Methods for extracorporeal development of autologous car-x cells for the treatment of non-malignant diseases

Abstract

The present disclosure relates to methods of treating autoimmune diseases and allergic disorders by generating chimeric antigen receptor (CAR) cells (e.g., T cells, NK cells, and / or myeloid cells) that target and promote clearance of, e.g., by killing, pathogenic cells. The methods described herein can be performed at the bedside in a subject-connected, closed-loop continuous-flow manner.

Description

METHODS FOR EXTRACORPOREAL DEVELOPMENT OF AUTOLOGOUS CAR-X CELLS FOR THE TREATMENT OF NON-MALIGNANT DISEASESCLAIM OF PRIORITYThe present application claims priority to U.S. Provisional Patent Application No. 63 / 523,434 filed on June 27, 2023. The entire contents of the foregoing application are incorporated herein by reference in their entirety.BACKGROUNDMethods for introducing a genetic payload into a desired cell type derived from a subject are useful in a number of biomedical fields. The creation of autologous cells directed to reduce levels of endogenous pathogenic cells in subjects has been fairly well established in both animals and subjects particularly using Chimeric Antigen Receptor (CAR) gene modifications. While a variety of ex vivo methods have been developed for the introduction of payloads such as nucleic acids into peripheral blood derived mononuclear cells (PBMCs), the cost, complexity and safety of these methods limit broad access and use in all but the most serious diseases. Recent efforts to address these issues involve introduction of the payload, for example, a gene cargo, and creation of therapeutic cells in the subject (gene therapy). However, these methods must contend with many challenges including delivery of the payload, biodistribution, off target impact, accurate dosing, immunogenicity to the delivery vehicle, and limitations in the ability to re-dose. As such, there is a need for new devices and methods to improve the delivery of payloads to peripheral blood derived cells, particularly for non-malignant conditions with diminished banners relative to malignant conditions including lower costs, the ability to re-treat, the use of non-integrating gene editing or introduction, and limited or no lymphodepletion.SUMMARYThe present disclosure features methods of providing a subject with a population of payload-associated cell complexes (PACCs) to treat a non-malignant disease in a subject, as well as related compositions thereof. In a first aspect, provided herein is a method of providing a subject with a population of payload-associated cell complexes (PACCs) to treat a non-malignant disease in the subject, comprising (i) providing a population of peripheral blood cells, e.g., apopulation of peripheral blood cells from the subject, wherein a peripheral blood cell in the population of peripheral blood cells includes a binding target; and (ii) cxtracorporcally contacting the population of peripheral blood cells with a payload including a chimeric antigen receptor (CAR) construct under conditions (e.g., time, temperature) sufficient for association of the payload with the cell including the binding target, wherein the conditions are not sufficient for entry of the payload into the cell with which it is associated, thus forming a population of PACCs; and (iii) introducing the population of PACCs into the subject, thereby providing subject with a population of PACCs for the treatment of a non-malignant disease. In an embodiment, the method comprises the creation of autologous cells useful for reducing the levels of endogenous pathogenic cells in the subject. In an embodiment, the non-malignant disease comprises an allergic, autoimmune, or fibrotic disease.In some embodiments of the foregoing aspect, the population includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1,000, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, or more PACCs. In some embodiments, the population includes at least 100,000 or more PACCs. In some embodiments, the population includes at least 1 million or more PACCs. In some embodiments, the population includes at least 10 million or more PACCs. In some embodiments, the population includes at least 100 million or more PACCs.In some embodiments of the foregoing aspect, the percentage of peripheral blood cells associated with the payload in the population is greater than 0.5%, 1%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the total cells in the population. In some embodiments, the percentage of peripheral blood cells associated with the payload in the population is greater than 50%, 60%, 70%, 80%, 90%, 95%, or more, or more of the total cells in the population. In some embodiments, the percentage of peripheral blood cells associated with the payload in the population is between 30%-90% total cells, e.g., 50%-80% total cells in the population. In some embodiments of the foregoing aspect, the population of cells is selected from monocytes, macrophages, neutrophils, basophils, eosinophils, stem cells, mast cells, and dendritic cells. In some embodiments, the population of cells is selected from B cells, T cells, effector or regulatory T cells, hematopoietic stem cells (HSCs), natural killer cells, NK T cells, g / d T cells, and plasma cells. In some embodiments, the peripheral blood cells may include T cells, B cells, natural killer cells, and / or myeloid cells.In some embodiments of the foregoing aspect, the conditions sufficient for association of the payload with a peripheral blood cell within the population include contacting the peripheral blood cells with the payload for between about 0 to about 10 hours, e.g., about 1 hour to about 9 hours, about 2 hours to about 8 hours. In some embodiments, the contacting of the peripheral blood cells with the payload is between about 5 minutes and 30 minutes. In some embodiments, the contacting of the peripheral blood cells with the payload is between about 30 minutes and 1 hour. In some embodiments, the contacting of the peripheral blood cells with the payload is between about 1 hour and 1.5 hours. In some embodiments, the binding of the payload to the peripheral blood cells occurs at a selected temperature 0 to 40 degrees Celsius (e.g., 4 °C, 37 °C). In some embodiments, the payload is disposed on the surface of a peripheral blood cell within the PACC.In some embodiments of the foregoing aspect, the payload includes a nucleic acid, a peptide, a polypeptide, or a small molecule. In some embodiments, the nucleic acid includes DNA or RNA.In some embodiments of the foregoing aspect, the PACCs are formulated in or on a delivery vehicle. In some embodiments, the delivery vehicle includes a lipid nanoparticle, viral vector, vesicle, or a liposome in which the payload is disposed. In some embodiments, the concentration of the delivery vehicle is higher than the concentration of peripheral blood cells in the sample. In some embodiments, the concentration of the delivery vehicle is lower than the concentration of peripheral blood cells in the sample. In some embodiments, the concentration of the delivery vehicle is optimized for binding to peripheral blood cells in the subject sample. In some embodiments, the delivery vehicle is a viral vector. In some embodiments, the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the LNP is a cationic LNP. In some embodiments, the delivery vehicle is a vesicle. In some embodiments, the vesicle is an extracellular' vesicle, exosome, a nanovesicle, or a microvesicle.In some embodiments, the delivery vehicle is disposed on the surface of a cell within the PACC. In some embodiments, the delivery vehicle is targeted to a surface molecule. In some embodiments, the delivery vehicle includes a targeting moiety to a surface molecule. In someembodiments, the surface molecule is a cell-specific surface protein. In some embodiments, the targeting moiety is an antibody.In some embodiments of the foregoing aspect, the association of the payload to the cell is covalent or non-covalent.In some embodiments of the foregoing aspect, the subject has or is diagnosed with having a disease or disorder, e.g., a non-malignant disease. In some embodiments, the non-malignant disease is an allergic, autoimmune, or fibrotic disease. In some embodiments, the non-malignant disease comprises systemic lupus erythematosus (SLE), myasthenia gravis (MG), pemphigus vulgaris (PV), coeliac disease, Crohn’s disease, Grave’s disease, Hashimoto’s thyroiditis, multiple sclerosis, rheumatoid arthritis, Sjogren’s disease, ulcerative colitis, vasculitis, allergic asthma, atopic dermatitis (e.g., eczema), atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, eosinophilic esophagitis (atopic and non-atopic), hay fever, severe eosinophilic asthma (SEA), and fibrosis. In some embodiments, the method further includes administering to the subject an additional agent, e.g., an immune- stimulatory agent.In some embodiments of the foregoing aspect, the introducing in step (iii) is carried out by a subject-connected closed- loop device. In some embodiments, between 1-10% of the cells in the population include the binding target. In some embodiments, the method further includes a) connecting a parenteral inlet to the subject, wherein the parenteral inlet is adapted to parenterally receive blood from the subject; b) permitting the blood, or a fraction thereof, from the subject to pass through the parenteral inlet to an extracorporeal cell binding (ECCB) module configured to allow extracorporeal formation of a PACC; c) maintaining conditions in the ECCB module such that cells from the subject’s blood and a payload form a PACC; and d) delivering the PACC to the subject via a parenteral outlet adapted to parenterally administer PACC to the subject.In some embodiments, the parental inlet, the ECCB module, and the parental outlet are in fluid connection. In some embodiments, each of the steps (a)-(d) occurs in a closed-loop system.In some embodiments of the foregoing aspect, i) a subject cell is taken from the subject, ii) the subject cell is contacted with a payload to form a PACC, and iii) the PACC introduced into the subject, and i-iii occur in less than 0.5, 1, 2, 4, 6, or 8 hours.In another aspect, the disclosure features a method of forming in a subject a cell that is transformed or transduced with a payload, including:introducing a population of extracorporeally formed PACCs into the subject under conditions sufficient for transformation or transfection of the cell of the PACC with the payload of the PACC in the subject; and allowing the transformation or transfection ^thereby forming in a subject with a cell transformed or transfected with a payload.In yet another aspect, the disclosure provides a subject-connected closed-loop device for use with any one of the method of any of the foregoing aspects.The present disclosure features a method of preparing a population of payload-associated subject-derived cell complexes (PACCs), as well as related methods of use thereof. In an embodiment, a PACC includes a cell (e.g., a cell derived from a subject) in contact with a payload (e.g., a therapeutic agent, e.g., a nucleic acid), wherein under certain conditions, the payload does not enter into the cell it is in contact with. In an embodiment, the cell is in direct contact with the payload. In an embodiment, the cell is indirectly in contact with the payload. In an embodiment, total PBMCs are bound to the payload. In an embodiment, the cell is selected from a B cell, T cell, hematopoietic stem cell (HSC), natural killer cell, and plasma cell. In an embodiment, the PACCs are introduced into the subject using a subject-connected closed-loop device.In another aspect, the present disclosure features a method of providing a subject with a population of PACCs, comprising providing a population of cells, e.g., a population of cells from the subject, wherein a cell in the population of cells comprises a binding target. In an embodiment, the method further comprises extracorporeally contacting the population of cells with a payload under conditions (e.g., time, temperature) sufficient for association of the payload with the cell comprising the binding target, wherein the conditions are not sufficient for entry of the payload into the cell with which it is associated, thus forming a population of PACCs. In another embodiment, the method further comprises introducing the population of PACCs into the subject.In another aspect, the present disclosure features a method of forming, in a subject, a cell that is provided with a payload, the method comprising introducing a population of extracorporeally formed PACCs into the subject under conditions sufficient for transformation or transfection of the cell of the PACC with the payload of the PACC in the subject; and allowing the transformation / transfection. In an embodiment, the method further comprises a) connecting a parenteral inlet to the subject, wherein the parenteral inlet is adapted to parenterally receiveblood from the subject; b) permitting the blood, or a fraction thereof, from the subject to pass through the parenteral inlet to an extracorporeal cell binding (ECCB) module configured to allow extracorporeal formation of a PACC; c) maintaining conditions in the ECCB module such that cells from the subject’s blood and a payload form a PACC; and d) delivering the PACC to the subject via a parenteral outlet adapted to parenterally administer PACC to the subject.In another aspect, the present disclosure provides a method of making a PACC, using methods described herein under conditions sufficient to prevent uptake or entry of the payload into the cells of the PACC.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limited.BRIEF DESCRPTION OF THE FIGURESThe skilled person in the art will understand that the drawings described below are for illustration purposes only.FIG. 1 is a schematic representing the general workflow of an exemplary system, combining an apheresis module with a binding module into a single extracorporeal closed-loop system.FIG. 2 is a schematic of an exemplary binding chamber module containing an agitation tray. The schematic illustrates how the agitation tray moves to gently rock the sample.FIG. 3 is a diagram of an exemplary extracorporeal closed-loop system. The extracorporeal closed-loop system comprises collection, binding, and reinfusion of peripheral blood derived mononuclear cells (PBMCs) into a single closed-loop procedure. The extracorporeal closed-loop system may comprise two connected components: (1) apheresis component collects apheresis material from a subject and returns washed particle-bound cells, and (2) particle binding component that is used to dose a sample, such as a lentivirus particle (e.g., PBMC Sample 1) to PBMCs in the binding chamber module and to clear unbound particles prior to infusion. The particle binding chamber module may enable administration of PBMC Sample 1 to a subject and may provide consistent and controlled contact with PBMCs duringincubation. Incubation under the conditions provided in the particle binding chamber module may enable target cell binding, c.g., T cell binding, which after infusion, may undergo in vivo genetic modification.FIG. 4 is a diagram of an exemplary extracorporeal patient-connected closed-loop system, e.g., the Lupagen Xynvivo™ System. Numbered elements of the system delineate key steps in the process: (1) PBMC collection, (2) preparation of PBMCs for binding, (3) addition of delivery vehicle comprising the payload (e.g., a nanoparticle or a lentiviral particle, e.g., PBMC Sample 1), (4) binding of the delivery vehicle to target cells, (5) removal of unbound particles by washing, and (6) reinfusion.FIG. 5 is a diagram illustrating binding of an exemplary sample, e.g., a lentiviral particle (e.g., PBMC Sample 1), to a target cell, e.g., a T cell, (e.g., a CD3+T cell). Key: Env: envelope, scFv: short chain variable fragment. The lentiviral particle, e.g., the PBMC Sample 1, may bind to a receptor, e.g., the CD3 T cell receptor, on the target cell.FIG. 6 is a graph illustrating that an exemplary sample, e.g., a lentiviral particle (e.g., PBMC Sample 1), may produce higher rates of target cell, e.g., T cell (e.g., CD3+T cell) activation and transduction in an in vitro culture relative to control samples, e.g., an empty vector control. 1 pL of the lentiviral particle, e.g., PBMC Sample 1, transduced approximately 90% of the target T cells, while 20 pL of the control particle, e.g., the empty vector control particle, transduced approximately 40% of target T cells.FIGs. 7 A and 7B are graphs illustrating that an exemplary sample, e.g., a lentiviral particle (e.g., PBMC Sample 1), may preferentially bind to target cells, e.g., T cells (e.g., CD3+T cells) (FIG. 7A). Following binding of the lentiviral particle, e.g., PBMC Sample 1, to target T cells, e.g., CD3+T cells, endocytosis may occur within between 2-8 hours (FIG. 7B).Endocytosis may increase at higher temperatures, e.g., 37°C, relative to lower temperatures, e.g., 43 °C.FIGs. 8A, 8B, and 8C are graphs illustrating that an exemplary sample, e.g., a lentiviral particle (e.g., PBMC Sample 1), may preferentially bind to target cells, e.g., T cells (e.g., CD3+T cells) at 24 and 48 hours (FIG. 8A). Target T cells, e.g., CD3+T cells, may be viable following binding of the lentiviral particle, e.g., PBMC Sample 1 (FIG. 8B). Within approximately 2 hours, 70% of target T cells, e.g., CD3- T cells, are bound by the lentiviral particle, e.g., PBMC Sample1 ; at approximately 24 hours, between 80-85% of target cells are bound by the lentiviral particle at both 4°C and 37°C (FIG. 8C).FIGs. 9A, 9B, 9C, and 9D are representative flow plots of gene expression in nonactivated target cells, e.g., T cells (e.g., CD3+T cells), following administration of an exemplary sample, e.g., a lentiviral particle (e.g., PBMC Sample 1), to a population of PBMCs (FIGs. 9C and 9D) relative to an untreated population of PBMCs (FIGs. 9A and 9B).FIGs. 10A and 10B are graphs depicting binding of an exemplary sample, e.g., a lentivirus particle (e.g., PBMC Sample 1), to target cells, for example, T cells, and that also provide information about cell viability. PBMC Sample 1-T cell binding (FIG. 10A) may be evaluated, e.g., by flow cytometry, for example by using labels, e.g., fluorescent labels, for the lentivirus particle and one or more cell markers, e.g., fluorescently labeled anti fusion glycoprotein (Cocal glycoprotein) and anti-CD3, to identify T cells (CD3+) and non-T cells (CD3‘). PBMC Sample 1-bound cells may be identified by positive co-staining for the lentivirus particle marker and the one or more cell markers, e.g., fusion glycoprotein and CD3. (FIG. 10B) To assess whether the payload-associated cell complexes (PACCs), e.g., PBMC Sample 1-bound cells may be suitable for administration, e.g., via reinfusion, into a subject, cell viability may be evaluated, e.g., by staining cells with acridine orange propidium iodide and performing imaging cytometry.FIGs. 11A and 11B are graphs depicting selective binding of an exemplary sample, e.g., a lentivirus particle (e.g., PBMC Sample 1), to target cells (FIG. 1 IB), for example, T cells, extracorporeally relative to control (FIG. 11 A). Appropriate imaging methods, e.g., flow cytometry, may be used to evaluate binding , e.g., by detecting expression of CD3 and fusion glycoprotein (e.g., fusion Cocal glycoprotein).FIGs. 12A and 12B are graphs illustrating that administration of an exemplary sample, e.g., a lentiviral particle, e.g., PBMC Sample 1, via the extracorporeal closed-loop system promotes target cell activation and transduction, e.g., T-cell activation and transduction, in an in vitro model of extracorporeal delivery. T cell activation and transduction may be evaluated, e.g., following extracorporeal incubation of PBMC Sample 1 with apheresis material, on the extracorporeal closed- loop system (e.g., at a multiplicity of infection of 2) for 1 hour followed by a wash. Cells may be collected and cultured in vitro, and subsequently may be harvested from the culture, e.g., on Days 3 and 8, and analyzed for expression of biomarkers and the CARpayload, e.g., by flow cytometry for CD25 (FIG. 12A) and anti-CD19 CAR expression (FIG. 12B). PBMC Sample 1 comprising the fusion glycoprotein, c.g., fusion Cocal glycoprotein, may selectively target and transduce CD3+T cells.FIGs. 13A and 13B are images illustrating that an exemplary sample, e.g., a lentiviral particle, e.g., PBMC Sample 1, preferentially transduces target cells, e.g., CD3+ T cells, to express a payload, e.g., an anti-CD19 CAR, following incubation on the extracorporeal closed- loop system (FIG. 13B). As a control, PBMC control cells may be circulated through the extracorporeal closed-loop system in the absence of the sample, e.g., PBMC Sample 1 (FIG. 13A). Cells may be harvested from in vitro culture, e.g., on Day 8 post-extracorporeal incubation, and expression of target cell-specific biomarkers, e.g., CD3, and the payload, e.g., the anti-CD19 CAR, may be detected by methods known in the art, e.g., by immunostaining.FIGs. 14A and 14B are graphs illustrating that, following extracorporeal incubation of an exemplary sample, e.g., a lentiviral particle (e.g., PBMC Sample 1), administration of the resulting PACCs to, for example, Nalm6 tumor-bearing immune compromised mice (e.g., NSG MHC I / II KO mice engrafted with 2.5 x 105 Nalm6-ffLuc tumor cells intravenously, e.g., 5 days prior to administration with PACCs), may produce efficient generation of functional CAR T cells. Apheresis material may be attached to the extracorporeal closed-loop device, washed, resuspended in binding buffer at a target of 100 x 106cells / mL in 10-20 mL total volume in the binding chamber module, and incubated with PBMC Sample 1 at a multiplicity of infection of 2 for one hour at room temperature. Following incubation, cells may be washed, harvested from the ECGD device, and administered to the tumor-bearing mice intraperitoneally, e.g., on Day 0. To determine whether T cell activation and transduction is dose-dependent, multiple doses (e.g., 25 x 106cells (FIG. 14A) and 15 x 106cells (FIG. 14B)) may be administered to the subjects, e.g., the tumor bearing mice. CAR T cells may be subsequently detected in the peripheral blood of the subjects, e.g., the Nalm6-tumor bearing mice, by detection methods known in the ail, for example flow cytometry. Key: data points represent the mean; error bars represent the standard error of the mean; n = 4 animals in the PBMC control groups; n = 7 animals in the PBMC Sample 1 dose groups.FIGs. 15A, 15B, and 15C are images and graphs illustrating that extracorporeal delivery of an exemplary sample, e.g., a lentiviral particle (e.g., PBMC Sample 1), may result in potent anti-tumor activity in a subject, e.g., Nalm6 tumor-bearing immune deficient mice (e.g., obtainedby the method described above). PACCs may be prepared as described above and administered to the tumor-bearing mice intraperitoneally, e.g., on Day 0. Tumor burden may be measured, e.g., by longitudinal monitoring of bioluminescence (FIGs. 15B and 15C) from the Nalm6 tumor cells in mice that received PBMC Sample 1 -bound PACCs from Donor 1 (left panels) and Donor 2 (right panels) (FIG. 15A). Key: data points represent the mean; error bars represent the standard error of the mean; n = 4 animals in the PBMC control groups; n = 7 animals in the PBMC Sample 1 dose groups.DETAILED DESCRIPTIONThe present patent application discloses a novel method to generate chimeric antigen receptor (CAR) cells (e.g., CAR-X cells), such as CAR-T, CAR-Natural Killer, or CAR-B cells for the treatment of antibody-driven diseases such as autoimmune diseases, allergic disorders, and fibrotic diseases. For example, methods described herein are beneficial for treating systemic lupus erythematosus (SLE), commonly known as lupus. Lupus is a complex autoimmune disorder characterized by aberrant immune system activation and subsequent attack on healthy tissues, resulting in chronic inflammation and organ damage. Current treatment options for lupus mainly focus on alleviating symptoms and controlling the disease progression; however, there is a pressing need for innovative therapeutic approaches that address the underlying causes. The use of CAR-T for the treatment of lupus has been described in animal models and clinical studies (Atisha-Fregoso, Y et al. 2021. Meant to B: B cells as a therapeutic target in systemic lupus erythematosus. The Journal of clinical investigation, 737(12), el49095; Lustgarten, J et al. 1995. Specific elimination of IgE production using T cell lines expressing chimeric T cell receptor genes. European journal of immunology, 25(10), 2985-2991; Mackensen, A et al. 2022. Anti- CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nature medicine, 28(10), 2124-2132). Thus, the targeting of B cells has proven efficacious in these subjects. However, the current modality of production of autologous CAR-T cells follows the manufacturing process used for the approved CAR-T products for B cell oncology. This production modality is expensive, is hard to scale to large number of subjects, is usually combined with a lymphodepletion regimen, is correlated with high lot failure rates, and has proved to be challenging to re-dose.CAR-X cells are known to be generated and used by a method comprising the following steps: (1) collecting cells from a subject, e.g., by aphcrcsis, (2) selecting for desired cell types (e.g., T cells, B cells, or myeloid cells), (3) engineering the selected cells to express CARs, (4) expand the CAR-modified cells, and (5) administer the expanded population of CAR-modified cells into the subject. The inventors surprisingly found that this process could be significantly shortened and robustly enhanced by splitting the 3rdstep and modifying the 4thand 5thsteps as follows: (3a) contact the selected cells with pay loads in conditions that allow for association of the pay loads and the cells but that do not allow for uptake or entry of the pay loads into the cells, (3b) administer (e.g., by infusion) the resulting complexes comprising the cells and the associated payloads to the subject, thereby removing the conditions preventing uptake or entry of the payloads into the cells, (4) generate CAR-X cells by allowing the endogenous cellular transcriptional and translational machinery to facilitate expression of the payloads, and (5) expand the CAR-X cells in the subject by relying on endogenous cell replication mechanisms. As steps (1) - (3b) of this method are facilitated to occur extracorporeally within a closed- loop system, it allows for a revolutionary bed-side treatment that can be completed within a short time frame. Furthermore, the CAR-X cells are generated in the subject, thereby (a) reducing the cost of the treatment and (b) decreasing the time between apheresis and CAR-X administration by removing the traditional extracorporeal CAR-X generation and CAR-X expansion steps.The present disclosure features devices and methods for the delivery of a payload to a cell extracorporeally to provide a population of payload-associated cell complexes (PACCs), useful in the prevention or treatment of a disease or disorder in a subject. The devices described herein provide for a method of transporting a population of cells, e.g., peripheral blood mononuclear cells (PBMCs), outside of a subject for a short time, contacting these cells with a payload (e.g., a payload enclosed within a delivery vehicle or associated with a delivery vehicle) to establish binding of the cells with the pay load forming PACCs, and then delivery of the PACCs back to the subject. In an embodiment, the subject is connected to the device throughout the entire process. The methods described herein may be carried out in conditions sufficient to allow for binding of the payload with the subject-derived cells, but not to allow for entry of the pay load into the cells. These methods provide many benefits over current ex vivo methods, including cost, speed, and simplicity by enabling in vivo payload delivery and activity. In addition, delivery of a payload to a cell extracorporeally to form PACCs has many benefits overnon -extracorporeal in vivo methods as the device and methods described herein may increase the control of dose, reduce residual free particles, limit off target impact and lower immunogenicity to the vehicle further enabling redosing, potentially improving safety and efficacy profile and enhancing the modes of therapeutic delivery which are not possible in vivo.DefinitionsUnless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.The articles “a” and “an” are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.As used herein, the terms “bead”, “microparticle”, “nanoparticle”, or “particle” is a structure of any shape and of any composition that is manipulatable or behaves according to predetermined principles and can act as a vehicle for pay load delivery. Such beads, microparticles, nanoparticles, or particles can be comprised of any suitable material, such as glass or ceramics, and / or one or more polymers, such as, for example, nylon, polytetrafluoroethylene, polystyrene, polyacrylamide, sepharose, agarose, cellulose, cellulose derivatives, agarose or dextran, a metal, a metal alloy (e.g., FeO), lipid, protein, polymer, lipopeptide, or other additional materials. In an embodiment, the particle is a lipid nanoparticle. In an embodiment, the particle comprises cationic lipids. In another embodiment, the particle comprises a targeting moiety (e.g., a ligand or an antibody targeting a cell-specific protein).As used herein, “closed-loop” refers to a system that is continuous, in which the contents of the system are connected to the subject for the duration of the method performed. For example, in the closed-loop system described herein, the population of cells derived from thesubject is kept in the system for the duration of the method, and can be associated with a payload cxtracorporcally then rc-introduccd to the subject, e.g., without the subject being moved off-line or disconnected. In an embodiment, the closed-loop systems described herein may allow for introduction of exogenous materials, such as buffers and payload, but the subject-derived cells are not removed from the system and manipulated externally before being returned to the subject. In an embodiment, the system may further be “subject-connected,” in which the system is connected to the subject for a duration