is endogenous to the parent cell that is enucleated to obtain the enucleated cell. In some embodiments, the transmembrane portion can be exogenous to the parent cell or the enucleated cell. In some embodiments, the transmembrane portion comprises a single transmembrane alpha-helix (a bitopic membrane protein). The transmembrane portion comprises a polymorphic transmembrane alpha helix protein. In some embodiments, the transmembrane portion comprises a polymorphic transmembrane beta sheet protein. In some embodiments, the transmembrane portion comprises a type I, type II, type III, or type IV transmembrane protein. Non-limiting examples of transmembrane proteins include any combination of CD4, CD14, glycophorin a (GPA), or integrins.

[0061] In some embodiments, a transmembrane moiety is added to an exogenous polypeptide by modification. For example, a transmembrane moiety can be added to the N-terminus or C-terminus of an exogenous polypeptide to insert the exogenous polypeptide into the cell membrane of an enucleated cell described herein. Non-limiting examples of modifications made to an exogenous polypeptide to add a transmembrane moiety can include the addition of an anchor molecule. The anchor molecule can be any molecule (e.g., a glycolipid) that can insert into and remain in the cell membrane. In some embodiments, the anchor molecule comprises glycosylphosphatidylinositol, farnesyl, palmitic acid, myristic acid, or a combination thereof.

[0062] In some embodiments, the enucleated cell comprises a cytokine. In some embodiments, the cytokine comprises an interleukin. In some embodiments, the cytokine comprises IL-12 (interleukin 12). In some embodiments, the cytokine comprises IL-15 (interleukin 15). In some embodiments, the exogenous cytokine or fragment thereof is conjugated to a transmembrane moiety. In some embodiments, the transmembrane moiety comprises a transmembrane polypeptide. In some embodiments, the exogenous cytokine or fragment thereof is conjugated to the N-terminus of the transmembrane polypeptide. In some embodiments, the exogenous cytokine or fragment thereof is conjugated to the 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, which modification anchors the exogenous cytokine or fragment thereof to the ectoplasmic or cytoplasmic side of the cell membrane of the enucleated cell. In some embodiments, the modification comprises conjugating the exogenous single domain antibody or fragment thereof to glycosylphosphatidylinositol, farnesyl, palmitic acid, myristic acid, or a combination thereof.

[0063] In some embodiments, the transmembrane domain comprises IL-12. In some embodiments, IL-12 is bound 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 include, but are not limited to, B7.1, B7.2, B7-H4, and B7-H3. In some embodiments, IL-12 is bound to a portion of the cell surface protein ligand. In some embodiments, IL-12 is bound only to the transmembrane portion of the cell surface protein ligand. In some embodiments, IL-12 is bound to the transmembrane portion of a B7 family member. In some embodiments, IL-12 is bound to the transmembrane portion of B7.1. As shown in FIG. 8A, the transmembrane (TM) region of B7.1 (B7) is linked to IL-12. The B7 TM is shown across the plasma membrane of the enucleated cell, and the IL-12 portion is linked to the B7 TM on the outer membrane side of the plasma 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 bond. In some embodiments, the IL-12 subunit alpha chain comprises two or more disulfide bonds. In some embodiments, the IL-12 subunit alpha chain comprises fewer than five disulfide bonds. In some embodiments, the IL-12 subunit alpha chain comprises four disulfide bonds. In some embodiments, the IL-12 subunit alpha chain comprises three disulfide bonds. In some embodiments, the IL-12 subunit alpha chain comprises a glycosylation modification. In some embodiments, the IL-12 subunit alpha chain comprises two or more glycosylation modifications. In some embodiments, the glycosylation modifications are N-linked.In some embodiments, the glycosylation modification is N-acetylglucosamine. In some embodiments, the glycosylation modification occurs 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 connecting it to the IL-12 subunit beta chain. In some embodiments, IL-12β comprises a signal peptide and interleukin-12 subunit beta. In some embodiments, IL-12β comprises interleukin-12 subunit beta. In some embodiments, the IL-12 subunit beta chain comprises a disulfide bond. In some embodiments, the IL-12 subunit beta chain comprises two or more disulfide bonds. In some embodiments, the IL-12 subunit beta chain comprises fewer than five disulfide bonds. In some embodiments, the IL-12 subunit beta chain comprises four disulfide bonds. In some embodiments, the IL-12 subunit beta chain comprises three disulfide bonds. In some embodiments, the IL-12 subunit beta chain comprises two disulfide bonds. In some embodiments, the IL-12 subunit beta chain comprises a glycosylation modification. In some embodiments, the IL-12 subunit beta chain comprises two 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 occurs 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 occurs on a tryptophan residue.