of the process (e.g., the entire process).As used herein, “continuous flow” refers to the flow of blood from a subject to the modular bedside system and back to the subject in which non-target cells (e.g red blood cells) generally return to the subject whereas target cells can be adapted by an association binding module in the device and then returned to the subject; all in a closed-loop, subject-connected manner and in real-time. The term “continuous flow” does not exclude operations within a closed-loop system that occur while a cell population or sub-population is shunted or deposited for some period of time, into a holding, or incubating chamber or receptacle within the system nor does it preclude small amounts of cells removed offline for sampling and analytical testing.As used herein, the term “extracorporeal” refers to outside of the subject. For example, the devices and methods described herein allow for associating a payload with subject-derived cells extracorporeally, or outside the subject. The extracorporeal devices and methods herein may form a closed-loop with the subject. In an embodiment, in the extracorporeal environment, blood flows from the subject to the extracorporeal system and back to the subject in a closed- loop. In an embodiment, in the methods described herein, in which non-target cells generally return to the subject without modification, whereas target cells are bound with or by the payload in the extracorporeal environment and then returned to the subject as PACCs.As used herein, the term “payload” is any agent that capable of being delivered to a subject-derived cell (e.g., a cell isolated from a subject described herein). Pay loads may include nucleic acids, peptides, proteins, lipids, small molecules, or any combination thereof. Exemplary payloads include DNA, RNA (e.g., mRNAs, modified mRNAs (mmRNAs), small interfering RNA (siRNA), micro RNA (miRNA)), oligonucleotides (e.g., antisense oligonucleotides), ribozymes, mini-circles, plasmids, immune-stimulating nucleic acids, antisense RNAs, antagomir, antimir, supermir, mini-circles, plasmids, vesicle (e.g., extracellular vesicles (EVs) and exosomes), small molecules, drug formulations, proteins, amino acids, DNA editingsystems, activation receptors, or other material. Payloads may be formulated in a manner such that they can enter and transform, position, or otherwise rcproducibly integrate DNA, RNA, mRNA siRNA, shRNA, mini-circles, plasmids, vesicles, EVs, exosomes, small molecules, drug formulations, proteins, amino acids, peptides, DNA editing systems, activation receptors, or other material in a target subject-derived cell. In some embodiments, payload is provided encapsulated or positioned in a vehicle as defined herein. In some embodiments, the payload is provided encapsulated or positioned in a vehicle and the vehicle is adapted to specifically bind or otherwise associate with a predetermined target cell. In some embodiments, the payload is provided encapsulated or positioned in a vehicle and the vehicle is adapted to specifically bind or otherwise associate with a predetermined target cell through the presence of a targeting molecule on the vehicle.A “payload-associated cell complex” (PACC), as used herein, comprises a cell and a payload, wherein the payload and the cell are incubated together extracorporeally with sufficient proximity or affinity for one another (e.g., prior to introduction into a subject) and in conditions such that, when the payload-associated cell is introduced into a subject, the payload and the cell with which it is associated such that the payload can enter the cell, e.g., traverse the cell membrane or enter the cell through a vesicle-mediated delivery mechanism. In an embodiment, the pay load is associated with the cell, e.g., through an interaction with the cell membrane or an entity associated with the cell membrane (e.g., ionic charge, hydrophobicity, a peptide, polypeptide, or lipid, e.g., an antibody or receptor). In an embodiment, the pay load is covalently associated with the cell, e.g., through a covalent linkage to the cell membrane or an entity associated with the cell membrane (e.g., a protein or lipid, e.g., an antibody or receptor). In another embodiment, the payload is associated with the cell through ligand binding to a target on the cell (e.g., an antibody binding to a cell-specific target). For clarity, a PACC does not include a cell into which the payload has already entered, e.g., extracorporeally. In an embodiment, the payload is disposed on or within a vehicle, such as a lipid or polymeric nanoparticle, extracellular’ vesicles, exosome, or a viral vector.A “subject” , as this term is used herein, is a human. In embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). The subject may be a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)). The subject may have a disease ordisorder, e.g., a non-malignant disease. In some embodiments, the non-malignant disease is an allergic, autoimmune, or fibrotic disease. In some embodiments, the non-malignant disease comprises systemic lupus erythematosus (SLE), myasthenia gravis (MG), pemphigus vulgaris (PV), coeliac disease, Crohn’s disease, Grave’s disease, Hashimoto’s thyroiditis, multiple sclerosis, rheumatoid arthritis, Sjogren’s disease, ulcerative colitis, vasculitis, allergic asthma, atopic dermatitis (e.g., eczema), atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil- associated diseases, eosinophilic esophagitis (atopic and non-atopic), hay fever, severe eosinophilic asthma (SEA), and fibrosis. The subject may display one or more symptoms or a disease or disorder, e.g., a non-malignant disease.As used herein, the term “CAR-X cell” refers to a cell, e.g., a modified cell (e.g., a modified subject-derived cell), that is capable of reducing (e.g., by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more) one or more target (e.g., pathogenic) cells. CAR-X cells are created by the methods described herein, e.g., by (1) isolating cells from a subject, (2) optionally, isolating one or more populations of cells (e.g., T cells) from the cells, (3) contacting the cells with a gene-editing payload under conditions sufficient to prevent uptake or entry of the payload into the cells, (4) washing the resultant cell-payload complexes, (5) administering (e.g., by infusion) the complexes to the subject, thereby removing the conditions preventing uptake or entry of the payload into the cell and subsequently allowing the endogenous cellular transcriptional and translational machinery to facilitate expression of the payload, giving rise to modified cells referred to herein as “CAR-X cells.” Exemplary CAR-X cells refer to modified immune cells described herein (e.g., subject-derived cells modified by the present methods). In some embodiments, the immune cells comprise T cells, B cells, or myeloid cells, e.g., monocytes, macrophages, dendritic cells, or granulocytes (e.g., neutrophils, mast cells, eosinophils, and / or basophils). Exemplary target cells (e.g., pathogenic cells) include T cells (e.g., CD8+ T cells, CD4+ T cells, Treg cells (e.g., gamma / delta T cells), stem cell memory T cells, lymphoid progenitor cells, HSCs, NK cells, and NKT cells), B or plasma cells (e.g., IgE producing cells, plasma B cells, or B cells producing autoantibodies), myeloid cells (e.g., monocytes, macrophages, dendritic cells, and granulocytes, e.g., neutrophils, mast cells, eosinophils, and basophils), macrophage cells (e.g., pro-fibrotic macrophage cells (MC3 macrophage cells)), and fibroblasts.As used herein “pathogenic cells” refer to cells that drive disease states, e.g., of autoimmune diseases, allergic disorders, or fibrotic diseases. Pathogenic cells may express autoantibodies or other inflammatory markers. Autoantibodies and inflammatory markers, e.g., of autoimmune diseases, have been extensively characterized in the art. A non-limiting list of exemplary autoantibodies includes, for example, ANA, SMA, LKM-1, LKM-2, LKM-3, HLA- DR52A, and HLA-DR3. A non-limiting list of exemplary inflammatory markers includes, for example, CSF, G-CSF, IFN-y, IL-1, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, TNF-a, and TNF- .As used herein, the term “vehicle” or a “payload delivery vehicle” refers to a payload carrier adapted to encapsulate, hold or otherwise at least partially contain the payload. Generally, a vehicle is adapted or formulated in a manner that it is capable of transporting the payload in the extracorporeal environment and / or the in vivo environment. Generally, the vehicle is formed of, contains or comprises, for example, a liposome, a virus (including, without limitation, viral components, viral particles, polypeptide sequences comprising a virus or component or particle thereof, and / or nucleic acids encoding a virus or component or particle thereof), polymer (including, without limitation, polymer-based or containing particles or nanoparticles), inorganic, lipid (including, without limitation, lipid-based or containing micelles, particles or nanoparticles), a hybrid lipid-polymer nanoparticle, an EV, an exosome, a dendrimer, a metal nanoparticle, a nanosphere, a silica nanoparticle, and / or a microbubble.As used herein, the terms “vehicle binding” or “bound vehicle” or “binding” are used to refer to a single time or reproducible interaction between the vehicle and a host cell, more specifically, the surface or outer membrane of the host cell. Vehicle binding may comprise, but is not limited to, ligand-targeted binding, binding facilitated by a chemical interaction, charge, receptor-mediated binding, hydrophobicity, or viral binding to a host cell.DeviceProvided herein are devices, methods, and compositions for the treatment of a disease or disorder comprising a closed-loop, continuous flow system. The devices and methods may allow for subject-connected bedside delivery of a payload to a subject extracorporeally in a single, convenient outpatient procedure, avoiding the complexity of traditional methods of delivering cellular therapies. The devices described herein comprise multiple modules, including at least anapheresis module and a binding module. The system may include cell processing to wash, buffer exchange and removal of free vehicle or payload before rcintroduction to the subject.Generally, in an aspect, the device described herein comprises an apheresis module capable of collecting peripheral blood mononuclear cells (PBMCs) in a process termed leukapheresis. The apheresis module may comprise an apheresis module known in the art or may be custom designed. The device described herein also comprises a binding module. In some embodiments, the apheresis module and the binding module operate as a single unit. In some embodiments, the apheresis module and the binding module are connected to form a single closed-loop fluid pathway. In some embodiments, the disposable set fluid pathway is connected to the subject’s circulatory system via venepuncture for the duration of the extracorporeal system procedure.In some embodiments, each module and / or chamber of the device is temperature- controlled. In some embodiments, the temperature may be between about 0°C to about 37 °C. In some embodiments, the temperature may be 4°C. In some embodiments, the temperature may be room temperature. In some embodiments, the temperature may be 37°C.Apheresis ModuleIn an embodiment, the subject is directly connected to the device, specifically the apheresis module. In an embodiment, the apheresis module comprises a leukapheresis system to separate peripheral blood mononuclear cells (PBMCs) (e.g., the Fresenius Kabi Amicus Separator™ or a Terumo Optia™) or subtypes of PBMCs (e.g., T cells, B cells, monocytes, and NK cells) from the plasma and red blood cells. The number of PBMCs or cells of a certain subtype of PBMC can be standardized by altering the processed blood volume based on the subject’s complete blood count (CBC) at time of collection.Binding ModuleThe devices described herein also comprise a binding module, in which the subject-derived cells are contacted with a pay load to form a population of PACCs. The binding module may facilitate binding of the cells to a payload. In an embodiment, the payload is packaged with or in a delivery vehicle, such as a lipid nanoparticle. In an embodiment, the delivery vehicle is a polymeric nanoparticlc. In an embodiment, the delivery vehicle is a viral vector. In an embodiment, the delivery vehicle is a vesicle including but not limited to an extracellular vesicle (EV), a microvesicle (MV), an exosome, or exosome-like vesicle (ELV). In an embodiment, thedelivery vehicle is introduced to the cell or binding module via a sterile port. In an embodiment, the delivery vehicle is introduced via a sterile port using a syringe.In an embodiment, the plasma is removed from the sample. In an embodiment, the plasma is exchanged with a binding buffer. In an embodiment, the binding buffer is a buffer that facilitates the binding of a payload delivery vehicle. In an embodiment, the binding buffer is a buffer that decreases the binding of a payload delivery vehicle to increase selective binding. The binding buffer utilized in the method as disclosed herein can be a binding buffer known in the art. In some embodiments, the binding buffer is optimized (e.g., pH, ionic strength, comprising proteins and / or peptides, tonicity) for efficient binding of a payload delivery vehicle to target cells.In certain embodiments, the concentration of cells in an apheresis sample is determined. In an embodiment, the cells are diluted in order to obtain a lower concentration of cells. In an embodiment, the cells are concentrated in order to obtain a higher concentration of cells. In an embodiment, the desired concentration of cells will be dependent on payload being delivered and the disease being treated. In an embodiment, the cells are concentrated within the apheresis module. In an embodiment, the cells are concentrated within an independent process step (e.g. spinning membrane, elutriation, centrifugation, absorbance, buoyancy, etc.). In an embodiment, the separated cells are shunted to a binding module.In an embodiment, the binding system comprises a binding chamber. The binding chamber contains the cell sample collected and separated during apheresis or sample collected and separated during apheresis that has been further prepared for binding. In an embodiment, the binding chamber is a medical grade blood collection bag. In an embodiment, the binding chamber is a cell culture chamber or flask. In an embodiment, the binding chamber is a beaker. In an embodiment, the binding chamber is set on a binding chamber platform. The binding chamber platform allows the cells to mix with the payload delivery vehicle. Mixing can be performed by shaking, tilting, rotating, spinning, or other mode to optimize vehicle contact and binding with cells. In an embodiment, the binding chamber platform controls the temperature of incubation of the cells and the payload delivery vehicle to optimize binding of the payload delivery vehicle and the cells, thereby forming stable PACCs.In an embodiment, incubation of the cells and the payload delivery vehicle within the binding chamber occurs for a sufficient amount of time that allows for the binding of the payload delivery vehicle and the cells, thereby forming stable PACCs.In an embodiment, the binding chamber platform mixes the cell and payload delivery vehicle mixture. In an embodiment, the mixing allows for binding of the payload delivery vehicle to the cell while preserving cell viability (e.g., not damaging the cells during mixing). In an embodiment, the platform is a rocker of a specific angle.In an embodiment, the binding chamber platform is a nutator mixer. In an embodiment, the binding chamber platform is an orbital mixer. In an embodiment, the binding chamber platform in a shaker mixer. In an embodiment, the rate of mixing and duration may be variable. In an embodiment, the binding chamber platform is an agitator. In an embodiment, the binding chamber platform facilitates mixing of the cells and the payload delivery vehicle during incubation in the binding chamber. In an embodiment, mixing of the cells and the payload delivery vehicle during incubation is constant. In an embodiment, mixing of the cells and the payload delivery vehicle during incubation is intermittent.In an embodiment, the apheresis module is directly linked to the binding module. In an embodiment, the apheresis module and binding module may be separated by one or more processing modules responsible for processing the sample, such as washing the sample, storing the sample. In an embodiment, the devices described herein comprises at least 1, 2, 3, 4, 5, 6 or more processing modules (e.g., washing modules).Processing ModulesIn an embodiment, the cells are washed in a washing module after incubation in the binding chamber. During this step, the cells may be washed and free pay load delivery vehicles (e.g., vehicles that are not bound to a cell) are removed from the cell sample. In an embodiment, the washing step comprises centrifugation of the cells. In an embodiment, the washing step comprises filtration of the cells. In an embodiment, the washing step comprises spinning membrane filtration. In an embodiment, the washing step comprises density gradient centrifugation. In an embodiment, the washing step comprises immuno-dcnsity cell isolation.In an embodiment, the binding buffer is exchanged with reinfusion buffer. In an embodiment, the reinfusion buffer is a saline buffer (e.g., 0.9% saline).In an embodiment, after the binding buffer is exchanged with reinfusion buffer and the cells are ready for reinfusion into the subject, the cells are reinfused into the subject. In an embodiment, the cells are reinfused into the subject through the same initial port. In anembodiment, the cells are reinfused into the subject through a different port. In an embodiment, the subject is connected to the system throughout the entirety of the process.In some embodiments, cell enrichment prior to payload introduction is contemplated. In certain embodiments, cells may be enriched based on physiological properties such as density, size, acoustics, charge, binding properties, etc. Methods and systems employing surface proteinbased target cell separation / purification techniques are often included and / or preferred. In such methods, a targeting moiety is utilized to specifically bind a cell surface protein on a known or predetermined cell population (including mixtures of cell populations sharing that cell surface protein). Often in such techniques a targeting moiety such as an antibody or binding fragment thereof is bound on or to a support (e.g., microbubble, magnetic particle, nanoparticle, bead, etc.) and the targeting moiety-bound support is introduced to a sample and permitted to mix within the sample and become bound with one or more target cells in the sample. Thereafter a process is used to separate or otherwise remove the support-bound cell from the remainder of the sample.In some embodiments, following cell enrichment, at least 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%, or more) of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof. For example, in some embodiments, following cell enrichment, at least 60% of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof. In some embodiments, following cell enrichment, at least 70% of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof. In some embodiments, following cell enrichment, at least 80% of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof. In some embodiments, following cell enrichment, at least 90% of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof. In some embodiments, following cell enrichment, at least 95% of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof. In some embodiments, following cell enrichment, at least 99% of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof. In some embodiments, following cell enrichment, more than 99% of the cells are bound to the targeting moiety, e.g., an antibody or binding fragment thereof.The present methods and systems contemplate an overarching requirement to not alter or harm the structure or function of the target cells nor adversely affect the medium in which they exist though that medium may be altered or replaced. Moreover, the present methods andsystems are directed to increasing the specificity and speed with which target cells are scparatcd / purificd.In any and all of the aspects as described herein, a subject is coupled to the system in a closed-loop fashion, with an inlet conduit coupled or connected to the subject to provide peripheral blood as input to the system. The inlet conduit receives blood directly from the circulation of the subject which is passed to the cell purification system. The cell separation module is designed, at a minimum, to perform collection of specific or known cell populations or collections present in the blood. This includes cell separation from blood components and optionally one or more enrichment steps. The cell purification systems contemplated herein are often adapted to purify cells from blood or other body fluids based on surface protein expression, size, shape, granularity, buoyancy, density, genetic identification, or any combination thereof. Mechanisms for cell separation / purification often according to the presently included methods and systems include buoyancy, centrifugal force, filtration, elutriation, sonic, electrical, magnetic, and / or acoustic properties.The systems and approaches described herein can also involve one or more steps of cell selection to isolate or enrich for cells that have been modified in a connected system.In some embodiments, the system can execute a program of cell purification in an automated manner with no or minimal operator input once the program is initiated.In conjunction with any or all of the techniques described in further detail below, filtration is or can be used as a method of reducing the volume of samples and / or separating sample components based on their ability to flow through or be retained by the filter. Sample centrifugation and / or leukapheresis (or aspects thereof) may be part of this sample treatment. Filtration, sample centrifugation and / or leukapheresis (or aspects thereof) may occur before and / or after target cell separation / purification.Timing is considered an important overall aspect of the system as disclosed herein. As the system is parenterally subject-connected, it is important that the time the system is parenterally connected with the subject be kept within tolerated periods of time. All contemplated system embodiments herein are configured with this aspect in mind. Tolerance to prolonged connection with the system is understood to be variable, with an understanding of the present inventors that reducing this total time period and maximizing the effectiveness of the present systems within that time period results in inter-module cycle time period reductions andbetter tolerance of the subjects to the treatment protocol delivered by the systems. As such the present systems arc configured to operate within target cycle times. This configuration relates to the purification technologies and modules utilized, the detector mechanisms and functionalities, the disposable aspects of the system, reagents, software, and / or the structural and / or functional arrangement of the system to ensure efficient and safe operation and quick passage of blood / sample / target cells within the systems and between or within modules.Overall, a treatment protocol, beginning with the presently described closed-loop parenteral connection of the subject with the system and ending with removal of the parenteral connection from the patent, will last between 1 to 6 hours. Most frequently this time period is between 2 to 4 hours. Within these ranges there are subranges as contemplated in the embodiments of the present systems and their operation. These ranges may be anywhere in the range of at or about 1 hour, at or about 1.5 hours, at or about 2 hours, at or about 2.5 hours, at or about 3 hours, at or about 3.5 hours, at or about 4 hours, at or about 4.5 hours, at or about 5 hours, at or about 5.5 hours, at or about 6 hours, or shorter than 2 hours. In certain limited embodiments the time period may be above 6 hours. It is noted that there are a variety of subject safety issues that are balanced in the process of the automated manner the present systems are configured and how they operate. In this regard, timing and safety considerations are balanced to ensure not only that the proper or desired treatment is provided in a quick and efficient manner, but also that safety protocols are rigorously adhered to in the system to ensure that no harm is caused by the operation of the system. Such issues related to the safety of modules, sample processing procedures, and reagents are discussed herein and integrated in the most frequent embodiments.In various embodiments, a cell washing unit can be integrated with the presently described systems, for example, placed between an apheresis module and the cell purification module and / or after the cell purification module, to remove one or more components of a cell suspension produced by the apheresis module or cell purification module, or in order to place the cells into a medium or solution better conducive to cell enrichment procedure. A cell washing unit generally includes a source of cell wash solution, such as a reservoir or bag of wash solution, connected via conduit or tubing to the washing unit. The flow of cell wash solution can be controlled by a valve controlled by a processor. A cell washing unit can include a centrifugal unit similar to the centrifugal unit in an apheresis device; cells enter the washing unit, are mixedwith wash solution introduced from a reservoir or other source of wash solution via conduit connected to the washing unit, and the suspension is spun to concentrate cells. One or more rounds of cell washing and re-suspension can be performed before the cells pass out of the cell washing unit. A cell washing unit can be interfaced with a detector configured to detect one or more properties of the cells or cell suspension, e.g., cell number, solution density, solution pH, solution ionic strength, etc.PACC Testing and VerificationIn an embodiment, samples before and after binding are obtained. In an embodiment, a sample is taken from the subject. In an embodiment, a sample is taken from the subject before apheresis. In an embodiment, the sample is assayed for cell type and cell health (e.g., cellularity (CBC), cell viability, cell count). In an embodiment, the sample is assayed for cell markers indicative of various immune cell subtypes (e.g., cell markers of B cells, monocytes, hematopoietic stem cells (HSCs), T cells, or NK cells). In an embodiment, a sample is taken after apheresis (e.g., leukapheresis). In an embodiment, a sample is obtained to determine cell counts. In an embodiment, cell counts are collected to determine cell concentration. In an embodiment, a sample is obtained to determine cellularity. In an embodiment, a sample is obtained to determine cell viability. In an embodiment, a sample is taken after the plasma is exchanged with the binding buffer. In an embodiment, samples are taken after introduction of a payload delivery vehicle to the cells. In an embodiment, the samples taken after introduction of a pay load delivery vehicle at various time points. In an embodiment, a sample is taken at the completion of the binding step. In an embodiment, the sample taken at the completion of the binding step may be assayed for cell count, cell viability, cell type (e.g., detection of cell markers), and binding efficiency. In an embodiment, a sample of PACCs may be placed in an incubator to mimic in vivo conditions to assess uptake of payload, transcription and / or translation of the payload (e.g., mRNA), cell viability or other parameters useful in assessing or predicting clinical outcomes. In an embodiment, a sample is taken after the cells arc washed and free pay load delivery vehicles (e.g., vehicles that are not bound to a cell) are removed from the cell sample. In an embodiment, a sample may be taken after the sample is washed and resuspended in an infusion buffer prior to return to the subject. In an embodiment a sample may be taken at various timepoints during the return to the subject. In an embodiment a post-procedure subject sample may be taken after the subject has been disconnected from the system.Subject-derived CellsIn one aspect, a payload delivery vehicle targets a cell isolated from a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the cell is isolated from a type of tissue. In some embodiments, the tissue is from a particular organ. In some embodiments, the organ or tissue is any of the following: brain, spinal cord, liver, blood, epidermis, neural, bone, kidney, cardiac, lung, endocrine, connective, muscle, or endothelial. In some embodiments, the organ or tissue is derived from peripheral blood. In some embodiments, the cell is an immune cell (e.g., a PBMC). In some embodiments, the immune cell is a T cell, e.g., a CD8+ T cell, a CD4+ T cell, a regulatory T cell (Treg) cell (e.g., a gamma / delta T cell), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell (HSC), a natural killer cell (NK cell), or a natural killer T cell (NKT cell). In some embodiments, the immune cell is a B cell, e.g., a plasma B cell. In some embodiments, the immune cell is a myeloid cell, e.g., a monocyte, a macrophage, a dendritic cell, or a granulocyte, e.g., a neutrophil, a mast cell, an eosinophil, and / or a basophil. Processing of Subject-derived CellsIn some embodiments, the methods include isolating cells (e.g., immune cells described above) from the subject, preparing, processing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more separation / preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separ ation, washing, and / or incubation and contacting of the payload vehicle to the cell. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.PayloadsNucleic AcidsIn some embodiments, the payload is a nucleic acid including, for example, messenger RNA (mRNA), antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antamir), messenger- RNA-interference complementary RNA (iRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), transfer RNA (tRNA), ribosomal RNA (rRNA), modified nucleic acids, and the like. In some embodiments, the nucleic acid is comprised in a nanoparticle to deliver said nucleic acid to a subject-derived cell to generate CAR-X cells that produce an exogenous polypeptide of interest, or to alter expression of an endogenous polypeptide of interest. In some embodiments, the nanoparticle can be any as described herein.In some embodiments, the nucleic acid is modified. In some embodiments, the modification comprises a non-canonical nucleotide. Certain non-canonical nucleotides, when incorporated into synthetic RNA molecules, can reduce the toxicity of the synthetic RNA molecules, in part by interfering with binding of proteins that detect exogenous nucleic acids. Non-canonical nucleotides that have been reported to reduce the toxicity of synthetic RNA molecules when incorporated therein include: pseudouridine, 5 -methyluridine, 2-thiouridine, 5- methylcytidine, N6-methyladenosine, and certain combinations thereof. However, the chemical characteristics of non-canonical nucleotides that can enable them to lower the in vivo toxicity of synthetic RNA molecules have, until this point, remained unknown. Furthermore, incorporation of large amounts of most non-canonical nucleotides, for example, 5-methyluridine, 2-thiouridine, 5-methylcytidine, and N6-methyladenosine, can reduce the efficiency with which synthetic RNA molecules can be translated into protein, limiting the utility of synthetic RNA molecules containing these nucleotides in applications that require protein expression. In addition, while pseudouridine can be completely substituted for uridine in synthetic RNA molecules without reducing the efficiency with which the synthetic RNA molecules can be translated into protein, in certain situations, for example, when performing frequent, repeated transfections, synthetic RNA molecules containing only adenosine, guanosine, cytidine, and pseudouridine can exhibit excessive toxicity.In some embodiments, the payload is an antisense oligonucleotide (ASO), or an antisense compound. In general, antisense compounds hybridize to a target nucleic acid and effectsmodulation of gene expression activity or function, such as transcription, translation or splicing. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi is a form of antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. This sequence- specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of any one of a variety of diseases.In some embodiments, the nucleic acid is a circular or circularized nucleic acid. In some embodiments, the circular or circularized nucleic acid comprises DNA. In some embodiments, the circular or circularized nucleic acid comprises RNA. Circular RNA is useful in the design and production of stable forms of RNA. The circularization of an RNA molecule provides an advantage to the study of RNA structure and function, especially in the case of molecules that are prone to folding in an inactive conformation (Wang and Ruffner, 1998). Circular RNA can also be particularly interesting and useful for in vivo applications, especially in the research area of RNA-based control of gene expression and therapeutics, including protein replacement therapy and vaccination. In some embodiments, the circular or circularized RNA can be, but not limited to, any as described in PCT / US2020 / 063494, PCT / US2020 / 034418, PCT / US2021 / 031629, or PCT / US2021 / 033276, which are incorporated herein by reference in their entireties.Antibodies and Antigen-Binding FragmentsIn some embodiments, the pay load is, or encodes, an antibody. An antibody is an immunoglobulin or immunoglobulin-like molecule, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and includes similar molecules produced during the immune response in any vertebrate (e.g., humans, goats, rabbits, and mice, and non-mammalian species such as shark immunoglobulins). The antibody can be an intact immunoglobulin and / or an antibody fragment or antigen binding fragment that specifically binds to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding toother molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M-l greater, at least 104M-l greater or at least 105M-l greater than a binding constant for other molecules in a biological sample). In some embodiments, the antibody is an engineered antibody, for example, a chimeric antibody (e.g., humanized murine antibodies). In some embodiments, the antibody is a heteroconjugate antibody (e.g., a bispecific antibody). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology 3rd unology, Ed., W.H. Freeman & Co., New York, 1997.In some embodiments, the payload is, or encodes, an antigen-binding fragment. In some embodiments, the antigen-binding fragment is a digestion fragment, specified portions, derivatives, and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and / or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. In some embodiments, the antigen-binding fragment is selected from the group of a single chain variable fragment (scFv), fragment antigen-binding region (Fab), F(ab’)2, Fab’, a monovalent fragment consisting of the VL, VH, CL, and CH domains, or a variable fragment (Fv). In some embodiments, the antigen-binding fragment is a single domain antibody (dAb) fragment (Ward et al. (1989) Nature 341:544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Any of the above-noted antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.Immune ReceptorsIn some embodiments, the payload is, or encodes, an immune receptor. Traditional cancer treatments mainly comprise surgery, radiotherapy, chemotherapy, and stem cell transplants.In some embodiments, the immune receptor comprises a target- specific binding element, e.g., an antigen binding domain. The choice of antigen binding element depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands for the antigen moiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease, alloimmune disease, fibrotic disease, and cancer cells.In some embodiments, the immune receptor is a T cell receptor (TCR). In some embodiments, the TCR is a wildtype TCR, a high affinity TCR, or a chimeric TCR. In some embodiments, the TCR is modified to have a higher affinity for the target cell (e.g., a cancer cell) antigen than a wildtype TCR. In some embodiments, the chimeric TCR may include chimeric domains, such as the TCR comprises a co-stimulatory signaling domain at a C terminal of at least one of the chains. In some embodiments, the TCR may include a modified chain, such as a modified alpha or beta chain. Such modifications may include, but are not limited to, N- deglycosylation, altered domain (such as an engineered variable region to target a specific antigen or increase affinity), addition of one or more disulfide bonds, entire or fragment of a chain derived from a different species, and any combination thereof.Techniques for engineering and expressing T cell receptors include, but are not limited to, the production of TCR heterodimers which include the native disulphide bridge which connects the respective subunits (Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S. Pat. No. 6,080,840).In some embodiments, the TCR comprises specificity to a target cell antigen. The target cell antigen may include any type of protein associated with a target cell. For example, the target cell antigen may be chosen to recognize a particular disease state of the target cell. Thus, examples of cell surface markers that may act as ligands for the antigen binding domain of the TCR including those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.In some embodiments, the immune receptor is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain comprises a target- specific binding domain (e.g., an antigen binding moiety) that binds to an antigen on a target cell. Examples of cell surface markers that may act as an antigen that binds to the antigen binding domain of the CAR include those associated with viral, bacterial and parasitic infections, autoimmune disease, alloimmune disease, fibrotic disease, and cancer cells. In some embodiments, the intracellular domain, or thecytoplasmic domain, comprises a costimulatory signaling region and an intracellular signaling region.In some embodiments, the target- specific binding domain comprises an antigen- specific antibody or fragment thereof. The target- specific binding domain can be directed to any desired antigen. In some embodiments, the target- specific binding domain may consist of an Ig heavy chain which may in turn be covalently associated with Ig light chain by virtue of the presence of CHI and hinge regions, or may become covalently associated with other Ig heavy / light chain complexes by virtue of the presence of hinge, CH2 and CH3 domains. In the latter case, the heavy / light chain complex that becomes joined to the chimeric construct may constitute an antibody with a specificity distinct from the antibody specificity of the chimeric construct. Depending on the function of the antibody, the desired structure and the signal transduction, the entire chain may be used or a truncated chain may be used, where all or a part of the CHI, CH2, or CH3 domains may be removed or all or part of the hinge region may be removed. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, multispecific antibodies, VHH, VNARs, and minibodies. In some embodiments, the CAR comprises a target- specific binding domain directed to a single target antigen. In some embodiments, the CAR comprises more than one target- specific binding domain directed to a single target antigen. In some embodiments, the CAR comprises more than one target-specific binding domain directed to more than one target antigen.In some embodiments, a CAR comprises an extracellular domain comprising an autoantigen or a fragment thereof. In some embodiments, the autoantigen binds to an autoantibody. In some embodiments, the autoantibody is expressed on a B cell. In some embodiments, the B cell is a memory B cell.In some embodiments, the CAR further comprises a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. In exemplary embodiments, the hinge region is capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as atumor cell. The flexibility of the hinge region permits the hinge region to adopt many different conformations.In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1 amino acid. For example, hinge regions include glycine polymers (G)n, glycine- serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142).In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789.The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge region.In some embodiments, the CAR comprises a transmembrane domain capable of spanning the plasma membrane of a cell (c.g., an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane.In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, a subject CAR comprises a CD8a transmembrane domain. In some embodiments, a subject CAR comprises a CD8a hinge domain and a CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. Tolerable variations of the transmembrane and / or hinge domain will be known to those of skill in the art, while maintaining its intended function.The transmembrane domain may be combined with any hinge domain and / or may comprise one or more transmembrane domains described herein.The transmembrane domains described herein, such as a transmembrane region of alpha, beta or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4- 1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9, can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains or intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR.In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.Preferred examples of intracellular signaling domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmicsignaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunorcccptor tyrosinc-bascd activation motifs or IT AMs. Examples of ITAM- containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD70a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule comprises a cytoplasmic signaling sequence derived from CD3 zeta. In some embodiments, the cytoplasmic domain can be designed to comprise the CD3 zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of a CAR. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, ICOS, CD2, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.In some embodiments, the cell comprising a CAR further comprises a transgenic payload (e.g., a fourth generation CAR). In some embodiments, the transgenic payload is an inducible payload, e.g., expression of the payload is inducible. In some embodiments, the transgenic payload is a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is IL-1, 11-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, or IL-23.In some embodiments, the CAR comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR component can be a transmembrane domain, a hinge domain, or a cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptor (KIR), e.g., KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, K1R2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural cytotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptor (FcR), e.g., CD16, and CD64; and Ly49 receptors, e.g.,LY49A, LY49C. The NKR-CAR described herein may interact with an adaptor molecule or intracellular signaling domain, e.g., DAP12. Exemplary configurations and sequences of CAR molecules comprising NKR components are described in International Publication No. WO2014 / 145252, the contents of which are hereby incorporated by reference.Gene editing systemsIn some embodiments, the payload is a nucleic acid, a polypeptide, or a combination, encoding a gene editing system. In some embodiments, the nucleic acid editing system edits DNA. In some embodiments, the nucleic acid editing system edits RNA. In some embodiments, the nucleic acid editing system is delivered to the target cell comprising a method comprising a vector comprising a nucleic acid sequence encoding the gene editing system. In some embodiments, the nucleic acid editing system is delivered to the target cell comprising a method comprising a polypeptide comprising the gene editing system. In some embodiments, the gene editing system is delivered to the target cell comprising a method comprising a nucleic and a polypeptide.In some embodiments, the gene editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas editing system, an engineered Transcription Activator-Like Effector Nuclease (TALEN) system, a zinc finger nuclease (ZFN), a meganuclease system, or a retrotransposon system (e.g., a PiggyBac transposon or Tcl / mariner-like system). These gene editing systems may be used according to any method known in the art.Other PayloadsIn another aspect, the payload described herein may be a small molecule, lipid, protein, or polypeptide. In some embodiments, the payload is a small molecule. Examples of small molecules include, but are not limited to, small organic molecules or compounds such as any conventional agent or drug known to those of skill in the art. Exemplary small molecules include natural products, therapeutic agents (e.g., chemotherapeutic agents, antibiotics, antivirals), amino acids, or derivatives or combinations thereof. In an embodiment, the small molecule may be therapeutically active itself or may be a prodrug, which become active upon further modification. In an embodiment, a small molecular derivative retains some or all of the therapeutic activity as compared to the unmodified agent, while in another embodiment, the small molecule is a prodrug that lacks therapeutic activity but becomes active upon furthermodification. In another aspect, the payload may be a lipid. In another embodiment, the payload may be a protein (c.g., an antibody, enzyme, cytokine).Delivery of Pay loadsIn some embodiments, the payload delivery vehicle associated with the cell is a lipid nanoparticle (LNP). LNPs are useful for the delivery of nucleic acids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc.In some embodiments, the LNP comprises a cationic lipid. Cationic lipids are amphiphilic molecules that generally contain a lipophilic region containing one or more hydrocarbon groups, and a hydrophilic region containing at least one positively charged polar head group. These lipids may become cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. Cationic lipids facilitate entry of macromolecules such as nucleic acids into the cytoplasm through the cell plasma membrane by forming a positively charged (total charge) complex with macromolecules, such as nucleic acids.In some embodiments, the cationic lipid is an amino lipid (or a pharmaceutically acceptable salts thereof (e.g., hydrochloride salt)). Suitable amino lipids useful in the invention include those described in WO 2012 / 016184, incorporated herein by reference in its entirety.In some embodiments, the payload delivery vehicle associated with the cell is an expression vector. A nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a retrovirus. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non- proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, e.g., a gammaretroviral vector. Agammaretroviral vector may include, e.g., a promoter, a packaging signal ( ), a primer binding site (PBS), one or more (c.g., two) long terminal repeats (LTR), and a transgcnc of interest, c.g., a gene encoding a CAR. A gammaretroviral vector may lack viral structural gens such as gag, pol, and env. Exemplary gammaretroviral vectors are described, e.g., in Tobias Maetzig et al., "Gammaretroviral Vectors; Biology, Technology and Application" Viruses. 2011 Jun; 3(6); 677- 713.In some embodiments, the retrovirus is a lentiviral vector. Lentiviral vectors are known in the ail, see Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873- 9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Pat. No. 6,013,516; and U.S. Pat. No.5, 994, 136, which are each incorporated herein by reference in its entirety. In general, these vectors are configured to carry the essential sequences for selection of cells containing the vector, for incorporating foreign nucleic acid into a lentiviral particle, and for transfer of the nucleic acid into a target cell.A commonly used lentiviral vector system is the so-called third-generation system. Third- generation lentiviral vector systems include four plasmids. The “transfer plasmid” encodes the polynucleotide sequence that is delivered by the lentiviral vector system to the target cell. The transfer plasmid generally has one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequences into the host genome. See Dull et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol. 72:8150-57.Third-generation systems also generally include two “packaging plasmids” and an “envelope plasmid.” The “envelope plasmid” generally encodes an Env gene operatively linked to a promoter. In some embodiments, the envelope plasmid comprises a promoter selected from a cytomegalovirus promoter and a synthetic promoter comprising the U3 region of a modified MoMuLV LTR conjugated to the myeloproliferative sarcoma virus enhancer (MND).The lentiviral particles produced generally include an RNA genome (derived from the transfer plasmid), a lipid-bilayer envelope in which the Env protein is embedded, and other accessory proteins including integrase, protease, and matrix protein. As used herein, the term “lentiviral particle” is intended to mean a viral particle that includes an envelope, has one or more characteristics of a lentivirus, and is capable of invading a target host cell.The lentiviral particles may comprise a glycoprotein. Glycoproteins are proteins comprising oligosaccharide chains covalently attached to amino acid side-chains, wherein the carbohydrate is attached to the protein in a cotranslational or posttranslational modification, e.g., by glycosylation. Glycoproteins are important integral membrane proteins and also comprise a large portion of secreted extracellular proteins. The most common types of glycoproteins are N- linked, wherein sugars are attached to nitrogen, usually on the amide side-chain of asparagine, and O-l inked glycoproteins, wherein sugars are attached to oxygen, typically on serine or threonine, but also on tyrosine or non-canonical amino acids such as hydroxylysine and hydroxyproline. In some embodiments, a lentiviral particle as used herein may comprise one or more (e.g., two, three, four, or more) glycoproteins. For example, in some embodiments, the lentiviral particle as used herein may comprise two or more glycoproteins. In some embodiments, the lentiviral particle as used herein may comprise three or more glycoproteins. In some embodiments, the lentiviral particle as used herein may comprise four or more glycoproteins. In some embodiments, the lentiviral particle as used herein may comprise more than four glycoproteins.In some embodiments, the glycoprotein is a naturally occurring glycoprotein. In some embodiments, the glycoprotein may comprise a modified glycoprotein, a synthetic glycoprotein or a recombinant glycoprotein. In some embodiments, the glycoprotein is a modified glycoprotein. In some embodiments, the modified glycoprotein may be a naturally occurring glycoprotein that is modified by enzymatic or chemical treatment. In some embodiments, the modified glycoprotein may comprise a modified sugar, e.g., a modified sugar or sugar derivative. In some embodiments, the modified sugar may comprise a modified thiol group, a halogen, a sulfonyloxy group, a halogenated acetamido group, a mercaptoacetamido group, or a sulfonated hydroxy acetamido group.In some embodiments, the glycoprotein is a synthetic glycoprotein. In some embodiments, the synthetic glycoprotein comprises an amino acid sequence that is non-naturally occurring.In some embodiments, the glycoprotein is a recombinant glycoprotein. In some embodiments, the recombinant glycoprotein comprises an amino acid sequence that differs from the sequence of a naturally occurring glycoprotein by a substitution, deletion, insertion, or addition of one or more (e.g., two, three, four, or more) amino acids. For example, in someembodiments, the recombinant glycoprotein comprises one or more (e.g., two, three, four, or more) substitutions. In some embodiments, the recombinant glycoprotein comprises one or more (e.g., two, three, four, or more) deletions. In some embodiments, the recombinant glycoprotein comprises one or more (e.g., two, three, four, or more) insertions. In some embodiments, the recombinant glycoprotein comprises one or more (e.g., two, three, four, or more) additions. In some embodiments, the recombinant glycoprotein comprises at least two or more substitutions, deletions, insertions, and / or additions. In some embodiments, the recombinant glycoprotein comprises an amino acid sequence comprising one or more fragments of amino acid sequences derived from naturally occurring glycoproteins. In some embodiments, the recombinant glycoprotein comprises an amino acid sequence derived from the sequences of at least two (e.g., at least three, four, five, or more) naturally occurring glycoproteins. For example, in some embodiments, the recombinant glycoprotein comprises an amino acid sequence derived from the sequences of at least three or more naturally occurring glycoproteins. In some embodiments, the recombinant glycoprotein comprises an amino acid sequence derived from the sequences of at least four or more naturally occurring glycoproteins. In some embodiments, the recombinant glycoprotein comprises an amino acid sequence derived from the sequences of at least five or more naturally occurring glycoproteins.In some embodiments, the glycoprotein is selected from the group consisting of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey virus (VSNJV), or Bas-Congo Virus (BASV), Cocal glycoprotein, EHV-1 gB, EHV-1 gC, EHV-1 gD, lubricin, and vesicular stomatitis virus envelope glycoprotein (VSV- G) (Humbert, O. et al. (2016). Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentivir al Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular therapy : the journal of the American Society of Gene Therapy, 24(7), 1237-1246. https: / / doi.org / 10.1038 / mt.2016.70). In some embodiments, the glycoprotein is selected from the group consisting of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), or Cocal Vesiculovirus (COCV), Cocal glycoprotein, and vesicular stomatitis virus envelope glycoprotein (VSV-G). In someembodiments, the glycoprotein is a viral fusion protein or a functional variant thereof, e.g., Cocal glycoprotein or a functional variant thereof.In some embodiments, the lentiviral particle as used herein may be one as described in U.S. Patent Applications 2021 / 0147871, 2022 / 0017920, International Patent Application No. PCT / US2022 / 013947, U.S. Patent Publication Nos. US20220017920, US20240141375, and International Patent Publication Nos. W02020106992A1 and WO2022164935A1, the disclosures of which are hereby incorporated by reference in their entireties. In other embodiments, the lentiviral particle as used herein is one described in any one of WO 2021 / 146627 or WO 2022 / 146891. In some embodiments, the lentiviral particle as used herein is one described in any one of U.S. Patent Publication Nos. US20220017920, US20240141375, and International Patent Publication Nos. W02020106992A1 and WO2022164935A1. In other embodiments, the payload is delivered using one of the methods described in WO 2014 / 011987, WO 2018 / 013918, WO 2017 / 079499, WO 2019 / 067425, or WO 2022 / 081694.In some embodiments, a polynucleotide encoding the lentiviral particle is provided to the host cell. In some embodiments, the polynucleotide encodes a polypeptide, e.g., a glycoprotein (e.g., a Cocal glycoprotein), or a functional variant thereof.In some embodiments, the protein sequence of the Cocal glycoprotein shares at least 75% (e.g., at least 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO: 1. For example, in some embodiments, the protein sequence of the Cocal glycoprotein shares at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the protein sequence of the Cocal glycoprotein shares at least 85% sequence identity with SEQ ID NO: 1. In some embodiments, the protein sequence of the Cocal glycoprotein shares at least 90% sequence identity with SEQ ID NO: 1. In some embodiments, the protein sequence of the Cocal glycoprotein shares at least 95% sequence identity with SEQ ID NO: 1. In some embodiments, the protein sequence of the Cocal glycoprotein shares at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the protein sequence of the Cocal glycoprotein shares at least 100% sequence identity with SEQ ID NO: 1.Table 1. Lentiviral Particle SequenceIn some embodiments, the vector is an adenoviral (Ad) vector. Adenoviral vectors are vectors which are based on or derived from the genome of a virus of the family Adenoviridae. There are at least 57 serotypes of human Adenoviruses, Adl -AD57, that form seven “species” A- G. All serotypes are similar in general structure and the functions of most proteins, but certain unique protein functions contribute to the unique properties of the serotype and the species. In some embodiments, the adenovirus is a human adenovirus from group A, B, C, D, E, F, or G. In some embodiments, the adenovirus is a human adenovirus from group B or C or D. In some embodiments, the adenoviral vector comprises a plurality of adenoviral early genes. The E1A proteins are the translation products of the first gene transcription events from the adenovirus genome within the nucleus at the E1A region.In some embodiments, the Ad vector is engineered to target specific cells based on chosen surface markers. In some embodiments, the Ad vector is equipped with a targeting adapter that recognizes a surface marker on a cell. In some embodiments, the targeting adapter is an antibody. In some embodiments, the target cells secrete a therapeutic molecule. In some embodiments, the target cell expresses a therapeutic molecule on the surface of the cell.In some embodiments, the viral vector is an adeno-associated viral vector. The properties of non-pathogcnicity, broad host range of infectivity, including non-dividing cells, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer.In some embodiments, the AAV vector comprises an intact AAV capsid from a single AAV serotype. In some embodiments, the AAV vector comprises an artificial capsid which contains one or more fragments of more than one AAV serotype (e.g., a chimeric capsid). In some embodiments, the AAV vector is selected from one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74 serotype. In some embodiments, the AAV vector is a pseudotyped AAV, such as a recombinant AAV (rAAV) 1 / 8 or rAAV2 / 9.The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigene, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.In some embodiments, the viral vector is provided at a multiplicity of infection (MOI) of between about 1 to 10 (e.g., between about 2 to 9, 3 to 8, 4 to 7, or 5 to 6). For example, in some embodiments, the viral vector is provided at a MOI of between about 2 to 9. In some embodiments, the viral vector is provided at a MOI of between about 3 to 8. In some embodiments, the viral vector is provided at a MOI of between about 4 to 7. In some embodiments, the viral vector is provided at a MOI of between about 5 to 6. In some embodiments, the viral vector is provided at a MOI of about 1. In some embodiments, the viral vector is provided at a MOI of about 1.5. In some embodiments, the viral vector is provided at a MOI of about 2. In some embodiments, the viral vector is provided at a MOI of about 4.In some embodiments, the payload delivery vehicle associated with the cell is a vesicle. In some embodiments, the vesicle is an exosome. A central feature of exosomes is their ability to contain biologically active payload within their interior space, or lumen. It is well known that exosomes contain endogenous payload including mRNA, miRNA, DNA, proteins, carbohydrates, and lipids, but the ability to direct specific loading of desired pay load is currently limited. Exosomes may be loaded by overexpressing desired payloads in a producer cell, but thisloading is often of limited efficiency due to stochastic localization of the payload to cellular cxosomc processing centersIn some embodiments, the vesicle is a nanovesicle. The nanovesicles may be produced by a method as described in US 2012 / 0177574, incorporated herein by reference, see in particular paragraphs