[0064] In some embodiments, the transmembrane domain comprises IL-15. In some embodiments, IL-15 can be linked to the transmembrane domain via a linker. In some embodiments, the transmembrane domain comprises IL-15 receptor alpha (IL-15Rα). In some embodiments, IL-15 can be linked to IL-15Rα on the ectoplasmic side of the plasma membrane of an enucleated cell. As shown in FIG. 10A, IL-15Rα spans the plasma membrane of an enucleated cell. The portion of IL-15Rα on the ectoplasmic surface is linked to IL-15 by a linker. At the end of IL-15 opposite the linker connecting it to IL-15Rα is a signal peptide. In some embodiments, IL-15Rα comprises a sushi domain and a transmembrane region. In some embodiments, IL-15Rα comprises a sushi domain, a linker, a proline / threonine-rich region, a transmembrane region, and a cytoplasmic domain. In some embodiments, IL-15Rα comprises a sushi domain, a transmembrane region, and any combination of a linker, a proline / threonine-rich region, or a cytoplasmic domain. In some embodiments, the sushi domain comprises two disulfide bonds. In some embodiments, the sushi domain can bind to IL-15. In some embodiments, IL-15Rα comprises a glycosylation modification. In some embodiments, the sushi domain comprises a glycosylation modificatio...

Claims

1. 1. A method for treating a disease or condition in a subject, comprising:

1. A method of treating a subject comprising administering to the subject enucleated cells comprising: (a) 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 cells on their cell surface in vivo for at least 24 hours.

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 to 3, wherein the enucleated cells induce an endogenous immune response in the subject at a site associated with the disease or condition.

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

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

7. 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. 5. The method of claim 4, wherein the site associated with the disease or condition comprises a lymphatic microenvironment, a liver microenvironment, a spleen microenvironment, a pancreatic microenvironment, an epidermal 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 cells associated with the disease or condition are cancer cells.

12. 11. 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 cells are white blood cells.

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

15. The method of any one of claims 1 to 12, wherein the disease or condition comprises cancer.

16. The method of any one of claims 1 to 15, wherein the IL-12 polypeptide is covalently attached to the cell surface of the enucleated cells for at least 24 hours.

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

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

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

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

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

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

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

24. 23. 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 to 24, wherein the enucleated cells induce the endogenous immune response in a microenvironment.

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

27. 26. The method of claim 25, wherein the microenvironment is a lymphatic microenvironment, a liver microenvironment, a splenic microenvironment, a pancreatic microenvironment, an epidermal microenvironment, or a combination thereof.

28. The method of any one of claims 22 to 24, wherein the enucleated cells induce the endogenous immune response in the vicinity of the cells.

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

30. 29. The method of claim 28, wherein the cell is an immune cell.

31. 31. The method of claim 30, wherein the immune cells are white blood cells.

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

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

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

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

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

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

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

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

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

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

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

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

44. 44. The method of any one of claims 1 to 43, wherein the enucleated cells further comprise an immune checkpoint inhibitor.

45. 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 CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.

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

47. 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. 48. The method of any one of claims 1 to 47, wherein the enucleated cells are depleted of immune recognition molecules.

49. 49. The method of claim 48, wherein the immune recognition molecule comprises an HLA antigen, a proteoglycan, a sugar moiety, a fetal antigen, or any combination thereof.

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

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

52. an exogenous mRNA encoding a cytokine polypeptide; an organelle for expressing said cytokine polypeptide, wherein said cytokine is expressed on the cell surface of said enucleated cell.

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

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

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

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

57. 57. The composition of any one of claims 52 to 56, wherein the cytokine polypeptide is expressed on the 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. 58. The composition of any one of claims 52-57, wherein the enucleated cells express 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 greater than the enucleated cells expressing an equivalent naturally occurring abundance of the cytokine polypeptide.

59. 59. The composition of claim 58, wherein the abundance of the cytokine polypeptide and the abundance of the equivalent naturally occurring cytokine polypeptide are determined by FACS.

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

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

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

63. 63. The composition of any one of claims 52 to 62, wherein the enucleated cells further comprise an immune checkpoint inhibitor.

64. 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 CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.

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

66. 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 to 66, wherein the enucleated cells are depleted of immune recognition molecules.

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

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

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

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

72. 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 CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.

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

74. 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. 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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