[0173] -

[0197] . A nanovesicle according to embodiments of the invention may have a size up to 500 nm, for example up to 300 nm, such as up to 250 nm or 200 nm. For example, the nanovesicles may have a size in the range of 100-200 nm. In some embodiments, the nanovesicles may have a size of about 100 nm. However, in some embodiments the nanovesicles may be smaller than 100 nm, for example at least 50 nm or at least 80 nm.In some embodiments, exosomes and other vesicles, e.g., extracellular vesicles (EVs), microvesicles, naturally produced and released by cells may also contain the payload and optionally also express a targeting molecule on its surface. Exosomes, EVs, microvesicles and artificial nanovesicles have several similarities but there are also important differences between these vesicles. Exosomes are produced through a natural process, involving inward budding of the cell membrane and are formed via multivesicular bodies. Exosomes are loaded with very specific materials, e.g., nucleic acids, which is not a random selection of cellular content.Artificial nanovesicles, produced by serial extrusions of cells through micro- and nanofilters, do contain any protein on their surface that the cell does not have, and also contains a random selection of the cytoplasm. Therefore, overexpression of targeting surface molecule in the cell membrane of an engineered cell will result in the presence of that molecule on the artificial nanovesicles. Similarly, a molecule overexpressed within the cell will also be present within the artificial nanovesicle in high concentrations. Most importantly, the yield of artificial nanovesicles is expected to be significantly higher than the yield of exosomes or other extracellular vesicles naturally released by a cell.In some embodiments, the vesicle comprises a targeting moiety. In some embodiments, the vesicle is produced by direct plasma membrane budding. In some embodiments, the vesicle is one as disclosed, for instance, in PCT / US2021 / 037053, incorporated by reference herein.In some embodiments, the payload delivery vehicle associated with the cell is a liposome. Liposomes, or lipid bilayer vesicles, have been used or proposed for use in a variety of applications in research, industry, and medicine, particularly for the use as carriers of diagnostic or therapeutic compounds in vivo. See, for example: Lasic, D. Liposomes: from physics toapplications. Elsevier, Amsterdam, 1993. Lasic, D, and Papahadjopoulos, D., eds. Medical Applications of Liposomes. Elsevier, Amsterdam, 1998. A liposome typically serves as a carrier of an entity such as, without limitation, a chemical compound, a combination of compounds, a supramolecular complex of a synthetic or natural origin, a genetic material, a living organism, a portion thereof, or a derivative thereof, that is capable of having a useful property or exerting a useful activity. For this purpose, the liposomes are prepared to contain the desired entity in a liposome-incorporated form.Ideally, such liposomes can be prepared to include the desired compound (i) with high loading efficiency, that is, high percent of encapsulated entity relative to the amount taken into the encapsulation process; (ii) high amount of encapsulated entity per unit of liposome bilayer material; (iii) at a high concentration of encapsulated entity, and (iv) in a stable form, i.e., with little release (leakage) of an encapsulated entity upon storage or generally before the liposome appears at the site or in the environment where the liposome-entrapped entity is expected to exert its intended activity.Properties of BindingIn some embodiments, the payload (e.g., or payload delivery vehicle) binds to a cell with an avidity or affinity so that the interaction between the payload (e.g., or payload delivery vehicle) and the cell is not disrupted through physical interference (e.g., as is described in U.S. Patent Application No. 63 / 421,342, incorporated herein by reference in its entirety). In an embodiment, the physical interference is at any step after the binding step and prior to infusion back into the subject. In some embodiments, the payload comprises a binding moiety that binds to a target moiety on the surface of a target cell.Binding of a payload (e.g., or payload delivery vehicle) and a cell may occur through any binding mechanism, including ionic interactions (e.g., cationic or anionic interactions), covalent binding, transient interactions, ligand-specific interactions, polar interactions, and others. In some embodiments, the binding moiety is an antibody or an antigen binding fragment thereof. In some embodiments, the binding moiety is a receptor. In some embodiments, the binding moiety is a ligand of a receptor. In some embodiments, the binding moiety is a lipid. In some embodiments, the binding moiety is a carbohydrate. In some embodiments, the binding moiety is a polypeptide. In some embodiments, the binding moiety binds to a target moiety on the cell surface of the target cell. In some embodiments, the binding moiety binds to a target moietywith an attenuated affinity. In some embodiments, binding moiety binds to a target moiety with high affinity (Kd).In some embodiments, the binding of the binding moiety to the target moiety is based on pH. In some embodiments, the binding moiety binds to the target moiety at acidic pH. In some embodiments, the binding moiety binds to the target moiety at basic pH. In some embodiments, the binding moiety disassociates with the target moiety at physiological pH. In some embodiments, physiological pH is about 7. In some embodiments, physiological pH is about 7.5. In some embodiments, physiological pH is about 7 to about 7.1, about 7.1 to about 7.2, about 7.2 to about 7.3, about 7.3 to about 7.4, or about 7.4 to about 7.5. In some embodiments, physiological pH is about 7.3. In some embodiments, physiological pH is about 7.35. In some embodiments, physiological pH is about 7.4. In some embodiments, physiological pH is about 7.45.In some embodiments, the binding moiety is a ligand of a receptor. In some embodiments, the binding moiety is a receptor which binds to a ligand. Some examples of ligand / receptor pairings include, but are not limited to, insulin / insulin receptor, low density lipoprotein / low density lipoprotein, Fc region / Fc receptor, transferrin / transferrin receptor, and GalNAc / Asialoglycoprotein receptor (ASGPR).In some embodiments, the payload delivery vehicle comprises a cationic lipid to facilitate binding to a cell. Cationic lipids are positively charged amphiphiles consisting of three basic chemical functional domains: a hydrophilic headgroup, a hydrophilic domain, and a linker bond that tethers the cationic headgroup and a hydrophobic tail domain. The headgroups can vary from primary, secondary, and tertiary aminos, or quaternary ammonium salts as well as phosphorus, guanidino, arsenic, imidazole, and pyridinium groups. In some embodiments, the cationic lipid is DMRIE-cholesterol, DOTIM-cholesterol, EDMPC-cholesterol, DC-cholesterol, DOTMA, DOSPER, DOSPA, GL-67, bPEI, PBAE, PDMAEMA, and PEG-b-PAMA, or variants thereof.In some embodiments, the payload delivery vehicle comprises an antibody or an antibody fragment thereof. In some embodiments, the antibody or antibody fragment thereof comprises a variable region that targets a moiety on the surface of a cell. In some embodiments, the moiety is a cell- specific marker. In some embodiments, the cell- specific marker is a marker for a peripheral blood mononuclear cell (PBMC). In an embodiment, the PBMC cell-specific markeris CD3. In an embodiment, the PBMC cell-specific marker is CD 16. In an embodiment, the PBMC is CD19. In an embodiment, the PBMC cell-specific marker is CD14.CAR-X CellsCAR-T cell therapy has emerged as a groundbreaking approach in the field of immunotherapy, revolutionizing the treatment of various malignancies (Sun Y et al. CARs: a new approach for the treatment of autoimmune diseases. Sci China Life Sci. 2023 Apr;66(4):711- 728.). CAR-T cells are genetically engineered immune cells that express a synthetic receptor, known as a CAR, on their surface. These receptors are designed to recognize specific antigens expressed on the surface of target cells. CAR-T cells have shown remarkable success in the treatment of hematological malignancies, such as leukemia and lymphoma, by redirecting the immune system to target and eliminate cancer cells. Building upon the success of CAR-T cell therapy in oncology, the instant application presents a novel application of CAR-T cell therapy for the treatment of antibody-mediated autoimmune diseases, allergic disorders, and fibrotic diseases.The concept underlying this invention involves the engineering of CAR-T cells to target pathogenic cells, e.g., autoreactive immune cells, implicated in disease pathogenesis. These autoreactive immune cells, including autoreactive B cells and certain subsets of T cells, play a crucial role in perpetuating the inflammatory response and promoting negative systems (e.g., organ damage in SLE). By genetically modifying CAR-T cells to express a CAR specific to the surface markers (e.g., autoantibodies or other inflammatory markers) expressed on these autoreactive cells, the engineered CAR-T cells can selectively recognize and eliminate the aberrant immune cells (e.g., pathogenic cells) while sparing the healthy cells of the body. The methods disclosed herein comprise, in part, generating CAR-X cells in the subject’s body.Following the steps of (a) facilitating the formation of complexes comprising the payloads and the subject-derived cells by contacting the subject-derived cells with the payloads described herein, but providing conditions sufficient to prevent uptake or entry of the payload into the cells, and (b) processing the complexes, e.g., by washing as described herein, to remove any impurities and unbound payload, the present method comprises the step of administering (e.g., by infusion) the resultant complexes to the subject. In an embodiment, selecting comprises isolating and / or purifying for the desired complexes, e.g., by washing the complexes as described herein. Once the complexes have been administered (e.g., by infusion) to the subject, theconditions preventing the payload from entering into the cells will no longer be present. Thus, the cell will uptake the payload (c.g., by cndocytosis) in vivo. Subsequently, the cell will be modified to express the payload by the endogenous transcriptional and translational machinery, thereby generating a CAR-X cell in the subject’s body from the administered payload-cell complex.In some embodiments, the steps of apheresis and extracorporeal gene delivery of the subject-derived cell pay load complexes are performed within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 10 hours. In some embodiments, the steps of apheresis and extracorporeal gene delivery of the subject-derived cell payload complexes are performed within 1 hour. In some embodiments, the steps of apheresis and extracorporeal gene delivery of the subject-derived cell payload complexes are performed within 2 hours. In some embodiments, the steps of apheresis and extracorporeal gene delivery of the subject-derived cell payload complexes are performed within 3 hours. In some embodiments, the steps of apheresis and extracorporeal gene delivery of the subject-derived cell payload complexes are performed within 4 hours. In some embodiments, the steps of apheresis and extracorporeal gene delivery of the subject-derived cell payload complexes are performed within 5 hours. In some embodiments, the steps of apheresis and extracorporeal gene delivery of the subject-derived cell payload complexes are performed within 10 hours. In some embodiments, extracorporeal gene delivery comprises delivery of a gene, an element of a gene (e.g., a regulatory element of a gene, e.g., a promoter), a portion of a gene, or any other genetic material commonly used in the art for therapeutic purposes (e.g., an siRNA).In some embodiments, one or more of the cells collected by apheresis will be cryopreserved. In some embodiments, the cryopreserved cells may be connected to the closed- loop system to participate in post-apheresis method steps described herein, such as, but not limited to, payload delivery, selection, and infusion.In some embodiments, the CAR-X cell is a T cell, a B cell, or a myeloid cell. In some embodiments, the T cell is a CD8+ T cell, a CD4+ T cell, a regulatory T cell (Treg) cell (e.g., a gamma / delta T cell), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell (HSC), a natural killer cell (NK cell), or a natural killer T cell (NKT cell). In some embodiments, the myeloid cell is a monocyte, a macrophage, a dendritic cell, or a granulocyte, e.g., a neutrophil, a mast cell, an eosinophil, or a basophil.T cellsIn some embodiments, the CAR-X cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumorinfiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art may be used.CAR-X FunctionCAR-X cells target pathogenic cells (e.g., cells that drive disease states in autoimmune diseases, allergic disorders, and fibrotic diseases) described herein by selectively binding to autoreactive cells (e.g., by recognition of autoantibodies or inflammatory markers) and eliminating such cells. By eliminating pathogenic cells, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more, thereby reducing systemic inflammation in the subject and alleviating one or more symptoms of the subject’s autoimmune disease, allergic disorder, or fibrotic disease. In some embodiments, CAR- X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 5%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 10%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 15%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 20%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 25%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by atleast 30%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, c.g., by at least 35%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 40%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 45%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 50%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 60%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 70%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 80%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 90%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 95%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 96%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 97%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 98%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 99%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 99.5%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by at least 99.9%. In some embodiments, CAR-X cells reduce the levels of pathogenic cells in the subject’s body, e.g., by more than 99.9%.Diseases and DisordersThe present invention provides compositions and methods for treating various diseases and disorders. In some embodiments, the disease or disorder is an autoimmune disease, an allergic disorder (e.g., an allergic, atopic, or hypersensitivity disorder), or a fibrotic disease. There are two basic categories of autoimmune disease: those predominantly caused by T cells, and those predominantly caused by B cells and the autoantibodies they produce. In some embodiments, the disease or disorder is an allergic, atopic or hypersensitivity disorder. Allergic disorders are typically caused by mast cells and eosinophil cells. Allergic, atopic, or hypersensitivity disorders are characterized by inappropriate or exaggerated immune reactions to foreign antigens, or alloantigens. This results in inappropriate immune reactions include thosethat are misdirected against intrinsic body components (self), leading to autoimmune disorders. In some embodiments, the disease or disorder is an alloimmunc disease or disorder. An alloimmune disease or disorder is an immune response to non-self antigens from members of the same species, which are called alloantigens or isoantigens. In some embodiments, the disease or disorder is a fibrotic disease, e.g., fibrosis.Non-limiting examples of autoimmune diseases include SLE, myasthenia gravis (MG), pemphigus vulgaris (PV), coeliac disease, Crohn’s disease, Grave’s disease, Hashimoto’s thyroiditis, multiple sclerosis, rheumatoid arthritis, Sjogren’s disease, ulcerative colitis, and vasculitis. Non-limiting examples of allergic disorders, atopic disorders, or hypersensitivity disorders include allergic asthma, atopic dermatitis (e.g., eczema), atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, eosinophilic esophagitis (atopic and nonatopic), hay fever, and severe eosinophilic asthma (SEA).SLE is a chronic autoimmune disease affecting multiple organs, including the skin, joints, kidneys, heart, and brain. The etiology of lupus involves complex interactions between genetic, hormonal, and environmental factors, leading to dysregulation of the immune system. In lupus, the immune system fails to distinguish between self and non-self, resulting in the production of autoantibodies that target various cellular components, such as DNA, RNA, and proteins. These autoantibodies form immune complexes that deposit in tissues, triggering inflammation and organ damage. Current treatments for lupus mainly involve the use of immunosuppressive drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids. However, these treatments often come with significant side effects and do not provide a definitive cure for the disease. SLE can be characterized by the presence and / or quantitative levels of certain biomarkers, e.g., Smith (Sm) antibody, B-cell activating factor (BAFF), antinuclear antibody (ANA), DNA antibody, and complement (2, 3, and 4), for example as described in Atisha-Fregoso, Y et al. (2021). Meant to B: B cells as a therapeutic target in systemic lupus erythematosus. The Journal of clinical investigation, 131(12), el49095; and in Mackensen, A et al. (2022). Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nature medicine, 28(10), 2124-2132.MG is an autoimmune neuromuscular junction disease that leads to varying degrees of skeletal muscle weakness. MG results from results from antibodies that inhibit nicotinic acetylcholine receptors (AChR) at the junction between the nerve and muscle. The most commonly affected muscles are those of the eyes, face, and swallowing, and symptoms mayinclude double vision, drooping eyelids, trouble talking, and trouble walking. MG can be characterized by the presence and / or quantitative levels of certain biomarkers, e.g., acetylcholine receptors (such as AChR) and muscle- specific kinase antibodies.PV is a rare chronic blistering skin disease that comprises the most common form of pemphigus, a rare group of blistering autoimmune diseases that affect the skin and mucous membranes. PV is classified as a type II hypersensitivity reaction in which antibodies are formed against desmosomes, causing acantholysis, or separation between the layers of skin that clinically resembles a blister. Over time, lesions increase in size and distribution throughout the body, behaving physiologically like a severe burn. PV can be characterized by the presence and / or quantitative levels of certain biomarkers, e.g., ANA, anti-smooth muscle antibodies (ASMAs), anti-parietal antibodies (APAs), PV auto-antigen, and anti-desmogleins (e.g., desmoglein 3, as described in Ellebrecht, C et al. (2016). Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science (New York, N.Y.), 353(6295), 179-184.).Atopy (also known as IgE atopy) is a disorder characterized by the tendency to produce an exaggerated IgE immune response to otherwise harmless substances in the environment. Atopy may have a hereditary component, and maternal psychological trauma in utero may also be a strong indicator for development of atopy. Atopy can be characterized by the presence and / or quantitative levels of certain biomarkers, e.g., IgE, relative to a paired normal sample (i.e., a level of IgE in a blood sample obtained from a normal, e.g., non-diseased, subject from the same age group as the atopy subject).Eosinophil-related diseases may comprise hypereosinophilic syndromes (HESs), a heterogeneous group of rare disorders characterized by peripheral eosinophilia and eosinophilic end organ complications (for example, as characterized in Klion A. (2018). Hypereosinophilic syndrome: approach to treatment in the era of precision medicine. Hematology. American Society’ of Hematology. Education Program, 2018(1), 326-331; and Klion, A. D., & Rothenberg, M. E. (2019). Advances in eosinophilic diseases in 2018. The Journal of allergy and clinical immunology, 144(6), 1490-1494). In some embodiments, eosinophil-related diseases may comprise one or more diseases selected from Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome), Eosinophilic Gastrointestinal Disorder, and Severe Eosinophilic Asthma. In some embodiments, eosinophil-related diseases may comprise one or more diseasesselected from Eosinophilic leukemia, hypereosinophilic syndrome, systemic mastocytosis, Mast cell leukemia (MCL), and mast cell sarcoma (MCS). Eosinophil-related disorders can be characterized by the presence and / or quantitative levels of certain biomarkers, e.g., EGF-like module containing mucin-like hormone receptor 1 (EMR1), as described in Legrand, F et al. The eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1): a novel therapeutic target for eosinophilic disorders. The Journal of Allergy and Clinical Immunology. 2014 May; 133(5): 1439-47, 1447.el-8; Siglec-6, as described in Patent Publication No. WO2005 / 124358A2 and in Schanin, J et al. (2022). Discovery of an agonistic Siglec-6 antibody that inhibits and reduces human mast cells. Communications biology, 5(1), 1226.; or Siglec-8, as described in Dellon, E et al. (2020). Anti-Siglec-8 Antibody for Eosinophilic Gastritis and Duodenitis. The New England journal of medicine, 383( T), 1624- 1634.SEA is a therapy-resistant respiratory condition with poor clinical control. SEA can be characterized by the presence and / or quantitative levels of certain biomarkers, e.g., IL-5, as described in Chen, S et al. (2022). Treatment of allergic eosinophilic asthma through engineered IL-5-anchored chimeric antigen receptor T cells. Cell discovery, 8(1), 80.In some embodiments, the disease or disorder is a refractory disease. A refractory disease may be any disease that resists or stops responding to treatment.Subject SelectionIn some embodiments, the subject is selected for treatment comprising the method as described herein based on the presence of biomarkers. In some embodiments, the biomarkers are autoimmune disease or disorder biomarkers. In some embodiments, the biomarkers are atopic or alloimmune disease biomarkers. In some embodiments, the biomarkers are autoimmune disease biomarkers. In some embodiments, the biomarkers are fibrotic disease biomarkers. In some embodiments, the biomarkers comprise one or more biomarkers described herein. In some embodiments, the biomarkers comprise one or more biomarkers commonly known in the art to be implicated in an autoimmune disease, allergic disorder, or fibrotic disease described herein.In some embodiments, the biomarker is a protein biomarker. In some embodiments, the protein biomarker is an antibody. In some embodiments, the protein biomarker is a cell surface protein. In some embodiments, the protein biomarker is an intracellular protein. In some embodiments, the protein biomarker is a soluble protein. In some embodiments, the proteinbiomarker is a secreted protein. In some embodiments, the biomarkers are nucleic acid biomarkers. In some embodiments, the nucleic acid biomarkers arc DNA or RNA. In some embodiments, DNA biomarkers comprise nuclear DNA and / or mitochondrial DNA. In some embodiments, the DNA biomarker comprises a modification to the DNA (e.g., methylation, phosphorylation, acetylation). In some embodiments, the RNA biomarkers comprise messenger RNA, transfer RNA, microRNA, short hairpin RNA, ribosomal RNA, and small interfering RNA. In some embodiments, the biomarker is a sugar molecule. In some embodiments, the sugar molecule is conjugated to a protein (e.g., a glycoprotein). In some embodiments, the biomarker is a lipid. In some embodiments, the biomarker is a metabolite.In some embodiments, the biomarker is identified and measured in a subject sample. In some embodiments, the subject sample comprises a hematological sample. In some embodiments, the hematological sample comprises red blood cells, peripheral blood mononuclear cells (e.g., T cells, B cells, NK cells, monocytes), platelets, and granulocytes (e.g., neutrophils, basophils, and eosinophils). In some embodiments, the hematological sample comprises plasma.In some embodiments, the subject has a disease or disorder (e.g., e.g., a non-malignant disease, such as an allergic, autoimmune, or fibrotic disease, e.g., systemic lupus erythematosus (SLE), myasthenia gravis (MG), pemphigus vulgaris (PV), allergic asthma, atopic dermatitis (e.g., eczema), atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, and fibrosis) or displays one or more (e.g., two, three, four, or more) symptoms of a disease or disorder. In some embodiments, the subject has a disease or disorder. In some embodiments, the subject displays one or more (e.g., two, three, four, or more) symptoms of a disease or disorder. For example, in some embodiments, the subject displays two or more symptoms of a disease or disorder. In some embodiments, the subject displays three or more symptoms of a disease or disorder. In some embodiments, the subject displays four or more symptoms of a disease or disorder. In some embodiments, the subject is diagnosed with a disease or disorder. In some embodiments, the subject is not diagnosed with a disease or disorder.The subject receiving the treatment described herein may be treatment naive or may have previously received treatment for the disease or disorder (e.g., a non-malignant disease). In some embodiments, the subject is treatment naive. A treatment naive subject may have previously received treatment for the disease or disorder. In some embodiments, the treatment naive subjectmay have previously received treatment for the disease or disorder, but the treatment was unsuccessful. A treatment may be considered unsuccessful if it did not or was not sufficient to alleviate the symptom(s) it was administered to treat and / or if it did not inhibit or reduce the progression of the disease or disorder. In some embodiments, the treatment naive subject has a refractory disease. In some embodiments, the treatment naive subject may have previously received treatment for the refractory disease, but the treatment was unsuccessful. In some embodiments, the treatment naive subjects may have previously received treatment for the disease or disorder, and the treatment was successful (e.g., the treatment alleviated the symptom(s) it was administered to treat and / or it inhibited or reduced the progression of the disease or disorder), but the symptom(s) has / have since recurred and / or the disease state has since progressed.Such treatment naive subjects have not received treatment for the disease or disorder within at least 6 months (e.g., at least 8 months, 10 months, 12 months, 24 months, or more) of being treated by the methods described herein. For example, in some embodiments, treatment naive subjects have not received treatment for the disease or disorder within at least 8 months of being treated by the methods described herein. In some embodiments, treatment naive subjects have not received treatment for the disease or disorder within at least 10 months of being treated by the methods described herein. In some embodiments, treatment naive subjects have not received treatment for the disease or disorder within at least 12 months of being treated by the methods described herein. In some embodiments, treatment naive subjects have not received treatment for the disease or disorder within at least 24 months of being treated by the methods described herein. In some embodiments, treatment naive subjects have not received treatment for the disease or disorder within more than 24 months of being treated by the methods described herein.In some embodiments, a treatment naive subject is currently being treated for the disease or disorder, but is not currently being treated according to the methods described herein. In some embodiments, a treatment naive subject is being treated for one or more (e.g., two, three, four, or more) symptoms of a disease or disorder (e.g., a non-malignant disease), but is not being treated for the underlying mechanism of the disease or disorder. In some embodiments, a treatment naive subject is being treated for one or more (e.g., two, three, four, or more) symptoms of a disease or disorder, but has not been diagnosed with the disease or disorder.In some embodiments, the subject has been previously treated with one or more therapeutics, c.g., directed to non-malignant diseases. In some embodiments, the subject has been previously treated with lymphodepleting therapy (e.g., fludarabine and cyclophosphamide). In some embodiments, the subject has been previously treated with an immunotherapy (e.g., antibodies, immune cell therapy, cytokine therapy, combination therapy). In some embodiments, the subject has been previously treated with a small molecule. In some embodiments, the subject has been previously treated with radiation or radiotherapy. In some embodiments, the subject has been previously treated with a pain medication. In some embodiments, the subject has been previously treated with chemotherapy. In some embodiments, the previous treatment with one or more therapeutics, e.g., directed to non-malignant diseases, was unsuccessful. In some embodiments, the previous treatment was successful, but the subject has experienced recurrence of the disease or one or more symptoms of the disease.In some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 25%, 50%, or more). For example, in some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 2%. In some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 3%. In some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 4%. In some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 5%. In some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 10%. In some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 25%. In some embodiments, treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by at least 50%. In some embodiments,treatment of a subject by the methods described herein can extend the subject’s life, improve the quality of the subject’s life, and / or extend the function of an organ by more than 50%.In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is an adult. In some embodiments, the subject meets inclusion and exclusion criteria as defined by the treatment for the specific disease or disorder. In some embodiments, the subject is tested for viral, bacterial, or fungal infections prior to treatment. In some embodiments, the subject has a recurrent disease or disorder. In some embodiments, the subject has a refractory disease or disorder. In some embodiments, the subject has more than one disease or disorder (e.g., one or more autoimmune diseases or disorders). In some embodiments, the subject has more than one disease or disorder at the same time. In some embodiments, the subject has had a previous disease or disorder. In some embodiments, the subject has an evaluable disease. Target Pathogenic CellsThe subject may be treated by the methods described herein. In some embodiments, the subject is treated by a multi-step method comprising extracorporeal gene delivery. In some embodiments, the extracorporeal gene delivery comprises administration of subject-derived cellpayload complexes to the subject. In some embodiments, following administration of the complexes to the subject, the subject-derived cells will facilitate uptake or entry of the payloads into the cells, e.g., by endocytosis. In some embodiments, following uptake or entry of the pay loads into the cells, the pay load is processed and expressed by the endogenous transcriptional and translation machinery, resulting in the generation of a modified subject-derived cell, herein referred to as a CAR-X cell.In some embodiments, the CAR-X cells target pathogenic cells (e.g., target cells) in the subject. Pathogenic cells are cells that are implicated in the pathogenesis of a disease (e.g., cells that drive a disease state). In preferred embodiments, the pathogenic cell is a mast cell, an eosinophil, or a B cell.In some embodiments, the pathogenic cell is a T cell, a hematopoietic stem cell (HSC), or a natural killer T cell (NKT cell). In some embodiments, the pathogenic cell is a B cell such as an IgE producing cell, e.g., a plasma B cell, or a B cell producing an autoantibody. In some embodiments, the B cell producing such autoantibodies drives a disease state such as SLE, PV, MG, Graves’ disease, Hashimoto’s thyroiditis, vasculitis, Sjogren’s disease, or an autoimmune disease, allergic disorder, or fibrotic disease described herein. In some embodiments, thepathogenic cells are myeloid cells, e.g., monocytes, macrophages (e.g., pro-fibrotic macrophages, e.g., MC3 macrophages as described in Fabre, T ct al. (2023). Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation. Science immunology, 8(82), eadd8945), dendritic cells, or granulocytes, e.g., neutrophils, mast cells, eosinophils, and / or basophils. In some embodiments, the pathogenic cells are fibroblasts.B cellsIn some embodiments, the target pathogenic cell is a B cell, E.g., a B cell producing an autoantibody or an IgE producing cell, e.g., a plasma B cell. In some embodiments, the target pathogenic cell is an IgE- or membrane-bound Ige (mlgE)- B cell, for example as described in Lustgarten, J et al. (1995). Specific elimination of IgE production using T cell lines expressing chimeric T cell receptor genes. European journal of immunology, 25(10), 2985-2991.Mast CellsA mast cell, also known as a mastocyte or a labrocyte, is a resident cell of connective tissue that contains granules rich in histamine and heparin. Specifically, mast cells are a type of granulocyte derived from myeloid stem cells that comprise part of the immune and neuroimmune systems. Although best known for their roles in allergy and anaphylaxis, mast cells play an important protective role as well, being intimately involved in wound healing, angiogenesis, immune tolerance, defense against pathogens, and vascular permeability (e.g., in brain tumors). In some embodiments, the target pathogenic cell is a mast cell.In some embodiments, mast cells may be targeted indirectly by targeting inflammatory cytokines released by mast cells or their receptors (e.g., IL-5, IL-5R, IL-4R, and / or tetrameric - tryptase) as described in Reber, L. et al. (2014). Targeting mast cells in inflammatory diseases. Pharmacology & therapeutics, 142( >), 416-435.EosinophilsEosinophils are a type of white blood cell. They are responsible for combating multicellular parasites and certain infections in vertebrates. Along with mast cells and basophils, they also control mechanisms associated with allergies and asthma. During hematopoiesis, eosinophils develop as granulocytes that eventually migrate into the blood, after which they become terminally differentiated and do not multiply. Eosinophils comprise large acidophilic cytoplasmic granules comprising enzymes including eosinophil peroxidase, RNases, DNases, lipase, plasminogen, and major basic protein, which can be released following activation of theeosinophil and are toxic to both parasite and host tissues. Tn certain embodiments, the target pathogenic cell is an eosinophil.Basophil CellsBasophils are a type of white blood cell. They release histamine and other chemicals that help fight off infections and parasites by recognizing foreign organisms that enter the host and phagocytosing the foreign organisms. Basophils also play a role in allergic reactions and inflammation by releasing the enzyme histamine, which dilates the proximal blood vessels, and by binding to IgE, which activates the basophils and induces their degranulation and synthesis of the cytokines interleukin 4 (IL-4) and IL- 13. In certain embodiments, the target pathogenic cell is a basophil cell.Fibroblast CellsFibroblasts are a type of cell that contributes to the formation of connective tissue. Fibroblasts are large, flat, elongated, spindle-shaped cells that possess processes extending out from the ends of the cell body. They secrete collagen proteins and also play an important role in wound healing. In certain embodiments, the target pathogenic cell is a fibroblast. Persistence of CAR-XCAR-X cells, as obtained and / or generated by the methods described herein, may persist in the subject’s body for at least 10 minutes, (e.g., at least 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 1 month, 6 months, or 1 year). For example, in some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 20 minutes. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 30 minutes. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 1 hour. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 2 hours. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 3 hours. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 4 hours. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 5 hours. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 6 hours. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 12 hours. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 1 day. In someembodiments, CAR-X cells described herein may persist in the subject’s body for at least 2 days. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 3 days. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 1 week. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 2 weeks. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 3 weeks. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 1 month. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 6 months. In some embodiments, CAR-X cells described herein may persist in the subject’s body for at least 1 year. In some embodiments, CAR-X cells described herein may persist in the subject’s body for longer than 1 year.Evaluation of Clinical EfficacyIn some embodiments, the subject may be treated by the methods described herein at least one time (e.g., two times, three times, four times, five times, or more). For example, in some embodiments, the subject may be treated by the methods described herein at least two times. In some embodiments, the subject may be treated by the methods described herein at least three times. In some embodiments, the subject may be treated by the methods described herein at least four times. In some embodiments, the subject may be treated by the methods described herein at least five times. In some embodiments, the subject may be treated by the methods described herein more than five times.In some embodiments, the subject may be treated by the methods described herein on a recurring schedule. For example, in some embodiments, the subject may be treated by the methods described herein daily. In some embodiments, the subject may be treated by the methods described herein weekly. In some embodiments, the subject may be treated by the methods described herein biweekly. In some embodiments, the subject may be treated by the methods described herein monthly. In some embodiments, the subject may be treated by the methods described herein bimonthly. In some embodiments the subject may be treated by the methods described herein every 3 months, every 6 months, or yeai’ly. In some embodiments, the subject may be treated by the methods described herein for a period of time, e.g., until alleviation of one or more symptoms of the subject’s disease is observed, followed by a wash-out period in which the subject receives no treatment, and optionally followed by another treatment period.In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase, c.g., as determined by measuring CAR-X cells in a blood sample obtained from the subject. In some embodiments, the level of CAR-X cells in the subject’s body may increase, e.g., by about 20%, 30%, 40%, 50%, or more. For example, in some embodiments, the level of CAR-X cells in the subject’s body may increase by about 20% or more. In some embodiments, the level of CAR-X cells in the subject’s body may increase by about 30% or more. In some embodiments, the level of CAR-X cells in the subject’s body may increase by about 40% or more. In some embodiments, the level of CAR-X cells in the subject’s body may increase by about 50% or more. In some embodiments, the level of CAR-X cells in the subject’s body may increase by more than 50%. In some embodiments, the level of CAR-X cells in the subject’s body may increase by about 80% or more.In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase, e.g., as determined by measuring CAR-X cells in a blood sample obtained from the subject, between about 1 day to 28 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase between about 2 days to 27 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase between about 3 days to 26 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase between about 4 days to 25 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase between about 5 days to 21 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase between about 7 days to 21 days.In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase between about 7 days to 14 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase within 7 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase within 14 days. In some embodiments, following treatment by the methods described herein, the level of CAR-X cells in the subject’s body may increase within 21 days.In some embodiments, following treatment by the methods described herein, the increased level of CAR-X cells in the subject’s body may persist for at least 1 day (e.g., at least 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, 28 days, or more). For example, in some embodiments, following treatment by the methods described herein, the increased level of CAR- X cells in the subject’s body may persist for at least 2 days or more. In some embodiments, following treatment by the methods described herein, the increased level of CAR-X cells in the subject’s body may persist for at least 3 days or more. In some embodiments, following treatment by the methods described herein, the increased level of CAR-X cells in the subject’s body may persist for at least 4 days or more. In some embodiments, following treatment by the methods described herein, the increased level of CAR-X cells in the subject’s body may persist for at least 7 days or more. In some embodiments, following treatment by the methods described herein, the increased level of CAR-X cells in the subject’s body may persist for at least 14 days or more. In some embodiments, following treatment by the methods described herein, the increased level of CAR-X cells in the subject’s body may persist for at least 21 days or more. In some embodiments, following treatment by the methods described herein, the increased level of CAR-X cells in the subject’s body may persist for at least 28 days or more. In some embodiments, following treatment by the methods described herein, the increased level of CAR- X cells in the subject’s body may persist for more than 28 days.In some embodiments, the subject may be treated by the methods described herein once the levels of CAR-X cells in the subject’s body, e.g., as determined by measuring CAR-X cells in a blood sample obtained from the subject, reduce beyond a threshold, e.g., reduce below 20%, 15%, 10%, 5% or less. In some embodiments, CAR-X cells can be measured by quantifying one or more biomarkers of CAR-X cells, e.g., a biomarker comprising the extracellular domain of the CAR.In some embodiments, treatment according to the methods described herein may be deemed to be effective or successful if one or more symptoms of the subject’s disease (e.g., autoimmune disease, allergic disorder, or fibrotic disease) are alleviated. In some embodiments, the treatment may be deemed to be effective or successful if the levels of one or more biomarkers of the subject’s disease (e.g., autoimmune disease, allergic disorder, or fibrotic disease) are changed (e.g., reduced or increased) from baseline. In some embodiments, the change in the one or more biomarkers of the subject’s disease cause the levels of the one or morebiomarkers in the subject to more closely match the levels of the one or more biomarkers in a normal (c.g., non-discascd) subject of the same age. For example, in some embodiments, the subject has SLE, and the blood titers of ANA decrease by about 40% relative to baseline levels, as determined by quantitation of ANA levels in blood samples obtained from the subject at day 0 and day 28, so the treatment is deemed effective.Other methods commonly used in the art may be utilized to determine the effectiveness of treatment according to the methods described herein. For example, in some embodiments, the subject has SLE, and the clinical endpoint used to assess the effectiveness of treatment comprises the SLE Disease Activity Index (SLED Al). Combination TherapiesIn one aspect, the PACCs as described or made by the methods described herein can be administered to a subject in combination with one or more additional therapeutics. In some embodiments, the PACCs are administered before the additional therapeutic. In some embodiments, the PACCs are administered after the additional therapeutic. In some embodiments, the PACCs are administered concurrently with the additional therapeutic. In some embodiments, the additional therapeutic is selected from a list including, but not limited to, such agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immuno-ablative agents such as CAMPATH, anti-CD3 antibodies, cytotoxin, fludarabine, cyclosporin, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun, 73:316-321 , 1991 ; Bterer et al., Curr. Opin. Immun. 5:763- 773, 1993). In some embodiments, the additional therapeutic is a B-cell ablative therapy, such as agents that react with CD20, e.g., Rituxan. In some embodiments, the additional therapeutic is a cancer vaccine.In some embodiments, the PACCs are obtained from a subject directly following treatment with an additional the additional therapeutic. In some embodiments, the additional therapeutic is administered to the subject following generation of the CAR-X cells (e.g., as determined by measurement of CAR-X- specific bio markers in a blood sample obtained from the subject).In some embodiments, the additional therapeutic is a therapeutic administered to a subject to address symptoms of the disease or disorder or side effects of another therapeutic agent. In some embodiments, the additional therapeutic is a pain medication. In some embodiments, the additional therapeutic is an anti-nausea therapeutic. In some embodiments, the additional therapeutic is a therapeutic administered to address disease-related cachexia. In some embodiments, the additional therapeutic is an anti-depressant. In some embodiments, the additional therapeutic is a vitamin or mineral. In some embodiments, the additional therapeutic is an anti-diarrheal medication. In some embodiments, the additional therapeutic is administered to address fatigue. In some embodiments, the additional therapeutic is administered to address a fever. In some embodiments, the additional therapeutic is administered to address nerve damage, e.g., peripheral neuropathy. In some embodiments, the additional therapeutic is administered to address chemotherapy-associated cardiovascular side effects.ENUMERATED EMBODIMENTSThe disclosure herein is further presented as a non-limiting list of numbered embodiments.1. A method of providing a subject with a population of payload-associated cell complexes (PACCs) for the treatment of a non-malignant disease, comprising:(i) providing a population of peripheral blood cells, e.g., a population of peripheral blood cells from the subject, wherein a peripheral blood cell in the population of peripheral blood cells comprises a binding target;(ii) extracorporeally contacting the population of peripheral blood cells with a payload comprising a chimeric antigen receptor (CAR) construct under conditions (e.g., time, temperature) sufficient for association of the payload with the cell comprising the binding target, wherein the conditions are not sufficient for entry of the payload into the cell with which it is associated, thus forming a population of PACCs; and(iii) introducing the population of PACCs into the subject, thereby providing subject with a population of PACCs for the treatment a non-malignant disease.2. The method of any of embodiment 1 , wherein the population comprises at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1,000, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, or more PACCs.3. The method of any of embodiment 2, wherein the population comprises at least 2 or more PACCs.4. The method of any of embodiment 2, wherein the population comprises at least 3 or more PACCs.5. The method of any of embodiment 2, wherein the population comprises at least 4 or more PACCs.6. The method of any of embodiment 2, wherein the population comprises at least 5 or more PACCs.7. The method of any of embodiment 2, wherein the population comprises at least 10 or more PACCs.8. The method of any of embodiment 2, wherein the population comprises at least 25 or more PACCs.9. The method of any of embodiment 2, wherein the population comprises at least 50 or more PACCs.10. The method of any of embodiment 2, wherein the population comprises at least 75 or more PACCs.11. The method of any of embodiment 2, wherein the population comprises at least 100 or more PACCs.12. The method of any of embodiment 2, wherein the population comprises at least 250 or more PACCs.13. The method of any of embodiment 2, wherein the population comprises at least 500 or more PACCs.14. The method of any of embodiment 2, wherein the population comprises at least 750 or more PACCs.15. The method of any of embodiment 2, wherein the population comprises at least 1,000 or more PACCs.16. The method of any of embodiment 2, wherein the population comprises at least 5,000 or more PACCs.17. The method of any of embodiment 2, wherein the population comprises at least 7,500 or more PACCs.18. The method of any of embodiment 2, wherein the population comprises at least 10,000 or more PACCs.19. The method of any of embodiment 2, wherein the population comprises at least 25,000 or more PACCs.20. The method of any of embodiment 2, wherein the population comprises at least 50,000 or more PACCs.21. The method of any of embodiment 2, wherein the population comprises at least 75,000 or more PACCs.22. The method of any of embodiment 2, wherein the population comprises at least 100,000 or more PACCs.23. The method of any of embodiment 2, wherein the population comprises at least 250,000 or more PACCs.24. The method of any of embodiment 2, wherein the population comprises at least 500,000 or more PACCs.25. The method of embodiments 1 or 2, wherein the population comprises at least 100,000 or more PACCs.26. The method of any one of the preceding embodiments, wherein the population comprises at least 1 million or more PACCs.27. The method of any one of the preceding embodiments, wherein the population comprises at least 10 million or more PACCs.28. The method of any one of the preceding embodiments, wherein the population comprises at least 100 million or more PACCs.29. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 0.5%, 1%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the total cells in the population.30. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 0.5% or more of the total cells in the population.31 . The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 1% or more of the total cells in the population.32. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 2% or more of the total cells in the population.33. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 10% or more of the total cells in the population.34. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 20% or more of the total cells in the population.35. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 30% or more of the total cells in the population36. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 40% or more of the total cells in the population.37. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 50% or more of the total cells in the population.38. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 60% or more of the total cells in the population.39. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 70% or more of the total cells in the population.40. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 80% or more of the total cells in the population.41. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 90% or more of the total cells in the population.42. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the pay load in the population is greater than 95% or more of the total cells in the population.43. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the payload in the population is between 30%-90% total cells, e.g., 50%-80% total cells in the population.44. The method of any one of the preceding embodiments, wherein the percentage of peripheral blood cells associated with the payload in the population is between 50-80% total cells in the population.45. The method of any one of the preceding embodiments, wherein the conditions sufficient for association of the payload with a peripheral blood cell within the population comprise:(i) contacting the peripheral blood cells with the payload for between about 0 to about 10 hours, e.g., about 1 hour to about 9 hours, about 2 hours to about 8 hours.46. The method of any one of the preceding embodiments, wherein the conditions sufficient for association of the payload with a peripheral blood cell within the population comprise:(i) contacting the peripheral blood cells with the payload for between about 1 hour to about 9 hours.47. The method of any one of the preceding embodiments, wherein the conditions sufficient for association of the payload with a peripheral blood cell within the population comprise:(i) contacting the peripheral blood cells with the payload for between about 2 hours to about 8 hours.48. The method of embodiment 45, wherein contacting the peripheral blood cells with the payload is between about 5 minutes and 30 minutes.49. The method of embodiment 45, wherein contacting the peripheral blood cells with the payload is between about 30 minutes and 1 hour.50. The method of embodiment 45, wherein contacting the peripheral blood cells with the pay load is between about 1 hour and 1.5 hours.51. The method of any one of the preceding embodiments, wherein the PACCs are formulated in or on a delivery vehicle.52. The method of embodiment 51 , wherein the delivery vehicle comprises a lipid nanoparticle, viral vector, vesicle, or a liposome in which the payload is disposed.53. The method of embodiment 52, wherein the delivery vehicle comprises a lipid nanoparticle in which the payload is disposed.54. The method of embodiment 52, wherein the delivery vehicle comprises a viral vector in which the payload is disposed.55. The method of embodiment 52, wherein the delivery vehicle comprises a vesicle in which the payload is disposed.56. The method of embodiment 52, wherein the delivery vehicle comprises a liposome in which the payload is disposed.57. The method of any one of embodiments 51-52, wherein the concentration of the delivery vehicle is higher than the concentration of peripheral blood cells in the sample.58. The method of any one of embodiments 51-52, wherein the concentration of the delivery vehicle is lower than the concentration of peripheral blood cells in the sample.59. The method of any one of embodiments 51-52 and 57-58, wherein the concentration of the delivery vehicle is optimized for binding to peripheral blood cells in the patient sample.60. The method of any one of the preceding embodiments, wherein the binding of the payload to the peripheral blood cells occurs at a selected temperature 0 to 40 degrees Celsius (e.g., 4 °C, 37 °C).61. The method of embodiment 60, wherein the binding of the pay load to the peripheral blood cells occurs at 0 °C.62. The method of embodiment 60, wherein the binding of the payload to the peripheral blood cells occurs at 4 °C.63. The method of embodiment 60, wherein the binding of the pay load to the peripheral blood cells occurs at 37 °C.64. The method of embodiment 60, wherein the binding of the payload to the peripheral blood cells occurs at 40 °C.65. The method of any one of the preceding embodiments, wherein the payload is disposed on the surface of a peripheral blood cell within the PACC.66. The method of any one of embodiments 57-65, wherein the delivery vehicle is a viral vector.67. The method of embodiment 66, wherein the viral vector is a lentiviral vector or an adeno- associated viral (AAV) vector.68. The method of embodiment 67, wherein the viral vector is a lentiviral vector.69. The method of embodiment 67, wherein the viral vector is an AAV vector.70. The method of any one of embodiments 57-65, wherein the delivery vehicle is a lipid nanoparticle.71. The method of embodiment 70, wherein the lipid nanoparticle is a cationic lipid nanoparticle.72. The method of any one of embodiments 57-65, wherein the delivery vehicle is a vesicle.73. The method of embodiment 72, wherein the vesicle is an extracellular vesicle, exosome, a nanovesicle, or a microvesicle.74. The method of embodiment 73, wherein the vesicle is an extracellular vesicle.75. The method of embodiment 73, wherein the vesicle is an exosome.76. The method of embodiment 73, wherein the vesicle is a nanovesicle.77. The method of embodiment 73, wherein the vesicle is a microvesicle.78. The method of any one of embodiments 57-77, wherein the delivery vehicle is disposed on the surface of a cell within the PACC.79. The method of any one of embodiments 57-78, wherein the delivery vehicle is targeted to a surface molecule.80. The method of any one of embodiments 57-79, wherein the delivery vehicle comprises a targeting moiety to a surface molecule.81. The method of any one of embodiments 79 or 80, wherein the surface molecule is a cellspecific surface protein.82. The method of embodiment 81, wherein the targeting moiety is an antibody.83. The method any one of the preceding embodiments, wherein the method comprises creating an autologous cell capable of reducing the level of a pathogenic cell (e.g., an endogenous pathogenic cell) in the subject.84. The method of any one of the preceding embodiments, wherein the population of cells is selected from monocytes, macrophages, neutrophils, basophils, eosinophils, stem cells, mast cells, and dendritic cells.85. The method of any one of the preceding embodiments, wherein the population of cells is monocytes.86. The method of any one of the preceding embodiments, wherein the population of cells is macrophages.87. The method of any one of the preceding embodiments, wherein the population of cells is neutrophils.88. The method of any one of the preceding embodiments, wherein the population of cells is basophils.89. The method of any one of the preceding embodiments, wherein the population of cells is eosinophils.90. The method of any one of the preceding embodiments, wherein the population of cells is stem cells.91. The method of any one of the preceding embodiments, wherein the population of cells is mast cells.92. The method of any one of the preceding embodiments, wherein the population of cells is dendritic cells.93. The method of any one of the preceding embodiments, wherein the population of cells is selected from B cells, T cells, effector or regulatory T cells, hematopoietic stem cells (HSCs), natural killer cells, NK T cells, g / d T cells, and plasma cells.94. The method of any one of the preceding embodiments, wherein the population of cells is B cells.95. The method of any one of the preceding embodiments, wherein the population of cells is T cells.96. The method of any one of the preceding embodiments, wherein the population of cells is effector T cells.97. The method of any one of the preceding embodiments, wherein the population of cells is regulatory T cells.98. The method of any one of the preceding embodiments, wherein the population of cells is hematopoietic stem cells (HSCs).99. The method of any one of the preceding embodiments, wherein the population of cells is natural killer cells.100. The method of any one of the preceding embodiments, wherein the population of cells is NK T cells.101. The method of any one of the preceding embodiments, wherein the population of cells is g / d T cells.102. The method of any one of the preceding embodiments, wherein the population of cells is plasma cells.103. The method of any one of the preceding embodiments, wherein the payload comprises a nucleic acid, a peptide, a polypeptide, or a small molecule.104. The method of any one of the preceding embodiments, wherein the payload comprises a nucleic acid.105. The method of any one of the preceding embodiments, wherein the payload comprises a peptide.106. The method of any one of the preceding embodiments, wherein the payload comprises a polypeptide.107. The method of any one of the preceding embodiments, wherein the payload comprises a small molecule.108. The method of embodiment 103 or 104, wherein the nucleic acid comprises DNA or RNA.109. The method of embodiment 108, wherein the nucleic acid comprises DNA.110. The method of embodiment 108, wherein the nucleic acid comprises RNA.111. The method of any one of the preceding embodiments, wherein the association of the payload to the cell is covalent or non-covalent.112. The method of any embodiment 111, wherein the association of the payload to the cell is covalent.113. The method of embodiment 112, wherein the association of the pay load to the cell is non- covalent.114. The method of any one of the preceding embodiments, wherein the subject has or is diagnosed with having a disease.115. The method of any one of the preceding embodiments, wherein the method further comprises administering to the subject an additional agent.116. The method of embodiment 115, wherein the additional agent comprises an immune- stimulatory agent.117. The method of any one of the preceding embodiments, wherein the introducing in (iii) is carried out by a patient-connected closed-loop device.118. The method of any one of the preceding embodiments, wherein between 1-10% of the cells in the population comprise the binding target.119. The method of embodiment 118, wherein between 2-9% of the cells in the population comprise the binding target.120. The method of embodiment 118, wherein between 3-8% of the cells in the population comprise the binding target.121. The method of embodiment 118, wherein between 4-7% of the cells in the population comprise the binding target.122. The method of any one of the preceding embodiments, further comprising: a) connecting a parenteral inlet to the subject, wherein the parenteral inlet is adapted to parenterally receive blood from the subject; b) permitting the blood, or a fraction thereof, from the subject to pass through the parenteral inlet to an extracorporeal cell binding (ECCB) module configured to allow extracorporeal formation of a PACC; c) maintaining conditions in the ECCB module such that cells from the subject’s blood and a payload form a PACC; and d) delivering the PACC to the subject via a parenteral outlet adapted to parenterally administer PACC to the subject.123. The method of embodiment 122, wherein the parental inlet, the ECCB module, and the parental outlet are in fluid connection.124. The method of any of embodiments 122-123, wherein each of the steps (a)-(d) occurs in a closed-loop system.125. The method of any of the preceding embodiments, wherein: i) a subject cell is taken from the subject, ii) the subject cell is contacted with a payload to form a PACC, and iii) the PACC introduced into the subject, and i-iii occur in less than 0.5, 1, 2, 4, 6, or 8 hours.126. The method of embodiment 125, wherein i-iii occur in less than 0.5 hours.127. The method of embodiment 125, wherein i-iii occur in less than 1 hour.128. The method of embodiment 125, wherein i-iii occur in less than 2 hours.129. The method of embodiment 125, wherein i-iii occur in less than 4 hours.130. The method of embodiment 125, wherein i-iii occur in less than 6 hours.131. The method of embodiment 125, wherein i-iii occur in less than 8 hours.132. The method of any one of the preceding embodiments, wherein the peripheral blood cells may comprise T cells, B cells, natural killer cells, and / or myeloid cells.133. The method of embodiment 132, wherein the peripheral blood cells comprise T cells.134. The method of embodiment 132, wherein the peripheral blood cells comprise B cells.135. The method of embodiment 132, wherein the peripheral blood cells comprise natural killer cells.136. The method of embodiment 132, wherein the peripheral blood cells comprise myeloid cells.137. The method of any one of the preceding embodiments, wherein the non-malignant disease is an allergic, autoimmune, or fibrotic disease.138. The method of embodiment 137, wherein the non-malignant disease is an allergic disease.139. The method of embodiment 137, wherein the non-malignant disease is an autoimmune disease.140. The method of embodiment 137, wherein the non-malignant disease is a fibrotic disease.141. The method of any one of the preceding embodiments, wherein the non-malignant disease comprises a disease selected from systemic lupus erythematosus (SLE), myasthenia gravis (MG), pemphigus vulgaris (PV), coeliac disease, Crohn’s disease, Grave’s disease, Hashimoto’s thyroiditis, multiple sclerosis, rheumatoid arthritis, Sjogren’s disease, ulcerative colitis, vasculitis, allergic asthma, atopic dermatitis (e.g., eczema), atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, eosinophilic esophagitis (atopic and nonatopic), hay fever, severe eosinophilic asthma (SEA), and fibrosis.142. The method of embodiment 141, wherein the non-malignant disease comprises systemic lupus erythematosus (SLE).143. The method of embodiment 141, wherein the non-malignant disease comprises myasthenia gravis (MG).144. The method of embodiment 141, wherein the non-malignant disease comprises pemphigus vulgaris (PV).145. The method of embodiment 141, wherein the non-malignant disease comprises coeliac disease.146. The method of embodiment 141, wherein the non-malignant disease comprises Crohn’s disease.147. The method of embodiment 141, wherein the non-malignant disease comprises Grave’s disease.148. The method of embodiment 141, wherein the non-malignant disease comprises Hashimoto’s thyroiditis.149. The method of embodiment 141, wherein the non-malignant disease comprises multiple sclerosis.150. The method of embodiment 141, wherein the non-malignant disease comprises rheumatoid arthritis.151. The method of embodiment 141, wherein the non-malignant disease comprises Sjogren’s disease.152. The method of embodiment 141, wherein the non-malignant disease comprises ulcerative colitis.153. The method of embodiment 141, wherein the non-malignant disease comprises vasculitis.154. The method of embodiment 141, wherein the non-malignant disease comprises allergic asthma.155. The method of embodiment 141, wherein the non-malignant disease comprises atopic dermatitis.156. The method of embodiment 141, wherein the non-malignant disease comprises eczema.157. The method of embodiment 141, wherein the non-malignant disease comprises atopy.158. The method of embodiment 141, wherein the non-malignant disease comprises chronic rhinosinusitis.159. The method of embodiment 141 , wherein the non-malignant disease comprises chronic sinusitis.160. The method of embodiment 141, wherein the non-malignant disease comprises an eosinophil-associated disease.161. The method of embodiment 141, wherein the non-malignant disease comprises atopic eosinophilic esophagitis.162. The method of embodiment 141, wherein the non-malignant disease comprises nonatopic eosinophilic esophagitis.163. The method of embodiment 141, wherein the non-malignant disease comprises hay fever.164. The method of embodiment 141, wherein the non-malignant disease comprises severe eosinophilic asthma (SEA).165. The method of any embodiment 141, wherein the non-malignant disease comprises fibrosis.166. The method of any one of the preceding embodiments, wherein the non-malignant disease is selected from systemic lupus erythematosus (SLE), an eosinophil-associated disease, and fibrosis.167. The method of embodiment 166, wherein the non-malignant disease is SLE.168. The method of embodiment 166, wherein the non-malignant disease is an eosinophil- associated disease.169. The method of embodiment 166, wherein the non-malignant disease is fibrosis.170. A method of forming in a subject a cell that is transformed or transduced with a payload, comprising: introducing a population of extracorporeally formed PACCs into the subject under conditions sufficient for transformation or transfection of the cell of the PACC with the payload of the PACC in the subject; and allowing the transformation or transfection; thereby forming in a subject with a cell transformed or transfected with a payload.171. A patient-connected closed-loop device for use with any one of the methods of any one of the preceding embodiments.All references and publications cited herein are hereby incorporated by reference.The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLESThe following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure. It will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the ail may be alternatively used.Example 1: Determining binding conditions for complexes comprising subject-derived cells and payloads in vitroThis example demonstrated the ability of the methods described herein to generate payload-subject derived cell complexes in vitro. The payload was designed to be encapsulated in a lipid nanoparticle (LNP). Hence, the binding of cells with the LNP vehicle was first evaluated. Next, binding of cells with payload-containing LNPs was evaluated. Finally, the potential for these pay load-cell complexes was assessed.Study 1In order to determine binding conditions of LNPs, small-scale cultures of peripheral blood mononuclear cells (PBMCs) were incubated with said LNPs loaded with DiIC18(5); 1,1'- dioctadecyl-3,3,3',3'- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) for 1 hour at room temperature with gentle rocking. After 1 hour at room temperature, the cells were then incubated for 24 hours and were sampled at 2, 4, 8, and 24 hours. Maximal binding at both 4°C and 37°C occurred in less than two hours with similar kinetics.Next, LNPs formulated with anti-CD3 antibody were used to determine if LNPs could be targeted to a specific subtype of PBMC, for example, T cells (FIG. 5). Various types of PBMCs (B cells, monocytes, NK cells, and T cells) were incubated with the following: DiD-loaded untargeted LNPs (formulated without anti-CD3 antibody), DiD-loaded LNPs with anti-CD3 antibody (CD3-LNPs), or were left untreated (FIG. 7A). The PBMCs were incubated at 4°C for 2 hours with the LNPs or were left untreated. The cells were then washed and incubated at 37 °C for 24 hours. After 24 hours, the cells were stained with antibodies to differentiate PBMC subtypes (anti-CD19 for B cells; CD14 for monocytes, CD16 for NK cells, and CD3 / CD4 or CD3 / CD8 for T cells) and were analyzed using flow cytometry. The data was normalized to untreated cells. CD3-LNPs were preferentially taken up by T cells compared to other lineages within the PBMC population (FIG. 7A). Moreover, negligible binding of the untargeted LNPs was observed in all cell subtypes, demonstrating binding was indeed mediated by anti-CD3 binding to the CD3 protein found on T cells.To investigate uptake of LNPs by T cells, T cells were incubated with CD3-targeted liposomes at either 4°C or 37°C to determine the rate and the timing of LNP endocytosis into the cell (FIG. 7B). Samples were taken at 0, 2, 4, 8, and 24 hours after incubation. Intracellular DiD signal peaked at 8 hours at 37°C while those incubated at 4°C did not show a change in intracellular signal after 2 hours.Next, cell viability was measured in order to determine if incubation with and binding of the LNPs was detrimental to the T cells. Cell viability was measured by flow cytometry (FIG. 9). Cells incubated with either non-targeted LNPs or CD3-targeted LNPs (FIGs. 9A and 9B) exhibited the same high viability as the untreated T cells (FIGs. 9C and 9D). Study 2The following study investigated the binding and uptake of polymeric nanoparticles by T cells.Jurkat cells were incubated for 4 hours at either 4°C or at 37°C with polymeric nanoparticlcs loaded with FITC. In order to distinguish between nanoparticlcs bound to the outside of the T cells from those that were endocytosed by the cells, Trypan blue was used to quench the signal from externally bound nanoparticles. Seven formulations of nanoparticles were used to compare binding: bPEI, bPEI: 2PBAE, PDMAEMA:4PBAE, PEG-b- PAMA:4PBAE, PBAE, PDMAEMA, and PEG-b-PAMA.After four hours, the cells and nanoparticles incubated at 4°C exhibited very little binding to up to about 30% binding. When the external signal was quenched using Trypan blue, most if not all of the signal was extinguished, indicated very little uptake of the nanoparticles by the T cells. In contrast, high levels of binding were observed after 4 hours of incubation at 37°C. After quenching with Trypan blue, high levels of internalizing were observed, particularly in cells incubated with nanoparticles formulated with PDMAEMA:4PBAE, PEG-b-PAMA:4PBAE, PBAE, PDMAEMA, and PEG-b-PAMA (FIG. 6). Nanoparticles formulated with bPEI and bPEI:2PBAE exhibited low percentages of internalization. These results indicate that binding and internalization of a payload using polymeric nanoparticles as a delivery vehicle can be affected by the makeup of the nanoparticle.Investigation was then earned out to determine whether the payload maintained functionality after internalization of the polymeric nanoparticle by the target cells. The polymeric nanoparticles were used to deliver a DNA molecule encoding a green fluorescent protein (GFP) so GFP expression could be measured. GFP expression was measured 24 hours and 48 hours post-transfection (FIGs. 8A, 8B, and 8C). For the majority of the polymeric formulations, expression could be measured after 48 hours, with the PEI nanoparticles being the exception. lurkat cells were then incubated with polymeric nanoparticles at different ratios (10 / 1, 20 / 1, 30 / 1, or 50 / 1). The nanoparticles were loaded with a DNA molecule encoding the luciferase gene. Subsequent expression of the luciferase protein was measured at each ratio for each polymeric formulation. The highest levels of expression were achieved using a nanoparticle comprising PDMAEMA:4PBAE at a 30 / 1 ratio. Significant expression was also found with nanoparticles comprising PDMAEMA:PBAE (20 / 1 and 30 / 1), PDMAEMA:2PBAE (20 / 1, 30 / 1, and 50 / 1), and PDMAEMA:4PBAE (50 / 1).Example 2. CAR-X cells generated in vivo demonstrate pathogenic B cell clearanceThis example demonstrates the ability of the CAR-X cells generated by the methods described herein to reduce a population of pathogenic cells (e.g., pathogenic B cells). Mouse modelFemale NOD. Cg-PrkdcscldIl2rgtmlw^ / SzJ mice (NSG) mice, 6-8 wk of age, were purchased from Jackson Laboratories. NSG mice were inoculated with 1.0 x 106luciferized Nalm-6 intravenously. SLE was defined in indicated animals as hair loss, edema in legs or around eyes, behavioral changes such as appetite loss, and clear decrease in health not attributable to Nalm-6 luciferase signal.MethodA device comprising an apheresis module, including washing and binding chambers of the closed-loop system described herein, was placed on an agitatable platform (FIG. 2) and subcutaneously attached to each indicated animal. Subject-derived cells were obtained by apheresis from a healthy donor. As the isolated cells moved through the chambers of the closed- loop system, the cells came into contact with an exemplary pay load (e.g., LNP containing mRNA encoding a CAR) under conditions sufficient to inhibit uptake or entry of the payload into the cells (FIG. 1). Control mice were provided either with the vehicle control (e.g., LNP with no payload) or with the payload control (e.g., LNP with scrambled RNA) in place of the payload. The resulting cell-payload complexes were washed (e.g., selected for) as they passed through the subsequent chamber and were injected into the mice by intraperitoneal infusion. ResultsFollowing the completion of the process, the mice were monitored and blood samples were collected every 4 days to evaluate expression (e.g., by qPCR and FACS) of the CAR-X cells and levels of autoantibodies and proinflammatory cytokines. The CAR-X cells were detected beginning at 4 days post-procedure. A decrease (e.g., of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or greater) in proinflammatory cytokines was observed in mice provided with the payload, but not in mice provided with the empty vehicle or the payload control. The decrease in proinflammatory cytokines persisted for 4 weeks.Example 3: Methods for Production of a Viral Vector to Deliver PACCsVector Production and Characterization.Lentiviral vectors were produced as described previously (Michels A, et al. Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted tomurine CDS. Mol Ther Methods Clin Dev. 2021 Oct 1 ;23:334-347. doi: 10.1016 / j.omtm.2021.09.014.). Briefly, a suspension of 293T cells was grown to a density of 2x106 cells / mL and transfected with relevant viral plasmids. Two days post transfection, lentiviral vector was harvested from the supernatant and clarified via centrifugation and 0.45um filtration. Clarified vector was purified via FPLCanion exchange chromatography (Akta Pure, Cytiva). Eluted samples were buffer exchanged by tangential flow filtration (Sartorius Vivaflow50, VF05P4) and sterile filtered with a 0.2uM PES syringe filter (Millipore). Vector titer was assessed using a SUP-T1 (ATCC, CRL-1942) transduction assay.Animals.Animals were housed and managed following an lACUC-approved protocol. The PBMC Sample 1 DP was surface engineered to selectively target human CD3+ T cells for binding, activation, co-stimulation, and transduction. The PBMC Sample 1 surface engineering, in particular the anti-CD3 scFv, did not cross react with other species commonly employed for pharmacology or toxicology modeling. Luciferase+ Nalm6 Tumor-bearing NSG MHCI / II KO mice were humanized by injecting 20xl06PBMCs into the intraperitoneal cavity. Mice received varying doses of VivoVec™ particles packaging of an anti-CD19 CAR transgene (FIGs. 14A and 14B).PBMC humanized mouse model (Michels A, et al. Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8. Mol Ther Methods Clin Dev. 2021 Oct l;23:334-347. doi: 10.1016 / j.omtm.2021.09.014.). Female NSG mice, 6-12 weeks of age, lacking expression of MHC I and II molecules (NSG DKO, stock number 025216), were purchased from the Jackson Laboratories. Animals were engrafted with 0.25E+06 Nalm-6 tumor cells expressing green fluorescent protein (GFP) and firefly luciferase (GFP::ffluc) via tail vein injection on study day 4. Animals were injected with 15 mg / kg of XenoLight D-Luciferin (Perkin Elmer, Waltham, Massachusetts) and evaluated after 25 min via the non-invasive In Vivo Imaging System (IVIS) Spectrum instrument (Perkin Elmer) (FIGs. 10A and 10B). Animals were humanized with 20E+06 PBMCs via intraperitoneal injection on day -1, and then dosed intraperitoneally with vehicle (10 mM Tris, 10% sucrose, 0.1% poloxamer 188 (v / v), pH 7.1), PBMC Sample 1 diluted in vehicle, or a Cocal pseudo-typed vector encoding an FMC63-RACR transgene (Cocal control) on day 0. Blood was collected for flow cytometry.Extracorporeal delivery using the extracorporeal closed-loop systemLucifcrasc+ Nalm6 Tumor-bearing NSG MHCI / II KO mice were infused with PBMCs that had been treated for 1 hour with viral delivery particles at a multiplicity of infection (MOI) of 2. Total anti-CD19 CAR+CD3+T cells found in the blood on Day 14 and tumor burden assessed over time (FIGs. 15B and 15C) as total flux and tracked over time using an In vivo imaging system (IVIS®) (FIG. 15A).The process steps performed on the extracorporeal closed-loop system are illustrated in FIG. 3 (the entire system is detailed in FIG. 4). The Operator first installed the single-use disposable sets onto each instrument and loaded ancillary solutions (i.e., anticoagulant, saline, binding buffer, and infusion buffer). The Operator then initiated the automated installation, performed integrity checks, and primed the disposable equipment. After successful installation and priming of the disposable sets, the two disposable sets were shunted together, using aseptic techniques, into a single closed-loop.The Operator then entered the whole blood volume to process, based on the animal / subject's complete blood count (CBC) and desired number of PBMCs. The subject was then connected to the extracorporeal closed-loop system via double-needle venipuncture. The subject-connected portion of the procedure started with a standard PBMC collection. After the PBMC product was collected, the return vein was kept patent during the following particle binding steps, and the draw line was sealed and removed.First, the collected PBMCs were washed to prepare the target cells for binding: where the PBMCs were concentrated, non-target cells (platelets, red blood cells) were reduced through the wash step, and the plasma was exchanged for a Binding Buffer using spinning membrane technology. The target cells were harvested at a pre-defined concentration or volume into the binding chamber where the PBMC Sample 1 was introduced via a sterile port. The extracorporeal closed- loop system provided a method to incubate the cells, PBMC Sample I, and binding buffer at a set temperature, angle, rate, and time duration while in the binding chamber. Lastly, the PBMC Sample 1 bound cells were washed using the same spinning membrane technology to prepare for reinfusion, the step where unbound PBMC Sample 1 particles were removed and the binding buffer was replaced with infusion buffer prior to being returned to the subject. The necessary duration for the procedure depended primarily on the required target total PBMCs collected, the starting CBC, and incubation duration. The activation and transduction oftarget cells (e.g., the endocytosis of the PBMC Sample 1 into T cells and subsequent translation of the payload, c.g., the CAR19 transgcnc) occurred in the subject following re-infusion.Example 4: Treating an Autoimmune Disease with PACCs of T CellsThis example characterizes the use of PACCs of T cells to treat an autoimmune disease (e.g., systemic lupus erythematosus (SLE) or pemphigus vulgaris (PV)).PACCs of T cells obtained by incubating T cells with polymeric nanoparticles comprising a lentiviral vector encoding DNA molecules encoding a T cell receptor (TCR) (e.g., a TCR useful for TCR-T therapy, e.g., a TCR capable of binding CD19 and / or CD20) were prepared and evaluated (e.g., for in vitro binding and cytotoxicity). PACCs were purified by washing.The resultant PACCs were then diluted in reinfusion buffer to obtain populations of cells comprising differing amounts of PACCs (e.g., 100 PACCs, 1,000 PACCs, 10,000 PACCs, 100,000 PACCs, 1,000,000 PACCs, 10,000,000 PACCs, or 100,000,000 PACCs). Each population of cells was administered at varying concentrations (e.g., diluted in 500 pL, 1 mL, or 2.5 mL reinfusion buffer) to mice (e.g., NZB / W Fl mice).Next, to assess the efficacy of PACCs of T cells in treating an autoimmune disease (e.g., SLE), PACCs of T cells were prepared according to the conditions described above and were administered to subjects having an autoimmune disease (e.g., SLE) and weighing at least 45 kg. Serum levels of PACCs (e.g., by RNA and / or protein quantification of the encoded TCR) were measured immediately prior to administration, 1 week-post administration, and 1 month postadministration. Symptoms were evaluated immediately prior to administration, 1 week postadministration, 1 month post-administration, and 1 year post-administration. Upon assessment of serum levels of PACCs, symptoms, and disease biomarkers (e.g., SLE biomarkers), a physician or other clinical practitioner may recommend re- administration (e.g., if serum levels of the SLE biomarkers remain constant or increase). Additional administrations may be performed as needed, e.g., as determined by a physician or other clinical practitioner, on a daily, weekly, biweekly, monthly, bimonthly, or yearly basis.Example 5: Treating an Atopic Disease with PACCs of T CellsThis example characterizes the use of PACCs of T cells to treat an atopic disease (e.g., eczema).PACCs of T cells obtained by incubating T cells with polymeric nanoparticles comprising a Icntiviral vector encoding DNA molecules encoding a T cell receptor (TCR) (e.g., a TCR useful for TCR-T therapy, e.g., a TCR capable of binding IgE) were prepared and evaluated (e.g., for in vitro binding and cytotoxicity). PACCs were purified by washing.The resultant PACCs were then diluted in reinfusion buffer to obtain populations of cells comprising differing amounts of PACCs (e.g., 100 PACCs, 1,000 PACCs, 10,000 PACCs, 100,000 PACCs, 1,000,000 PACCs, 10,000,000 PACCs, or 100,000,000 PACCs). Each population of cells was administered at varying concentrations (e.g., diluted in 500 L, 1 mL, or 2.5 mL reinfusion buffer) to mice (e.g., Adaml7fl / fl Sox9-Cre mice).Next, to assess the efficacy of PACCs of T cells in treating an atopic disease (e.g., eczema), PACCs of T cells were prepared according to the conditions described above and were administered to subjects having an atopic disease (e.g., eczema) and weighing at least 45 kg. Serum levels of PACCs (e.g., by RNA and / or protein quantification of the encoded TCR) were measured immediately prior to administration, 1 week-post administration, and 1 month postadministration. Symptoms were evaluated immediately prior to administration, 1 week postadministration, 1 month post-administration, and 1 year post-administration. Upon assessment of serum levels of PACCs, symptoms, and disease biomarkers (e.g., atopic disease biomarkers), a physician or other clinical practitioner may recommend re-administration (e.g., if serum levels of the atopic disease biomarkers remain constant or increase). Additional administrations may be performed as needed, e.g., as determined by a physician or other clinical practitioner, on a daily, weekly, biweekly, monthly, bimonthly, or yearly basis.Example 6. Nonclinical in vivo pharmacology model demonstrating POC for extracorporeal administration of PBMC Sample 1Extracorporeal incubation and PBMC Sample 1 T-cell binding.PBMC Sample 1 -extracorporeal closed-loop system proof-of-concept studies demonstrated consistently high levels (greater than 85%) of CD3+ T cell binding by PBMC Sample 1. PBMC Sample 1 particles preferentially bound CD3-T cells; particles were retained on the cell surface during the ECGD device wash procedure (FIG. 4A). Cell viability was maintained at high levels throughout the extracorporeal particle binding procedure and wash (FIG. 10B). To evaluate PBMC Sample 1 particle binding to PBMCs, PBMC Sample 1 was injected into the binding chamber of the extracorporeal closed-loop system at an MOI of 2,following loading of the PBMCs from in-line wash of apheresis material, and incubated for 1 hour. Following incubation, cells were washed, harvested, and evaluated for particle binding based on flow cytometry detection of cellular with bound viral fusion glycoprotein.The selectivity for PBMC Sample 1 binding to T cells was illustrated both by the frequency of fusion glycoprotein+CD3+cells, as well as the magnitude of fusion glycoprotein flow cytometry staining measured as the geometric mean fluorescence intensity (MFI) of the fluorescent anti-fusion glycoprotein antibody (FIGs. 11 A and 1 IB). There was a clear positive shift both in frequency and intensity of fusion glycoprotein staining in the CD3+ T cell population compared to the CD3' population (FIG. 5A). The magnitude of PBMC Sample 1 particle binding to the CD3+and CD3" populations was compared quantitatively by calculating the ratio of fusion glycoprotein MFI in the CD3+to the CD3 populations to further reinforce the preferential binding to the CD3+population (FIG. 10A).PBMC Sample 1 bound to conventional CD4 and CD8 T cells as well as NKT cells during the extracorporeal incubation. Some NK cells also bound to PBMC Sample 1 due to their expression of CD2, although to a lesser extent than the CD3’ expressing cell types (FIG. 12). This binding pattern reflected the specificity and avidity afforded by the PBMC Sample 1 surface engineering (CD58 BD-CD58 ECD-anti CD3 scFv-CD80 BD-CD80 ECD) to direct specific binding to cells with cognate ligands / receptors. Because NKT cells express both CD3 and CD2 and NK cells express CD2, the small fraction of NKT and NK cells bound to PBMC Sample 1 was not indicative of off-target binding. Indeed, PBMC Sample 1 binding to NKT, NK cells, and monocytes in addition to the target T cells would be beneficial to the intended anti-tumor function, as those cells, once activated and transduced, would work in combination with the CAR T cell population generated from the conventional CD4 and CD8 T cell populations.T cell activation and transduction in vitro following PBMC Sample 1 incubation and wash in the extracorporeal closed-loop system. Following completion of the extracorporeal closed-loop procedure, PBMC Sample 1-bound washed material was cultured in vitro at 37°C to model return to the patient to evaluate T cell activation and transduction. T cell activation, as measured by upregulation of CD25, was observed in the PBMC Sample 1-bound washed material on Days 3 and 8 post ECGD procedure (FIG. 12A). Anti-CD19 CAR expression was detected on Day 3 and further increased on Day 8 post PBMC Sample 1 binding to cells in the extracorporeal closed-loop system (FIG. 12B).In vitro evaluation of transduced cell populations following extracorporeal incubation demonstrated that the PBMC Sample 1 surface engineering and MOA resulted in selective targeting and transduction of CD3+ T cells. CD3+ T cells were the only CAR-expressing cell type quantified in the in vitro culture (FIG. 13).Representative flow cytometry analysis of PBMCs cultured in vitro following extracorporeal incubation with PBMC Sample 1 on the extracorporeal closed-loop system. PBMC control cells were circulated through the extracorporeal closed-loop system in the absence of PBMC Sample 1. Cells were harvested from in vitro culture on Day 8 post extracorporeal incubation and stained with reagents to detect cell surface CD3 and the anti-CD19 CAR (FIGs. 13A and 13B).Next, a tumor-bearing immune-deficient mouse model was employed to evaluate proof- of-concept for the PBMC Sample 1 mechanism of action in the context of extracorporeal delivery. The NSG MHCI / II double knockout (NSG-(KbDb)null (IA)null, NSG-(Kb Db)null (lAnull)) mouse strain was engrafted with the Nalm6 B-ALL tumor cell line engineered to express firefly luciferase (ffluc) to enable in-life quantitative measure of tumor burden. The studies described herein employed the extracorporeal closed-loop system to generate the PBMC Sample 1 -bound human T cells generated from separately collected apheresis material from 2 healthy donors.Extracorporeal incubation of PBMC Sample 1 with healthy donor apheresis material (at an MOI of 2) at full-scale on the ECGD device resulted in rapid CAR T cell generation in vivo in Nalm6 tumor-bearing immune-deficient mice following administration of PBMC Sample 1- bound cells. Cells derived from the post-extracorporeal incubation and washed material were administered intraperitoneally (IP). The CAR T cell numbers peaked at Day 14 and then waned (FIGs. 14A and 14B) concomitant to the reduction in CD 19+ tumor burden (FIGs. 15B and 15C). Animals that received Donor 1 cells at the higher cell number exhibited increased CAR T cell numbers on Day 7 in comparison to mice that received Donor 2 cells, however the CMAX and TMAX were consistent between donors.The generation of CAR T cells resulted in potent anti-tumor activity as demonstrated by a consistent and dramatic reduction in tumor burden across all animals that received PBMC Sample 1 -bound cells following extracorporeal incubation on the extracorporeal closed-loop system (FIGs. 15A, 15B, and 15C).The number of putative CAR T cells infused per animal was estimated by multiplying the in vitro transduction frequency measured on Day 7 post extracorporeal incubation on the extracorporeal closed-loop system by the total number of cells infused per animal (data not shown). Dosing based on lentiviral particle concentration together with a target cell number and concentration on the extracorporeal closed-loop system appeared to be most effective for transducing T cells to express the CAR.

Claims

CLAIMS1. A method of providing a subject with a population of payload-associated cell complexes (PACCs) for the treatment of a non-malignant disease, comprising:(i) providing a population of peripheral blood cells, e.g., a population of peripheral blood cells from the subject, wherein a peripheral blood cell in the population of peripheral blood cells comprises a binding target;(ii) extracorporeally contacting the population of peripheral blood cells with a payload comprising a chimeric antigen receptor (CAR) construct under conditions (e.g., time, temperature) sufficient for association of the payload with the cell comprising the binding target, wherein the conditions are not sufficient for entry of the payload into the cell with which it is associated, thus forming a population of PACCs; and(iii) introducing the population of PACCs into the subject, thereby providing subject with a population of PACCs for the treatment a non-malignant disease.

2. The method of any of claim 1, wherein the population comprises at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1,000, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, or more PACCs.

3. The method of claim 1, wherein the population comprises at least 100,000 or more PACCs.

4. The method of claim 1, wherein the population comprises at least 1 million or more PACCs.

5. The method of claim 1, wherein the population comprises at least 10 million or more PACCs.

6. The method of claim 1, wherein the population comprises at least 100 million or more PACCs.

7. The method of claim 1 , wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 0.5%, 1%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the total cells in the population8. The method of claim 1, wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 50%, 60%, 70%, 80%, 90%, 95%, or more, or more of the total cells in the population.

9. The method of claim 1, wherein the percentage of peripheral blood cells associated with the payload in the population is between 30%-90% total cells, e.g., 50%-80% total cells in the population.

10. The method of claim 1, wherein the conditions sufficient for association of the payload with a peripheral blood cell within the population comprise:(i) contacting the peripheral blood cells with the payload for between about 0 to about 10 hours, e.g., about 1 hour to about 9 hours, about 2 hours to about 8 hours.

11. The method of claim 10, wherein contacting the peripheral blood cells with the payload is between about 5 minutes and 30 minutes.

12. The method of claim 10, wherein contacting the peripheral blood cells with the pay load is between about 30 minutes and 1 hour.

13. The method of claim 10, wherein contacting the peripheral blood cells with the pay load is between about 1 hour and 1.5 hours.

14. The method of claim 1, wherein the PACCs are formulated in or on a delivery vehicle.

15. The method of claim 14, wherein the delivery vehicle comprises a lipid nanoparticle, viral vector, vesicle, or a liposome in which the payload is disposed.

16. The method of claim 14, wherein the concentration of the delivery vehicle is higher than the concentration of peripheral blood cells in the sample.

17. The method of claim 14, wherein the concentration of the delivery vehicle is lower than the concentration of peripheral blood cells in the sample.

18. The method of claim 14, wherein the concentration of the delivery vehicle is optimized for binding to peripheral blood cells in the subject sample.

19. The method of claim 1, wherein the binding of the pay load to the peripheral blood cells occurs at a selected temperature 0 to 40 degrees Celsius (e.g., 4 °C, 37 °C).

20. The method of claim 1, wherein the pay load is disposed on the surface of a peripheral blood cell within the PACC.

21. The method of claim 14, wherein the delivery vehicle is a viral vector.

22. The method of claim 21, wherein the viral vector is a lentiviral vector or an adeno- associated viral (AAV) vector.

23. The method of claim 14, wherein the delivery vehicle is a lipid nanoparticle.

24. The method of claim 23, wherein the lipid nanoparticle is a cationic lipid nanoparticle.

25. The method of claim 14, wherein the delivery vehicle is a vesicle.

26. The method of claim 25, wherein the vesicle is an extracellular vesicle, exo some, a nanovesicle, or a microvesicle.

27. The method of claim 14, wherein the delivery vehicle is disposed on the surface of a cell within the PACC.

28. The method of claim 14, wherein the delivery vehicle is targeted to a surface molecule.

29. The method of claim 14, wherein the delivery vehicle comprises a targeting moiety to a surface molecule.

30. The method of any one of claims 28 or 29, wherein the surface molecule is a cell-specific surface protein.

31. The method of claim 30, wherein the targeting moiety is an antibody.

32. The method of claim 1, wherein the method comprises creating an autologous cell capable of reducing the level of a pathogenic cell (e.g., an endogenous pathogenic cell) in the subject.

33. The method of claim 1, wherein the population of cells is selected from monocytes, macrophages, neutrophils, basophils, eosinophils, stem cells, mast cells, and dendritic cells.

34. The method of claim 1, wherein the population of cells is selected from B cells, T cells, effector or regulatory T cells, hematopoietic stem cells (HSCs), natural killer cells, NK T cells, g / d T cells, and plasma cells.

35. The method of claim 1, wherein the pay load comprises a nucleic acid, a peptide, a polypeptide, or a small molecule.

36. The method of claim 35, wherein the nucleic acid comprises DNA or RNA.

37. The method of claim 1, wherein the association of the pay load to the cell is covalent or non-covalent.

38. The method of claim 1, wherein the subject has or is diagnosed with having a disease.

39. The method of claim 1, wherein the method further comprises administering to the subject an additional agent, e.g., an immune-stimulatory agent.

40. The method of claim 1, wherein the introducing in (iii) is carried out by a subject- connected closed-loop device.

41. The method of claim 1, wherein between 1-10% of the cells in the population comprise the binding target.

42. The method of claim 1, further comprising: a) connecting a parenteral inlet to the subject, wherein the parenteral inlet is adapted to parenterally receive blood from the subject; b) permitting the blood, or a fraction thereof, from the subject to pass through the parenteral inlet to an extracorporeal cell binding (ECCB) module configured to allow extracorporeal formation of a PACC; c) maintaining conditions in the ECCB module such that cells from the subject’s blood and a payload form a PACC; and d) delivering the PACC to the subject via a parenteral outlet adapted to parenterally administer PACC to the subject.

43. The method of claim 42, wherein the parental inlet, the ECCB module, and the parental outlet are in fluid connection.

44. The method of claim 42, wherein each of the steps (a)-(d) occurs in a closed-loop system.

45. The method of claim 42, wherein: i) a subject cell is taken from the subject, ii) the subject cell is contacted with a payload to form a PACC, and iii) the PACC introduced into the subject, and i-iii occur in less than 0.5, 1, 2, 4, 6, or 8 hours.

46. The method of claim 42, wherein the peripheral blood cells may comprise T cells, B cells, natural killer cells, and / or myeloid cells.

47. The method of claim 1, wherein the non-malignant disease is an allergic, autoimmune, or fibrotic disease.

48. The method of claim 1, wherein the non-malignant disease comprises a disease selected from systemic lupus erythematosus (SLE), myasthenia gravis (MG), pemphigus vulgaris (PV), coeliac disease, Crohn’s disease, Grave’s disease, Hashimoto’s thyroiditis, multiple sclerosis, rheumatoid arthritis, Sjogren’s disease, ulcerative colitis, vasculitis, allergic asthma, atopic dermatitis (e.g., eczema), atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, eosinophilic esophagitis (atopic and non-atopic), hay fever, severe eosinophilic asthma (SEA), and fibrosis.

50. The method of claim 1, wherein the non-malignant disease is selected from systemic lupus erythematosus (SLE), an eosinophil-associated disease, and fibrosis.

51. The method of claim 1 , wherein the subject has a disease or disorder, wherein the disease or disorder is a non-malignant disease.

52. The method of claim 51, wherein the non-malignant disease is an allergic, autoimmune, or fibrotic disease.

53. The method of claim 52, wherein the non-malignant disease is selected from systemic lupus erythematosus, myasthenia gravis, pemphigus vulgaris, allergic asthma, atopic dermatitis, atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, and fibrosis.

54. The method of claim 1, wherein the subject is treatment naive, wherein treatment naive may comprise one or more of the following:(i) the subject is currently undergoing treatment for the non-malignant disease, but is not currently being treated according to the claimed method;(ii) the subject is currently being treated for one or more symptoms of the non-malignant disease, but is not being treated for the underlying mechanism of the disease;(iii) the subject is being treated for one or more symptoms of a non-malignant disease, but has not been diagnosed with the disease;(iv) the subject has previously received treatment for the non-malignant disease, but the treatment was insufficient to alleviate the symptom(s) it was administered to treat and / or the treatment did not inhibit or reduce the progression of the disease;(v) the subject has previously received treatment for the non-malignant disease, and the treatment was successful, but the symptom(s) has / have since recurred and / or the disease state has since progressed; or(vi) the subject has never been treated for the non-malignant disease or for any symptoms of the non-malignant disease.

55. The method of claim 1, wherein the subject has been previously treated with one or more therapeutics directed to non-malignant diseases.

56. A method of forming in a subject a cell that is transformed or transduced with a payload, comprising: introducing a population of extracorporeally formed PACCs into the subject under conditions sufficient for transformation or transfection of the cell of the PACC with the payload of the PACC in the subject; and allowing the transformation or transfection; thereby forming in a subject with a cell transformed or transfected with a payload.

57. A subject-connected closed- loop device for use with any one of the methods of any one of the preceding claims.

58. A viral vector for use with any one of the methods of any one of the preceding claims, wherein the viral vector may be selected from an adenoviral vector, an adenoviral-associated vector, a lentiviral vector, or a virus-like particle.

59. The viral vector of claim 58, wherein the viral vector is an adenoviral vector or an adenoviral-associated vector.

60. The viral vector of claim 58, wherein the viral vector is a lentiviral vector.

61. The viral vector of any one of claims 58-60, wherein the viral vector expresses a glycoprotein.

62. The viral vector of claim 61, wherein the glycoprotein is a Cocal glycoprotein.

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